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New Mode (Molecular-Sensing) of Heinz Body Formation Mechanisms Inherent in Human Erythrocytes: Basis for Understanding of Clinical Aspects of Drug-Induced Hemolytic Anemia and the Like
ISSN: 1948-593X
Journal of Bioanalysis & Biomedicine

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New Mode (Molecular-Sensing) of Heinz Body Formation Mechanisms Inherent in Human Erythrocytes: Basis for Understanding of Clinical Aspects of Drug-Induced Hemolytic Anemia and the Like

Yoshiaki Sugawara*, Yuki Shigemasa, Yuko Hayashi, Yoko Abe, Ikumi Ohgushi and Eriko Ueno

Department of Health Science, Prefectural University of Hiroshima, Hiroshima 734-8558, Japan

*Corresponding Author:
Yoshiaki Sugawara
Department of Health Science
Prefectural University of Hiroshima
Hiroshima 734-8558, Japan
Tel: +81-82-251-9783
Fax: +81-82-251-9405
E-mail: [email protected]

Received May 02, 2013; Accepted Date: June 12, 2013; Published Date: June 15, 2013

Citation: Sugawara Y, Shigemasa Y, Hayashi Y, Abe Y, Eriko Ueno IO (2013) New Mode (Molecular-Sensing) of Heinz Body Formation Mechanisms Inherent in Human Erythrocytes: Basis for Understanding of Clinical Aspects of Drug-Induced Hemolytic Anemia and the Like. J Bioanal Biomed 5: 036-056. doi: 10.4172/1948-593X.1000078

Copyright: © 2013 Sugawara Y, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

The human hemoglobin (Hb) molecule (α2β2) has two types of α–β interface (i.e., α1–β1[and α2–β2] and α1–β2[and α2–β1]). The latter α1–β2(and α2–β1) interface is associated with cooperative O2 binding, and exhibits principal roles if the molecule goes from its deoxygenated to oxygenated quaternary structure. The role of the former α1–β1(and α2–β2) interface has been unclear for a long time. In this regard, important and intriguing observations have been accumulating, so that a new gaze can be focused on the α1–β1(and α2–β2) interface. Our most recent findings suggest that the α1–β1(and α2–β2) interface may exert delicate control of the intrinsic tilting capability of the distal (E7) His residues (i.e., α58His (E7) in the α chain and β63His (E7) in the β chain) depending on internal and external conditions of the erythrocyte to lead to degradation of Hb to hemichrome, and subsequent clustering of Heinz bodies within the erythrocyte. In the spleen, rigid intra-erythrocytic hemichrome inclusions (Heinz bodies) act as “sticking points”, so Heinz body-containing red cells become trapped and undergo hemolysis. In this article, we first provide our necessary basic experimental findings that led us to grasp molecular biosensing mechanisms inherent in human erythrocytes for the appreciation of aging and determination of their lifespan, and summarize their roles in physiology. We then discuss how these accomplishments contribute to deeper understanding of clinical aspects of drug-induced hemolytic anemia, defects in the intra-erythrocytic reducing system and unstable Hb disease, in which the mechanisms for acute hemolytic crisis cannot be explained on the basis of conventional views.

Keywords

Red cell aging; Hemichrome formation; Heinz-body clustering; Drug-induced hemolytic anemia; Defects in the intraerythrocytic reducing system; Unstable hemoglobin disease

Introduction

Human red blood corpuscles survive in the circulation for an average of 120 days. Removal of aged and damaged red cells from the blood circulation is essential for its homeostasis. How do human erythrocytes appreciate aging and determine their lifespan? In a series of studies, we have attempted to examine the relationship between the oxidative behavior of human hemoglobin (Hb) molecules (i.e., oxidation of HbO2 by the bound O2 to the ferric met-form), hemichrome emergence and the formation of Heinz bodies within the erythrocytes.

Heinz bodies are intra-erythrocytic inclusions of hemichrome formed from oxidized or denatured hemoglobin (Hb). Heinz bodies are typically formed in aged red cells [1]. However, they are rarely mentioned in the context of normal Hb or normal erythrocytes. Heinz bodies have been characterized in drug-induced hemolytic anemia, defects in the intra-erythrocytic reducing system (e.g., glucose 6-phosphate dehydrogenase [G-6-PD] deficiency) and in unstable Hb disease [2,3]. Nevertheless, Heinz bodies in normal erythrocytes are of interest because they (or their related intra-erythrocytic inclusions) are involved in the recognition mechanisms in the spleen responsible for the removal of non-functional erythrocytes from the circulation. The rigid intra-erythrocytichemichrome inclusions are known to act as “sticking points”, and hence Heinz body-containing red cells become trapped and undergo hemolysis [4].

Hemichrome is rarely found in erythrocytes in situ, even though the reaction dynamics of Hb with molecular oxygen (O2) make them particularly suitable O2 carriers. Hb can bind O2 in the ferrous state to carry out its physiological functions. During this reversible binding of O2, the oxygenated form of Hb (HbO2) is known to be oxidized by the bound O2 to the ferric met form (metHb), which cannot be oxygenated, and is thus physiologically inactive. The resultant met Hb is reduced back to the ferrous state by an intra-erythrocyticnicotinamide adenine dinucleotide (NADH)-dependent reducing system [5-7]. However, it has been suggested that its oxidation (autoxidation) can be followed by transformation of the oxidized molecule (high-spin Fe3+) into a species absorbing as a low-spin compound, i.e., hemichrome, the formation of which can result in the accumulation of soluble and insoluble hemichromes as well as precipitation [8-13]. Despite these findings, direct evidence of hemichrome formation in normal erythrocytes is lacking.

Compared with the tetrameric parent Hb, hemichrome formation is enhanced in separated α and β chains [8,13-16]. Following the method of Brunori et al. [14], our ultraviolet/visible (UV/VIS) spectroscopic study [17] showed that human adult Hb (HbAO2 or simply designated as HbO2) from healthy donors tended to degrade to produce hemichrome even at close-to-physiological temperatures and pH. However, its occurrence was a function of pH, temperature and the progress of autoxidation of ferrous HbO2 to the ferric met form through oxidation by bound O2.

Cellular life is reliant upon rapid and efficient responses to internal and external conditions. The basic molecular events associated with these processes are the structural transitions of the proteins involved [18]. Therefore, understanding of the structural basis of protein allostery is of paramount importance to characterize these processes. The human Hb molecule (α2β2; alternatively, a dimer of αβ protomers), whose α and β chains contain 141 and 146 amino-acid residues, respectively, holds a special position in these structural transitions. This is due to the achievements of Perutz, whose pioneering studies led to the identification of two distinct Hb structures, the tense (T) and relaxed (R) state, which are associated with the deoxygenated and oxygenated form of the protein, respectively [19-21]. Hb has two types of αβ interface (i.e., α1β1 [and α2β2] and α1β2 [and α2β1]). The latter α1–β2(and α2–β1) interface is known to be associated with cooperative dioxygen (O2) binding, and exhibits important roles if the molecule goes from its deoxygenated to the oxygenated quaternary structure. However, the role of the former α1–β1(and α2–β2) interface has been unclear for a long time.

A representative set of successive O2-binding constants regarding human Hb is given in terms of mmHg–1as follows: K1 = 0.0188, K2 = 0.0566, K3 = 0.407, K4 = 4.28 in 0.1 M buffer (pH 7.4) containing 0.1 M KCl at 25°C [22]. In this reaction, by comparing their X-ray crystal structures, major differences have been defined between deoxygenated and oxygenated forms. These include: movement of the iron atom into the heme plane with a simultaneous change in the orientation of the proximal (F8) His; rotation of the α1β1 dimer relative to the other α2β2 dimer about an axis P by 12–15°; and a translation of one dimer relative to the other along the P axis by ~1 Å (10–1 nm). These changes are accompanied by sequential breaking of “salt bridges” by C-terminal residues [23-27]. When HbO2 goes from the deoxygenated to the oxygenated quaternary structure, the α1–β2(and α2–β1) interface undergoes the principal changes associated with cooperative oxygen binding, so it is named the “sliding contact” that involves 19 residues, including mainly helices C and H and the FG corner [25,28].

Conversely, negligible changes are found with respect to the crystal structure examined for the α1–β1(and α2–β2) interface that associates 35 residues including B, G, and H helices as well as the GH corner. Understanding of subunit interactions between the four Hb chains (and how these explain cooperative O2 binding) has been the primary focus in Hb research. This has been coupled with a tendency for structural analyses to focus on the changes at the proximal side of the heme and at the α1–β2(and α2–β1) interface, as mentioned above. This is despite the fact that the configuration of the residues lining the distal side of the heme pocket (where molecular O2 binds) are also altered by oxygenation, and are thought to have a role in controlling access of the ligand to the heme pocket [29]. Hence, the possibility of subunit interactions originating from or being transmitted via distal side effects has, for the most part, been neglected.

In this regard, important and intriguing observations have been accumulating while paying attention to the α1–β1(and α2–β2) interfaceas well as distal-side perturbations. Hence, a new gaze could be focused on the α1–β1(and α2–β2) interface and the distal-side perturbations of the heme pocket. With respect to the α1–β1(and α2–β2) interface, a new role has been attributed to stabilizing the HbO2 tetramer against acidic autoxidation. That is, the α1–β1(and α2–β2) interface produces a conformational constraint in the β chain whereby the distal (E7) histidine (His) residue is tilted slightly away from the bound O2 so as to prevent proton-catalyzed displacement of O2– by a solvent water molecule [30,31]. The β chains thus acquire pH-dependent delayed autoxidation in the HbO2 tetramer. The next role was suggested by our studies searching for similar phenomena in normal human erythrocytes under mild heating [17,32,33]. It seemed that tilting of the distal (E7) His in turn triggers degradation of the Hb molecule to hemichrome, and subsequent clustering of Heinzbodies within the erythrocyte. In the spleen, Heinz body-containing red cells become trapped while traversing small apertures in the basement membranes separating the cords from the sinusoids (where the spaces are sufficiently small to require extreme deformation of red cells) and undergo hemolysis [4]. Thus, it was suggested that the Hb molecule controls removal of erythrocytes from the blood circulation by triggering degradation of the Hb molecule to hemichrome, and subsequent clustering of Heinz bodies within the erythrocytes depending on the internal and extraneous conditions of the erythrocyte (including pH and temperature). Hence, in our recent publication [34], we reviewed and summarized current interpretations of the oxidative behavior of human Hb and the related results. We emphasized the correlation between hemichrome emergence and the formation of Heinz bodies within the erythrocytes. In relevance to a clinical aspect, we also suggested that a quite large number of unstable Hbs are resulted from substitutions affecting on the β chain, especially in the α1–β1(and α2–β2) interface including B, G, and H helices: Tacoma [β30(B12)Arg → Ser], Abraham Lincoln [β32(B14)Leu → Pro], Peterborough [β111(G13)Val → Phe], Madrid [β115(G17)Ala → Pro], J. Guantanamo [β128(H6)Ala → Asp], Leslie [β131(H9)Gln → deleted] and so on.

In this article, we first provide an overview of our necessary basic analytical and bioanalytical methods and their findings that led us to grasp molecular biosensing mechanisms inherent in human erythrocytes for the appreciation of aging and determination of their lifespan, and summarize their roles in physiology, whereby the intrinsic tilting capability of distal (E7) His residues (their bis-histidyl coordination proficiency to the heme iron) and the α1–β1(and α2–β2) interface of the Hb molecule has major responsibility. Along with these lines of accomplishments and by integrating the most recent state of Hb research, we attempt to form a new mode of Heinz body formation mechanisms, in which built-in sensors in Hb molecule (i.e., bis-histidyl coordination proficiency of distal (E7) His residues and the α1–β1(and α2–β2) interface) can control degradation of the Hb molecule to hemichrome depending on internal and external conditions in the erythrocyte, and subsequent clustering of Heinz bodies within the erythrocyte. Granting this new mode (molecular sensing) of Heinz body formation mechanisms to be basic premise, we then examine how these new views contribute to deeper understanding of clinical aspects of hemoglobinopathies and hemolytic anemia and its associated acute blood loss, in which the mechanisms for acute hemolytic crisis cannot be explained on the basis of conventional views.

Experimental Accomplishments

Hemichrome formation (Degradation of the Hb molecule to Hemichrome) observed in Human Hb (HbO2) even at physiological pH and temperature: UV/VIS spectroscopic observations

The dynamics of the reaction of human Hb with O2 provide them particularly suitable O2 carriers. In the O2 binding process of Hb, the iron remains in the ferrous state so that the reaction is an oxygenation, not an oxidation. However, once the molecule is oxygenated, the resultant species (HbO2) must undergo oxidation by the bound O2 to the ferric met-form. If HbO2 is oxidized (autoxidized), the product (ferric metHb) cannot be oxygenated in its present form.

In this regard, it has been suggested that the autoxidation process of Hb may be associated with transformation of the oxidized molecule (high-spin Fe3+) into hemichrome, the formation of which can result in the accumulation of soluble and insoluble hemichromes as well as precipitation [8-13]. Formation of hemichrome is known to be enhanced in separated α and β chains [9,13-17]. So, we deal first the innate molecular instability of Hb, which triggers degradation of the molecule to hemichrome. This process is detected readily through UV/VIS spectroscopic observation during the entire process of autoxidation of human HbO2 [17].

HbO2 was prepared from freshly drawn samples of human blood (total, 50–80 mL) obtained from healthy donors. The details can be referred to Sugawara et al. [35]. The autoxidation of HbO2 was measured by UV/VIS spectroscopic means in 0.1 M buffer over a wide pH range (5.3–10.5) at 37°C. Two milliliters of solution containing 0.2 M buffer was placed in a test tube and incubated in a water bath maintained at 37 ± 0.1°C using a NESLAB temperature control system (Model RTE-100 or 111 or 210; NESLAB Instruments, Inc., Portsmouth, NH, USA). The reaction was started by adding the same volume of fresh HbO2 solution (50–125 μM in heme contents). The reaction mixture was then quickly transferred to a spectrophotometric cell (Spectrocell, Type Inject-A-Cell; Funakoshi Co., Tokyo, Japan) with a screwcap stopper. Changes in absorption at 450–650 nm were recorded on the same chart at measured time intervals. Spectra were recorded using an UV/VIS spectrophotometer (JASCO, Model Ubest-50 or V-560 or V-570; Japan Spectroscopic Co., Tokyo, Japan), equipped with a thermostatically controlled (within ± 0.1°C) cell holder. At the final state of each run, Hb molecules were all completely converted to the ferric met form by addition of potassium ferricyanide. The buffers used were: acetate for pH 4.5–5.5, 2-(N-morpholino) ethanesulfonic acid monohydrate (MES) for pH 5.0-6.75, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) for pH 6.55-8.3, 2-(cyclohexylamino) ethanesulfonic acid (CHES) for pH 8.2-10.2, and 3-cyclohexylaminopropanesulfonic acid for pH 10.0-10.5.

Figure 1 indicates how autoxidation is inseparably related to the instability of the Hb molecule and its degradation to hemichrome. Figure 1(a) shows the spectrophotometric changes over time from 450 nm to 650 nm when fresh HbO2 was placed in 0.1 M MES buffer (pH 5.0) at 37°C. HbO2 was autoxidized to its ferric met form (metHb) without showing any hemichrome formation during the entire process. Contrary to this, the situations demonstrated in Figures 1b-1d are very different. Figure 1(b) illustrates the observed UV/VIS spectra with time for hemichrome formation during autoxidation of HbO2 when HbO2was incubated in 0.1 M MES buffer (pH 6.5) at 40°C. HbO2 was autoxidized to its ferric met form. However, sudden disruption of the spectra was observed during the late stage of the reaction whereby hemichrome formation could bedetected by elevation of the baseline and a shift in isosbestic points caused by precipitation. Similarly it took place at the intermediate stage in 0.1 M HEPES buffer (pH 8.0) at 40°C in the case of Figure 1(c), while at the initial stage in the case of Figure 1(d) in which immediately after fresh HbO2 was transfer to the cuvette in 0.1 M HEPES buffer (pH 7.0) at 45°C.

bioanalysis-biomedicine-hemichrome-redrawn

Figure 1: Hemichrome formation associated with human HbO2 autoxidation, redrawn from Sugawara et al. [17]. UV/VIS spectral changes over time for hemichrome formation associated with human HbO2 autoxidation in which the reaction was monitored (a) in 0.1 M MES buffer (pH 5.0) at 37°C and scanning interval = 15 min; (b) in 0.1 M MES buffer (pH 6.5) at 40°C and scanning interval = 150 min; (c) in 0.1 M HEPES buffer (pH 8.0) at 40°C and scanning interval = 150 min; (d) in 0.1 M HEPES buffer (pH 7.0) at 45°C and scanning interval = 60 min. Conditions: HbO2 concentration was 235 μM (in heme contents).

In brief, occurrence of pH- and temperature-dependent hemichrome formation could be readily detected in HbO2 at every stage during the course of autoxidation (i.e., during the initial, intermediate, and final stages) while varying the temperature of the solution from 35°C to 55°C and the pH from 4.5 to 10.5. In Figure 2a,we therefore attempted to categorize the phenomenon into the following four cases in terms of [HbO2]t = E.P./[HbO2]0, as itsemergence was a function of not only the pH and temperature of the solution, but also of the progress of the autoxidation of HbO2:

(1) t= 0 or [HbO2]t = E.P./[HbO2]0 = 1 ≤ tE.P.< [HbO2]t = E.P./[HbO2]0 = 0.75;

(2) [HbO2]t = E.P./[HbO2]0 = 0.75 ≤ t E.P< [HbO2]t = E.P./[HbO2]0 = 0.25;

(3) tE.P. ≤ [HbO2]t = E.P./[HbO2]0 = 0.25;

(4) no hemichrome formation during the entire process.

bioanalysis-biomedicine-hemichrome-temperature

Figure 2: Hemichrome formation associated with human HbO2 autoxidation. (a) pH−temperature diagram for HbO2 autoxidation. (b) pH−temperature diagrams for the autoxidation of isolated α and β chains. The symbols used are as follows: ●− hemichrome formation noticeable at the initial stage during the course of autoxidation; ▲− at the intermediate stage; △− at the final stage; ○− autoxidation reaction proceeded with no hemichrome formation during the entire process.

Here E.P. is the observed emergence point of hemichrome formation in each run. [HbO2]t = E.P./[HbO2]0 is the ratio of HbO2 concentration after time t = E.P. to that at time t = 0 and can be monitored by the absorbance ratio of (At – A)/(A0 – A) at 576 nm (α-peak of HbO2). [HbO2]t = E.P./[HbO2]0 = 0.5 represents equal mixtures of HbO2 and metHb, i.e., the midpoint of the autoxidation reaction. Hence, case (1) means that hemichrome formation was noticeable at the initial stage of autoxidation. Accordingly, case (2) indicates its occurrence at the intermediate stage, and case (3) at the final stage. In Figure 2a, the symbols used correspond to: ● for case (1), ▲ for case (2), △ for case (3) and ○ for case (4). To determine if the phenomenon was represented by case (3) or case (4), the reaction mixture was converted to metHb by the addition of small amounts of ferricyanide and maintained at the given temperaturefor 2 days to see if hemichrome precipitation occurred.Thus, the findings suggested that human HbO2 was highly susceptible to hemichrome formation, even under physiological pH and temperature. The broken lines in Figure 2a show that the threshold for this susceptibility in relation to pH and temperature.

On the other hand, Figure 2b manifests that the isolated α (αp-MB) and β (βp-MB) chains might have much higher susceptibilities to hemichrome formation, when compared with the tetrameric parent molecule,and possess individual susceptibility as detailed in their pH–temperature diagrams. Isolation of α and β chains from the parent molecules was made using sodium p-hydroxymercuribenzoate (p-MB) by a one-column method using carboxymethyl (CM)-cellulose and alternatively by a two-column method (the details of which can be referred to in Sugawara et al. [17]).

Hence, the occurrence of hemichrome of human HbO2 can be described as follows:

(1)

Along with this line, the question may be arisen as to whether the monomeric Mb (MbO2) have similar propensity of human HbO2 to hemichrome. Regarding to this issue, a series of experiments using UV/VIS spectroscopic observation for hemichrome emergence (hemichrome emergence tests) were undertakenfor monomeric bovine heart MbO2. At first, these tests were conducted in 0.1 M buffer over the same range of pH (pH 4.5 to 10.5) and temperature (35 to 55°C). However, our attempts failed to recognize any hemichrome emergence in this pH and temperature range. In order to further check this point, we made an attempt to expand our tests to temperature region exceeding 55°C. In this respect, we employed thermal denaturation measurement for MbO2 using differential scanning calorimetry (DSC). Ultimately we engaged in these tests over a wide temperature range for bovine heart MbO2 from physiological to temperatures just before thermal unfolding [17].

Mbs (involving MbO2 and met Mb) were isolated from bovine heart muscle, in which the essential step was chromatographic separation of MbO2 from metMb on a DEAE-cellulose column.Thermal denaturation of bovine heart Mb was assessed by differential scanning calorimetry in terms of Td, the temperature at which the transition is half complete. A non-adiabatic calorimeter (DSC, Seiko I &E DSC-10/SSC-570 Thermal Controller Model, Tokyo, Japan) was used at a heating rate of 1°C·min-1 and at protein concentrations of 1.5-5.5 mg·ml-1. After treatment of the raw DSC data, in which correction of the sample curve for changes of slope observed in a run with buffer in each cell was made by subtracting the buffer curve from the protein curve point by point, the linear portions of the corrected curve corresponding to pre- and posttransition base lines were extrapolated to Td. Td was also determined by successive area measurements until the vertical line drawn at Td divided the peak area into two halves. The area under heat capacity curve was determined by means of a planimeter.

Figure 3a is an example of DSC scanning for MbO2 in 0.1 M HEPES buffer at pH 7.4. In terms of Td, that is, the temperature at which the thermal unfolding is half complete, Figure 3b gives an upper limit temperature to examine whether hemichrome formation associated with autoxidation can be observed in monomeric MbO2 or not, although the concentration (0.91 mM) used for DSC measurement was 100 times higher than that of our hemichrome emergence test.

bioanalysis-biomedicine-hemichrome-monomeric

Figure 3: Hemichrome formation test for monomeric bovine heart Mb. (a) Tracing of DSC curve for unfolding of bovine heart MbO2 in 0.1 M HEPES buffer at pH 7.4. Protein concentration was 2.35 mg ml-1 (0.91 mM). (b) By means of DSC, the properties of thermal unfolding of bovine heart MbO2 and metMb were depicted as a function of pH and temperature in terms of Td (i.e., the temperature at which the transition is half complete). Protein concentration was 1.5 −5.5 mg ml-1 (0.91 to 5.81 mM) ○: MbO2, △: metMb. (c) Temperature dependence of first-order rate constants, kA of autoxidation of bovine heart MbO2. The autoxidation was measured in 0.1 M buffer at a given pH and temperature under air saturated condition. The resulting data of pH 5.0 in 0.1 M MES buffer (○), 6.0 in 0.1 M MES buffer (△), 7.0 in 0.1 M HEPES buffer (equation) and 9.0 in 0.1 M CHES buffer (equation) were plotted against a given temperature. MbO2 concentration was 97μM. (d) Arrhenius plot of kA, measured in 0.1 M HEPES buffer at pH 7.4.

Considering an upper limit temperature in each run, our search for detecting possible hemichrome emergence was carried out by varying pH of the solution from 4.5 to 10.5 and varying the temperature from the physiological level to the level just before thermal unfolding. Typical examples are shown in Figure 3c and 3d), where kA represents the observed first-order rate constant of autoxidation of MbO2 at a given pH and temperature. In air-saturated buffers, MbO2 is oxidized by the bound O2 to its ferric met form (metMb) with generation of superoxide anion radical (O2−) as essentially in the same manner as HbO2:

(2)

(3)

In the hemichrome emergence tests, the ratio of MbO2 concentration after time t to that at time t = 0 was monitored by absorbance change, for instance, at α-peak (581 nm) of MbO2. In each run, kA in hr-1 was determined from the slope of linear plots of –ln([MbO2]t/[MbO2]0) versus time and plotted against temperature of the solution in Figure 3c and against the inverse of the absolute temperature, that is Arrhenius plots in Figure 3d. In these figures, our experimental data gave a single straight line against the temperature examined at constant pH of the solution.This implies that there was no hemichrome emergence (formation) under these circumstances. This point was also confirmed by the resulting activation energy, EA. From the slope of each straight line on Arrhenius plots shown in Figure 3d, EA was calculated and appeared to be almost constant, i.e., 121.55 ± 14.38 KJ·mol-1 (29.08 ± 3.44 Kcal·mol-1) over the pH range of 4.5 to 10.5 and at the temperature range greater than or equal to 35°C. These were in good agreement with those observed by Goto and Shikama [36], where EA is almost constant with the value of 26.5 Kcal·mol-1 (110.77 KJ·mol-1) over pH range of 5 to 10 using bovine heart MbO2 based on the measurements of the rate at 0, 15, 25, and 30°C.

We, therefore, concluded that the monomeric bovine heart Mb (MbO2) did not show any propensity for hemichrome formation over a wide pH range of 4.5 to 10.5 and over a wide temperature range from physiological to temperatures just before thermal unfolding. Contradictorily, higher susceptibilities to hemichrome formation as well as occurrence of hemichrome emergence even under physiological pH and temperature was pronounced to be the most significant feature of the tetrameric human HbO2 that is O2–carrying protein in the blood.

Oxidative behavior of human Hb (HbO2): pH-Dependent biphasic autoxidation curves seen in acidic solutions

Since the early works of Brooks [37,38], the oxidative process (autoxidation) of human Hb has been investigated by several researchers. Among others, Monsouri and Winterhalter [39] and Tomoda et al. [40] observed that the rate of autoxidation was strongly dependent upon the pH values of solutions. The study of Monsouri and Winterhalter [39] also showed that autoxidation of human HbO2 exhibited a biphasic nature, with an initial fast (kAf) reaction followed by a second slower (kAs) reaction, when observed in acidic solutions. Following the method of Monsouri and Winterhalter [39], we here focus on the biphasic nature of the autoxidation seen in acidic solutions on the basis of our previous report [35].

As previously shown, Figure 1(a) is a representative example, in which HbO2 was autoxidized to its ferric met form through the tendency of the bound O2 to oxidize ferrous heme iron (II) with generation of superoxide anionradical (O2−), as in the same fashion in MbO2 autoxidation mentioned above:

(4)

Here kA represents the observed rate constant of the reaction at a given pH and temperature. The reaction process was followed in each experimental run by the ratio ([HbO2]t/[HbO2]0) of HbO2 concentration after time t to that at time t=0 that was monitored by the absorbance ratio of (At – A)/(A0 – A) at 576 nm (α-peak of HbO2). The ratio was then plotted against time. Each curve shown in Figure 4a represents the –ln([HbO2]t/[HbO2]0) versus time plot (first-order plot) for this phenomenon. In acidic-to-neutral solutions, the reaction was biphasic and can be described by first-order kinetics containing two rate constants as follows:

(5)

This equation was originally proposed by Monsouri and Winterhalter [39], where kAf and kAs represent first-order rate constants for the initial fast oxidation and the second slower oxidation, respectively, and P is the molar fraction of the initial fast component. In the Figure, solid lines represent the best fitting derived by the leastsquares method using the equation to each run. However, when the pH of solutions is more than8, a plot of –ln([HbO2]t/[HbO2]0) versus time showed a single-phase reaction with a single first-order rate constant, although curve fittings could be made using the same equation under conditions setting P=0.5 and kAf=kAs. Thus, it was shown that the biphasic behavior seen in acidic solutions gradually disappeared with increasing pH of solutions and completely disappeared if the pH of solutions was more than 8.

bioanalysis-biomedicine-oxidative-behavior

Figure 4: Oxidative behavior of human Hb (HbO2) in various buffer conditions. (a) A−ln([HbO2]t/[HbO2]0) versus time plot (first−order plot) for human HbO2 autoxidation. Two−phase autoxidation with an initial fast reaction (kAf) followed by a second slower reaction (kAs) seen in acidic−to−neutral pH ranges. Solid lines represent the leastsquares fitting to experimental data in each run. Conditions: HbO2 concentration was 50−125 μM (in heme contents); and the reaction proceeded in 0.1 M buffer at 37°C. (b) A kAskAf versus pH plot for human HbO2 autoxidation. The solid line stands for the best-suited fitting under the assumption that a single, dissociable group, AH with Ka, can be involved in the reaction. This was achieved with the setting pKa= 7.4.

In this way, the kinetic parameters and molar fraction of the initial fast component (P) for the biphasic nature of human HbO2 autoxidation were established in each run in 0.1 M buffer at 37°C (Table 1). It was evident that the values of P varied from 0.47 to 0.53. This means that HbO2 was autoxidized with half of the component via the reaction process with the initial fast reaction with kAf and the other half via the procedure with the second slower rate (kAs). Thus, autoxidation of human HbO2 may be written as follows:

(6)

pH P kAf
(h−1)
kAs
(h−1)
kAs/kAf
5.3 0.47 0.438 0.078 0.178
5.9 0.48 0.270 0.030 0.111
6.2 0.48 0.134 0.0154 0.115
6.55 0.49 0.070 0.010 0.142
6.9 0.53 0.0465 0.0073 0.157
9.15 0.50 0.0095 0.0095 1.0
9.5 0.50 0.0184 0.0184 1.0
9.7 0.50 0.0381 0.0381 1.0
10.2 0.50 0.080 0.080 1.0
10.4 0.50 0.157 0.157 1.0

Table 1: Summary of kinetic parameters for the autoxidation of human HbO2 in 0.1 M buffer obtained by least-squares fitting to the experimental data in each run, redrawn from Sugawara et al. [34].

To clarify the processes behind this phenomenon, the obtained values of kAs/kAf were plotted against the pH of solutions (Figure 4(b)). In the Figure, the solid line shows that the computed curve obtained by the least-squares method fitted the experimental data over the whole range of pH 5-10.5, where a single dissociable group, AH with Ka, was assumed to be involved in the reaction:

(7)

For neutral amino acids, acid–base equilibrium is usually given by the dissociation constant: AH ↔A+ H+ and thus Ka = [A]·[H+]/[AH]. The best fitting was achieved when pKa= 7.4 (at 37°C). In this regard, Hermans and Rialdi [41,42] reported microcalorimetric ionization data on amino-acid residues for sperm whale myoglobin (Mb) in which they assigned the His residue with values of pKa=6.62, the standard Gibbs energy (ΔG°)=37.7 kJ·mol–1, the enthalpy (ΔH)=29.7 kJ·mol–1, and the entropy (ΔS°)=–26.8 J·mol–1·K–1. It is unwise to identify a dissociation group only by its pKa value because of the anomalies often found in proteins. Hence, we confirmed that AH must be a His residue assuming that the primary and tertiary structures of α and β subunits are remarkably similar to each other and to Mb. This fact provides information for the existence of a pH-sensed molecular device that can manifest the biphasic autoxidation of the HbO2 tetramer via participation of a single amino-acid residue with pKa=7.4 (at 37°C).

In order to not only elucidate the biphasic characteristics of human HbO2 autoxidation but also to clarify how the Hb molecule can prompt the range of fast (kAf) and slow (kAs) components against pH values of the solutions, chain separation of the constituted chains from the parent molecules and rate measurements for the separated chains were conducted. Chain separation was made using p-MB [17], as mentioned above.

Figure 5 shows the obtained pH profiles for the autoxidation rate of the isolated αp-MB and βp-MB chains. With reference to the rates (kAf and kAs) of the parent molecules calculated from biphasic autoxidation curves, the isolated chains were shown to be oxidized much more rapidly to the ferric met-form over the entire pH range (5-10.5) because the reaction followed simple, first-order kinetics as follows:

(8)

Where Hbsub represents each subunit of the Hb molecule. It was also shown that the rate of the initial first component (kAf) of the parent molecules was found to lie closer to the values of kA for the isolated αp-MB and βp-MB chains if the pH of solutions was less than 6. Similar pH profiles were obtained for p-MB-removed chains, i.e., the αSH and βSH chains. p-MB can be removed from the αp-MB and βp-MB chains by incubation with 2-mercaptoethanol, as described elsewhere [30]. In separated-chain solutions, the protein is known to exist in equilibrium of α↔α2 or β↔β4, respectively. Under our experimental conditions, the monomeric form (87%) was present in the separated α chain, whereas the tetrameric form (99%) was predominant in the β chain. This estimation was made on the basis of the results by McDonald et al. [16]. Accordingly it became evident that the isolated α and β chains could be oxidized much more readily over the measured pH range (5– 10.5) when compared with the respective rates of the parent molecules as a reference. With respect to the difference in the rate between the isolated individual chains, this seemed to be within reasonable experimental errors in the values of αp-MB and βp-MB chains, and the αSH and βSH chains.

bioanalysis-biomedicine-logarithmic-autoxidation

Figure 5: Logarithmic values of first-order rate versus pH plot for autoxidation of the isolated α (αp-MB) and β (βp-MB) chains with a reference to the relevant values (kAf and kAs) of the parent molecules, redrawn from Sugawara et al. [34]. Conditions: the concentration of isolated α and β chains was 50 μM (in heme contents); the reaction rate was measured in 0.1 M buffer at 37°C.

Thus, the obtained findings put forward an idea that the individual chains have acquired considerable resistance to oxyheme autoxidation in their tetrameric protein architecture. The same appears to be the case in which human HbO2 has acquired a pH-sensitive molecular device, whereby participation of a single dissociation group of amino-acid residue (probably a His residue) with pKa=7.4 (at 37°C) plays a key role, as a consequence of development of the tetrameric protein architecture. We will see later that this issue was the starting point that led us to uncover a new mode of subunit interaction of the Hb molecule that can be emerged from participation of the α1–β1(and α2–β2) interface via distal side perturbations within the heme pocket relying upon inherent tilting capability of the distal (E7) His residues (i.e., α58His (E7) in the α chain and β63His (E7) in the β chain).

We must be careful when evaluating the data of the isolated chains shown in Figure 5. Because of the instability of Hb molecules and their relationship to hemichrome, the first-order rate constant for the isolated chains was established in each experimental run using only the initial slope of the–ln([HbsubO2]t/[HbsubO2]0) versus time plot. Once hemichrome precipitation arose, one could not get the endpoint spectrum (the ferric met form) of the reaction, whereby Hb molecules in the reaction mixture should be all completely converted to the ferric met form by addition of potassium ferricyanide. Hence, measurement must be confined within the initial range, in which existence of isosbestic points of the spectra that means non-occurrence of hemichrome formation during the entire reaction process was surely guaranteed.

Hemichrome emergence and subsequent formation of heinz bodies within erythrocytes: observation in normal human erythrocytes during mild heating

Herein we consider the results of our study investigating the formation of Heinz bodies in normal human erythrocytes [32-34]. Aliquots of freshly drawn venous blood from healthy donors were subjected to mild heating at desired temperature above 37°C for 30 min to investigate hemichrome formation and subsequent Heinz-body formation in normal human erythrocytes. Heinz bodies were then visualized by exposing blood smears to acetylphenylhydrazine and staining with crystal violet. Changes within the erythrocytes were observed using light microscopy under oil immersion.

Prior to the acetylphenylhydrazine test [42,43], blood samples were subjected to mild heating in vitro. A 2-mL sample was placed in a test tube and incubated in a water bath maintained at each desired temperature (± 0.1°C) above 37°C for 30 min using a NESLAB temperature control system (Model RTE-100 or 111 or 210 or 221). In each experimental run, we employed a temperature-untreated reference as a control. The blood sample used as a temperatureuntreated reference was kept at a low temperature (0–4°C) for as long as possible after withdrawal from the donor, and never exposed to temperatures above room temperature.

β-Acetylphenylhydrazine and crystal violet (research grade for pathology) were used (Wako Pure Chemical, Co., Osaka, Japan) for the procedure, as described by Bauer [43]. A 0.1-mL aliquot of blood sample was suspended in 2 mL of acetylphenylhydrazine solution, in which 100 mg of acetylphenylhydrazine and 200 mg of glucose in 100 mL of 0.067 M phosphate buffer were dissolved at pH 7.6. Using a “blowout” pipette, the solution was aerated twice or thrice by drawing it up into the pipette and blowing it out, together with a small quantity of air. This mixture was incubated at 37°C for 2 h. Aeration was repeated halfway through and immediately after the 2-h incubation. A drop (approximately10 μL) of the resulting mixture was placed on a coverslip, which was then inverted onto a microscope slide containing 30 μL of crystal violet solution. The smear was allowed to stand for 20 min in wet preparation at room temperature and subsequently examined under a light microscope (Nikon Model-FXA; Nikon Co., Tokyo, Japan) equipped with a digital camera (Olympus Model-DP-70; Olympus Co., Tokyo, Japan). Photomicrographs were taken under oil immersion at 1,000× magnification. Subsequently, the number of Heinz bodies per cell was counted in 100 red cells per view.

Figure 6 shows representative images of Heinz bodies within normal red corpuscles (obtained from a healthy donor) during mild heating to a given temperature with using a temperature-untreated reference as a control. Heinz bodies were observed in each smear. Figure 7 puts on view about an entire image of changes of the number of the Heinz bodies per cell in each smear. Here histograms of samples treated at 37°C, 40°C, 42°C and 45°C are displayed with a temperatureuntreated sample as a reference based on the number of Heinz bodies contained in each of 100 red cells per view. These results confirmed that Heinz-body formation increased with increasing temperature of blood samples treated at temperatures more than 37°C. Red cells in blood samples exposed to 48°C were hemolyzed. Some of the changes are also shown in Figure 8(a); histograms of the samples treated at 37°C and 42°C are laid out with a temperature-untreated sample as a reference.

bioanalysis-biomedicine-microscopic-bodies

Figure 6: Microscopic views of Heinz bodies after mild heating of blood samples obtained from one healthy donor, redrawn from Sugawara et al. [32]. Using a temperature-untreated sample as a control, aliquots of freshly drawn venous blood were subjected to mild heating at 37°C, 40°C, 42°C, 45°C and 48°C for 30 min. Heinz bodies were then visualized by exposure to acetylphenylhydrazine and dyeing with crystal violet. The changes that occurred within erythrocytes were observed by light microscopy under oil immersion.

bioanalysis-biomedicine-histograms-microscopic

Figure 7: Histograms obtained from the microscopic views as shown in Figure 6, redrawn from Sugawara et al. [32]. On the basis of the number of Heinz bodies contained in each of 100 red cells per view, samples treated at (a) 45°C, (b) 42°C, (c) 40°C and (d) 37°C are displayed with a temperature-untreated sample as a reference (e). The results demonstrate that hemichrome emergence and subsequent formation of Heinz bodies within the erythrocytes increased with increasing temperature treated of blood samples over 37°C.

The other histograms shown in Figure 8b-8f represent the results from another five healthy donors, with a temperature-untreated sample as a reference. Although there was considerable inter-individual variation, temperature-dependent Heinz-body formation was apparent in all preparations. Arithmetic summation was used for all histograms to reduce variability and allow better assessment of temperaturedependent Heinz-body formation. The results of this summation are shown in Figure 9. Solid lines represent the curves derived using the least-squares method using a Gaussian curve. Each computed curve showed satisfactory-to-good agreement with the experimental data over the entire range. The following is a summary of the Gaussian constants resulting from the least-squares fitting: a (peak height) = 101, b (peak width at half of maximum) = 5.0 and c (peak location) = 4.0 for blood samples treated at 42°C; a=215, b=3.5 and c=1.0 for blood samples treated at 37°C; and a=215, b=3.0 and c=0.8 for temperatureuntreated blood samples.

bioanalysis-biomedicine-histograms-representing

Figure 8: Histograms representing Heinz bodies detected in blood samples after mild heating at >37°C, redrawn from Sugawara et al. [32]. The histograms for 37°C and 42°C are displayed using a temperature-untreated sample as a reference. One-hundred red cells were chosen, and the number of Heinz bodies contained in each red cell counted. (a) was constructed from the microscopic views (Figure 6) and the concerned histograms shown in Figure 7, whereas (b−f) represent the results from another five healthy donors.

bioanalysis-biomedicine-histograms-obtained

Figure 9: Histograms obtained from the arithmetic summation of all histograms shown in Figure 8, redrawn from Sugawara et al. [32]. The solid lines show the computed curve obtained by the least-squares method using a Gaussian curve. The resulting Gaussian constants are summarized in the text.

In vitro evaluation of blood fluidity during mild heating using a micro-channel array flow analyzer (MC-FAN) to provide an index of erythrocyte deformability

In 1992, Kikuchi et al [44] described a tool for measuring the fluidity or rheology of blood [45]. This has subsequently been implemented in numerous studies [45-52]. A MC-FAN includes a characteristic V-shaped groove array in an integral circuit with 8,736 flow paths (width, 7 μm; length, 30 μm; depth, 4.5 μm) engraved on a 15×15×0.5-mm single-crystal silicon substrate using an anisotropic etching technique. The array is housed in a cylinder. This equipment enables observation of red cells passing through individual microchannel arrays by use of an inverted metallographic microscope, as well as to evaluate blood fluidity through the groove array in terms of the transit time of the blood sample for a given transit sample volume. Flow rate can be determined by timing when the meniscus of the blood sample crosses graduation marks at 10-μL intervals from 0 μL to 100 μL. The transit sample volume is therefore equivalent to the loss of the blood sample within the cylinder. Here we look into our in vitro findings on blood fluidity during mild heating using a MC-FAN (HR300; Hitachi Haramachi Electronics Co., Ibaraki, Japan) [32,33].

Donors were male or female student volunteers from the Prefectural University of Hiroshima (Hiroshima, Japan) and were aged 18-22 years. All volunteers were aware of the aims and procedures of the study. They gave their written informed consent to participate. The study protocol was approved by the Ethical Review Board of the Prefectural University of Hiroshima. Nine aliquots of samples of freshly drawn venous blood (10 mL in total) were obtained from healthy donors and mixed with one aliquot of 3.2% sodium citrate (Na3C6H5O7·2H2O) or 3.8% sodium citrate (Na3C6H5O7·5H2O). Blood samples were centrifuged at 400×g for 10 min at 0-4°C to eliminate the possibility of interference with blood fluidity measurements caused by platelet aggregation onto microchannel flow paths. The supernatant (platelet-rich plasma (PRP)) was then discarded using a narrow Teflon-lined capillary connected to a waterjet pump. The remainder (PRP-removed blood) was subjected to mild heating prior to application to the MC-FAN. A 2-mL aliquot was placed in a test tube and incubated in a water bath maintained at each desired temperature (± 0.1°C) above 37°C for 30 min using a NESLAB temperature control system (Model RTE-100 or 111 or 210 or 221).

Using a 1-mL disposable syringe and a thin catheter, 200 μL of each temperature-treated blood sample were introduced into a groove array via the cylinder house, which was connected to the inlet hole. The sample was allowed to flow through the cylinder house by applying a pressure difference of 20 cm H2O. Red cells passing through individual micro-channel arrays were monitored using an inverted metallographic microscope, a video camera, and a video-recorder system. As shown in Figure 10a and 10b, microscopic images revealed good erythrocyte deformability in temperature-untreated samples subjected to MC-FAN. However, a marked decline in erythrocyte deformability was observed in blood samples treated at temperatures greater than 37°C for 30 min. An example is shown in Figure 10c. Temperature-treated samples also demonstrated increased transit time for low transit sample volumes.

bioanalysis-biomedicine-microscopic-samples

Figure 10: Microscopic details of blood samples passing through individual micro-channel arrays of the micro-channel array flow analyzer (MC-FAN), redrawn from Sugawara et al. [33]. (a) View of a temperature-untreated sample with a transit time = 12.6 s and transit sample volume = 25 μL. (b) Identical temperature-untreated with a transit time = 27.8 s and transit sample volume = 50 μL; (c) View of an identical blood sample to that in (a) and (b) but subjected to mild heating at 45°C for 30 min prior to application to MC-FAN with a transit time = 120.7 s and transit sample volume = 30 μL.

Figure 11 illustrates some examples of blood fluidity of temperaturetreated samples measured as transit time versus transit sample volume. These graphs show that erythrocyte deformability decreased with increasing temperature at temperatures above 37°C, although there was considerable inter-individual variation. Blood was therefore obtained from a further six healthy donors, and temperature-treated samples compared with a temperature-untreated sample as a reference. The results were similar to those in Figure 11. Both sets of results (n=10) are summarized in Table 2. The temperature-dependent decrease in erythrocyte deformability induced by mild heating was evaluated using the Student’s t-test on the total sum of the difference in transit times between a given temperature-treated sample and each temperatureuntreated reference, with varying transit sample volumes (20, 40 and 60 μL). Significant differences (p< 0.001) between treated and control samples were evident for all samples treated at 45°C with transit sample volumes of 20, 40 and 60 μL, and all samples treated at 42°C with transit sample volumes of 20, 40 and 60 μL. To adequately show the results of Figure 11, significant differences (p<0.001) of the samples treated at 45°C (i.e., two-point lines) and 42°C (i.e., one-point lines) against the control samples (i.e., solid lines) are shown. Furthermore a triplicate-experiment was conducted for each of three healthy donors (n=3) in terms of transit time versus transit sample volume, whereby blood fluidity was assessed by the measurements each being allowed an interval of two to three months (Figure 12). On the basis of these findings, we concluded that erythrocyte deformability decreased with increasing temperature of blood samples treated at temperatures more than 37°C.

bioanalysis-biomedicine-representative-fluidity

Figure 11: Representative examples (n = 4) of blood fluidity measured in terms of transit time against transit sample volume, redrawn from Sugawara et al. [33]. Solid lines represent the temperature-untreated control; broken lines represent samples treated at 37°C; one-point lines represent samples treated at 42°C; two-point lines represent samples treated at 45°C.

bioanalysis-biomedicine-transit-triplicate

Figure 12: Transit time versus transit sample volume plots obtained by a triplicate experiment. Blood fluidity was measured in three stages concerning three healthy donors ((a); (b); (c)), an interval of two to three months being allowed between measurements. Solid lines represent the temperature-untreated control; broken lines represent samples treated at 37°C; one-point lines represent samples treated at 42°C; two-point lines represent samples treated at 45°C.

The mean ± standard deviation of transit time of blood samples (s) for a given transit sample volume (μL)
  Transit blood sample volume (μL)
20 μL 40 μL 60 μL
Temperature-untreated reference 8.0 ± 1.08 16.3 ± 2.24 25.1 ± 3.50
Temperature-treated at 37 °C 7.8 ± 1.35 16.4 ± 2.57 26.4 ± 3.42
Temperature-treated at 42 °C 8.5 ± 1.38 ** 18.3 ± 2.94 ** 36.3 ± 6.32 **
Temperature-treated at 45 °C 9.3 ± 2.46 ** 21.2 ± 5.99 ** 41.8 ± 13.23 **

Table 2: Summary of the mean ± standard deviation of transit time of blood samples (s) for a given transit sample volume (μL),redrawn from Sugawara et al. [33]. Prior to application to MC-FAN, blood samples were subjected to mild heating at 37°C, 42°C and 45°C for 30 min, respectively. The number of subjects was 10. Significant differences (p<0.001) of the temperature-treated samples against the temperature-untreated reference were shown as **.

New Mode of Molecular Biosensing Mechanisms Inherent in Human Erythrocytes for Appreciation of Cell Aging and Determination of their Lifespan

Human red blood corpuscles survive in the circulation for an average of 120 days. Removal of aged and damaged red cells from the blood circulation is essential for its homeostasis. This prompts the question: how do human erythrocytes appreciate aging and determine their lifespan?

Through a series of studies described in the preceding section, we drew seven main conclusions. First, autoxidation of human HbO2 (the reaction of which is inevitable in nature for all O2–binding proteins and which has been dealt with in the periphery of Hb research) seemed to be inseparably related to the instability of the Hb molecule and its degradation to hemichrome. Second, in terms of pH- and temperature-dependent hemichrome emergence observed by UV/VIS spectroscopy, HbO2 was supposed to be highly susceptible to hemichrome formation (even under physiological pH and temperature). Third, in contrast to the tetrameric human HbO2 as O2–carrying protein in the blood, the monomeric bovine heart Mb (MbO2) did not show any propensity for hemichrome formation over a wide pH range of 4.5 to 10.5 and over a wide temperature range from physiological to temperatures just before thermal unfolding. Fourth, in terms of pH-dependent biphasic autoxidation (kAf for the initial fast oxidation and kAs for the second oxidation) seen in acidic solutions, participation of a single dissociation group of an amino-acid residue (probably a His residue) with pKa=7.4 (at 37°C) appeared to have a key role in how the Hb molecule can prompt the range of fast (kAf) and slow (kAs) components against the pH values of the solutions. Fifth, the isolated α and β chains were oxidized much more readily over the measured pH range (5–10.5) when compared with the respective rates of the parent molecules used as a reference. Sixth, using freshly drawn venous blood from healthy donors, exposure of red cells to acetylphenylhydrazine and subsequent staining with crystal violet revealed a greater abundance of Heinz bodies with increasing temperature if blood samples were subjected to mild heating at temperatures greater than 37 °C for 30 min. Finally, in vitro evaluation of blood fluidity during mild heating using a MCFAN demonstrated a marked decline in erythrocyte deformability with increasing temperature of blood samples if treated at temperatures more than 37°C for 30 min.

In this section, we start to deal with the oxidative behavior (autoxidation) of the human HbO2 molecule. This is because autoxidation is inseparably related to the instability of the molecule and its degradation to hemichrome, relying upon the intrinsic tilting capability of the distal (E7) His residues (their bis-histidyl coordination proficiency to the heme iron). In Hb research, understanding of subunit interaction between the four Hb chains (how these explain cooperative O2 binding) has been the primary focus [22-28]. Most of the emphasis is based on the distinct differences between deoxygenated and oxygenated quaternary structures as determined by X-ray diffraction [23-28]. The configuration of the residues lining the distal side of the heme pocket (where O2 binds and where geographical alteration can be made by oxygenation) is thought to play a part in controlling access of the ligand to the heme pocket. Nevertheless, the possibility of subunit interactions originating from or being transmitted via distal effects has (for the most part) been neglected [29]. Relatively little attention has been paid to the autoxidation of HbO2 (oxidation of ferrous heme iron by bound O2), even though autoxidation is inevitable in nature for all O2-binding heme proteins. It has been customary to deal with the “periphery” in Hb research.

Shikama [54-58] evaluated various mechanisms proposed for autoxidation involving MbO2 and HbO2. He demonstrated clearly that the autoxidation reaction does not simply involve the dissociative loss of O2− from HbO2, but is instead caused by the nucleophilic displacement of O2− from HbO2 by a water molecule or a hydroxyl ion that enters the heme pocket from the surrounding solvent. The iron is thus converted to the ferric met form, and the water molecule or hydroxyl ion remains bound to Fe(III) at the sixth coordinate position to form the aqua- or hydroxide-met species. A generalized pathway for this SN2 mechanism can be written using Mb (MbO2) as an example:

Here, kA represents the rate constant of anion-induced autoxidation with nucleophilic anion displacement. N can be SCN, F, OCN, N3, or CN and, in vivo, it can be H2O or OH. Anion-induced autoxidation with nucleophilic anion displacement of O2 results in an intermediate ferrous heme/anion complex that acts as an electron donor to displace oxygen. Shikama [55,58] also demonstrated that, in vacuo, oxyhemeis inherently stable and unlikely to dissociate O2− spontaneously. Because O2 is a poor one-electron acceptor, a considerable thermodynamic barrier exists for such an electron transfer. In aqueous media (i.e., in contrast to in vacuo), oxyheme is subject to the nucleophilic attack of an entering water molecule (with or without proton catalysis) and to the attack of an entering hydroxide anion. These can cause irreversible oxidation of oxyheme to met-species with generation of O2. Mb and Hb have therefore evolved with a globin moiety that can protect the Fe(II)O2 center from the easy access of a water molecule (including its conjugate ions OH and H+,/).

In an aqueous protein-free system, Kao and Wang [58] reported the oxidation of dipyridine-ferrohemochrome by O2 using a stoppedflow technique. The main pathway was interpreted to involve one O2 molecule replacing one of the pyridine molecules in dipyridineferrohemochrome to form an oxyheme and then undergoing decomposition of ferrohemochrome to ferrihemochrome and O2. Unfortunately, the rate constant for this oxidation reaction could not be obtained because the concentration term of pyridine was always involved in its rate equation in a complicated manner. By numerical calculations, however, it follows that oxyheme autoxidation can proceed with a rate constant that is much higher than 1 s–1 in 0.1 M buffer, pH 8.5, at 25°C. If such an oxyheme is placed in a protein matrix, it would be protected against the nucleophilic attack of the solvent water molecule or hydroxyl ion so as to reduce its autoxidation by a factor of approximately 103. This was the case in our study with denatured MbO2 in 8 M urea (the details of which can be referred to in Sugawara et al. [59]). A globin moiety can act as a “breakwater” in aqueous media even in denatured conditions with 8 M urea. Furthermore, if an oxyheme is embedded in the native Mb architecture, MbO2 acquires remarkable stability against oxyheme autoxidation by a factor of approximately 106.

With regard to oxyheme stability in human HbO2, the half-life (t1/2) was only 1.5 d in 0.1 M buffer and at physiological pH and temperature [34]. This is despite the fact that the content of metHb in normal red cells in situ [60,61] has been maintained to be less thanor equal to 1% as a consequence of a NADH-dependent reducing system that can reduce the ferric-metHb resulting from autoxidation to deoxy-ferrous Hb [5-7]. However, as compared with the rates of monomeric mammalian Mbs, human HbO2 seemed to be 2.2–3.6 times more stable against autoxidation (probably owing to its tetrameric architecture). Even though the autoxidation rate is a function of pH and temperature, the observed first-order rate constant was 0.0023 h–1 (12.6 d for t1/2) for human HbO2 autoxidation under the conditions of 0.1 M buffer at pH 7.2 and 25°C in our study [35] because this value was calculated from the first rapid phase. Conversely, the relevant values of the monomeric mammalian Mbs were 0.0082 h–1 (3.5 d for t1/2) for human MbO2, 0.0072 h-1 (4.0 d for t1/2) for bovine MbO2, and 0.0050 h–1 (5.8 d for t1/2) for sperm-whale MbO2 under the same conditions used for human HbO2.

In our experiments, tetrameric human HbO2 showed a biphasic autoxidation curve in acidic solutions in terms of the –ln([HbO2]t/ [HbO2]0) versus time plot (Figure 4(a)); an initial rapid reaction (kAf) could be followed with a slower second phase (kAs). The difference in the rates between kAf and kAs decreased with increasing pH values of solutions, and finally disappeared (i.e., kAf equal to kAs) if the pH of the solutions was more than 8 and became kAf equal to kAs. This finding leads to certain questions: how does the Hb molecule prompt the range of fast (kAf) and slow (kAs) components in acidic solutions? How does the molecule provoke kAfequal to kAs if the pH of solutions is more than 8? Does the biphasic autoxidation seen in acidic pH regions reflect a different rate owing to the individual Hb chains or the presence of valency hybrid intermediates such as (α2+β3+)2 and (α3+β2+)2?

A plot for the obtained values of kAf/kAs versus pH indicated that the reaction involves a single dissociation group of amino-acid residue with pKa=7.4 (at 37°C) (Figure 4(b)), probably a His residue. In addition, study of rate measurement for the isolated αp-MB and βp-MB chains (and αSH and βSH chains) disclosed that once the constituted chains were isolated from the parent molecule, both separated chains could be oxidized much more rapidly to the ferric met-form over the measured pH range (5–10) when compared with the respective rates (kAfand kAs) of the parent molecules calculated from biphasic autoxidation curves as a reference (Figure 5). Also, there were no practical differences regarding the rate between the isolated individual chains (Figure 5). These findings may indicate that the individual chains have acquired considerable resistance to oxyheme autoxidation in their tetrameric protein architecture. It was also suggested that human HbO2 seems to have acquired a “pH-sensitive molecular device” as a consequence of development of the tetrameric protein architecture. Hence, the molecule shows remarkable stability against oxyheme autoxidation more than in cases of isolated individual chains from the parent molecules and monomeric mammalian Mbs.

Moreover, our recent measurements (UV/VIS spectrophotometry, isoelectric-focusing electrophoresis, and polyacrylamide gel electrophoresis) for autoxidation of human HbO2 [33] revealed that only two valency hybrids, i.e., (α3+β2+)2 and (α2+β3+)2, emerged in the tetrameric HbO2 autoxidation, even though, in theory, seven species of valency hybrids can be in existence during the reaction time course. These species are (α2+α2+β3+β2+), (α3+α2+β2+β2+), (α2+β3+)2, (α3+β2+)2, (α2+α3+β2+β3+), (α2+α3+β3+β3+) and (α3+α3+β3+β2+). These had satisfactory-to-good consistency with those reported by Tomoda et al. [15,62-67]. Computer simulations on the basis of these measurements (UV/VIS spectrophotometry, isoelectric-focusing electrophoresis, and polyacrylamide gel electrophoresis) indicated that the relevant concentration progress curves derived from the subsequent tentative model could explain not only aspects of the spectrophotometric changes in terms of the–ln([HbO2]t/[HbO2]0) versus time plot, but also aspects of the associated isoelectric-focusing electrophoretic profiles and polyacrylamide gel electrophoretic profiles, respectively, as shown below.

equation

In an autoxidation reaction monitored in 0.05 M phosphate buffer (pH 5.0) at 37°C in the presence of 20% (v/v) glycerol, the following kinetic constants and molar fraction of the initial fast component (P) were established by least-squares fitting: kAf = 0.148 h–1 (4.7 h for t1/2), kAs = 0.0208 h–1 (1.4 for t1/2), and P=10.51. These findings led us to conclude that kAf represents intrinsic α-chain oxidation and kAs reflects intrinsic β-chain oxidation, and that the β chain manifests delayed autoxidation in human HbO2, which could be much more evident in acidic solutions.

Figure 13(a) shows our proposal with respect to this issue as reported in 1998 [30] and 2002 [31] in which we suggested a new function to the α1–β1(and α2–β2) interface for stabilizing the HbO2 tetramer against acidic autoxidation. That is, the α1–β1(and α2–β2) interface produces a conformational constraint in the β chain whereby the distal (E7) His at position 63 is tilted slightly away from the bound O2 so as to prevent the proton-catalyzed displacement of O2− from the Fe(II)O2 center by entrance of a water molecule. The β chain thus acquires a remarkably delayed oxidation rate in the HbO2 tetramer. This is the origin of the chain heterogeneity seen in acidic solutions of HbO2 autoxidation.

bioanalysis-biomedicine-schematic-representation

Figure 13: Schematic representation of the role of the α1−β1 (and α2−β2) interface in human HbO2.(a) The leftfigure shows a molecular dyad axis of Hb tetramer (which is perpendicular to the plane of the figure) relating the α1β1 dimer to the α2β2 dimer and consisting of the two different types of αβ contacts: one is the α1−β1 (and α2−β2) (packing contact); and the other is the α1−β2 (and α2−β1) (sliding contact). The right figure illustrates that the α1−β1 (and α2−β2) produces in the β chain a tilting of the distal (E7) His residue, thereby preventing the proton-catalyzed displacement of O2− by a solvent water molecule. (b) The figure demonstrates that depending on internal and extraneous circumstances of the erythrocyte including pH and temperature, the α1−β1 (and α2−β2) interface produces a conformational constraint in the constituted chains via tilting of the distal His (E7) residues so as to cause degradation of the Hb molecule to hemichrome, and subsequent Heinz-body clustering within the erythrocyte. In the spleen, Heinz body-containing red cells become trapped and hence undergo hemolysis.

The most recent refinement of the crystallographic structure of human HbO2 (where the O2molecule is clearly visible in the highresolution electron density maps) demonstrated that the hydrogen bond made by His E7β (i.e., β63His) is much weaker than that made by His E7α (i.e., α58His). However, it also substantiated that the geometry of the ligand and distal histidine is slightly different in the two subunits, with the O2 atom lying 2.7 ± 0.1 Å (10−1 nm) from the Nε atom of the distal histidine in the α subunits, and 3.0-Å away in the β subunits [68]. However, negligible changes were found for the α1–β1(and α2–β2) interface with respect to examination of the crystal structure.

Similar phenomena suggesting participation of the α1–β1(and α2–β2) interface (i.e., participation of the inherent tilting capability of the distal (E7) His residues of the Hb molecule) garnered our interest. Our UV/VIS spectroscopic study suggested that human HbO2 was highly susceptible to hemichrome formation even under physiological pH and temperature (Figure 1 and 2(a)) and, once isolated from the tetrameric parent molecule, the α (αMp-MB) and β (βp-MB) chains showed much higher susceptibilities to hemichrome when compared with the parent molecule (Figure 2(b)). In this regard, one must note that monomeric bovine heart Mb (MbO2) did not show a propensity for hemichrome formation over a wide range of pH (4.5-10.5) and temperature from physiological to temperatures just before thermal unfolding (Figure 3).

Heinz bodies are intra-erythrocytic inclusions of hemichrome formed as a result of Hb oxidation. They have been detected and characterized in drug-induced hemolytic anemia, defects in the intra-erythrocytic reducing system (e.g., G-6-PD deficiency), and in unstable Hb disease [2,3]. Using unstable Hb disease as an example, several unstable Hbs have been reported. Regarding the molecular pathogenesis of unstable Hbs, it is known that the instability of labile Hb variants in patients can be attributed to amino-acid substitutions (or deletions). These disrupt and perturb the Hb structure via interference with α-helix formation, disruption of heme binding, or altered α1–β1(and α2–β2) or α1–β2(and α2–β1) contacts [2]. The consequent changes in circulating red cells in patients with unstable Hb disease include an inherent tendency towards irreversible denaturation of Hb or globin due to a defect in the amino-acid composition of labile Hb molecules, a continuous tendency toward hemichrome formation, precipitation or aggregation of Hb molecules resulting in buildup of the molecules to form Heinz bodies, and hemolysis.

Exposure of red cells to acetylphenylhydrazine and subsequent staining with crystal violet also revealed a greater abundance of Heinz bodies within G-6-PD-deficient cells compared with that in normal cells [69,70]. In addition, Sear et al. [71] and Campwala and Desforges [72] reported that Heinz bodies often appeared in normal-aging red cells, and that this age-related appearance of Heinz bodies was particularly pronounced in splenectomized individuals [73]. According to several authors [1,74,75], aged or damaged red cells affected by drugs may be “filtered off” by the spleen irrespective of whether or not they contain Heinz bodies in a similar manner to the filtering-off of red blood cells in patients with unstable Hb caused by unstable Hb hemolytic anemia.

Using freshly drawn venous blood from healthy donors, we found that the number of Heinz bodies formed in red cells increased with increasing temperature when blood samples were subjected to mild heating at temperatures greater than 37°C for 30 min (Figures 6-9). Under identical conditions of mild heating, we measured blood fluidity using a MC-FAN [32,33] because it involves a characteristic V-shaped groove array with micro flow paths (width, 7 μm; length, 30 μm; depth, 4.5 μm) engraved on a single-crystal silicon substrate in an integral circuit. When red cells passing through individual micro-channel arrays were monitored using an inverted metallographic microscope, images revealed good erythrocyte deformability when temperatureuntreated samples were subjected to the MC-FAN (Figure 10a and 10b)). Conversely, a marked decline in erythrocyte deformability was observed in blood samples treated at temperatures more than 37°C for 30 min (Figure 10(c)). Temperature-treated samples also demonstrated an increased transit time for low transit sample volumes (Figure 11 and 12, Table 2).We therefore concluded that erythrocyte deformability decreased with increasing temperature of blood samples treated at temperatures above 37°C. The combination of our experimental findingsand in situ observations suggests that instability leading to hemichrome formation is not only a peculiarity of labile Hb variants but is also an innate characteristic of physiologically normal Hb molecules.

In our experiments, the occurrence of hemichrome can be described as follows:

(9)

Taking into account the accepted framework of the SN2 mechanism for autoxidation [54-58] and the accepted framework for hemichrome formation [13] in physiological conditions, hemichrome can form as follows:

In this scheme, Hbsub represents each subunit of the Hb molecule. Nucleophilic displacement of O2− by entry of a water molecule or a hydroxyl ion should be the rate-limiting step. Also, the subsequent conversion of the met form into hemichrome by a heme ligand (N) endogenous to the protein must proceed very quickly with the kinetic relationship kN>>kA. The most probable candidate for N in the HbA molecule is the Nε-nitrogen of the distal His (E7) (i.e., the only aminoacid side chain in the ligand pocket) of each subunit because Nε-nitrogen is located more than 4 Å (10−1 nm) from the iron in Hb, and is therefore not expected to coordinate in native Hb [13,76].

As described above, the content of metHb in normal erythrocytes [61,62] has been reported to be less than or equal to1% by a NADHdependent enzyme system [5-7]. This fact throws into question how erythrocytes can elicit the range of responses to hemichrome with such a small amount of metHb. We suggest that this can be achieved by intramolecular anion-induced nucleophilic displacement of molecular dioxygen via an intermediate ferrous heme/anion complex (a low-spin hemichrome) [17]. Such a reaction would follow the scheme:

In this reaction, nucleophilic displacement can be caused within the heme pocket by the Nε-nitrogen of the distal His (E7) instead of nucleophilic incursion of the water molecule or the hydroxyl ion from outside the molecule. As indicated above, hemichrome formation occurs at every stage during the autoxidation of HbO2 (i.e., initial, intermediate, and final) as a function of the pH and temperature of the solution. While the reaction proceeds along this scheme, hemichromes may be derived not only from HbO2 species, but also from deoxyHb and metHb (Figure 13(b)). Vital hemichromes in erythrocytes in situ might arise from this intramolecular anion-induced nucleophilic displacement of O2via an intermediate ferrous heme/anion complex.

Our electron paramagnetic resonance (EPR) measurement study [30] showed a low-spin spectrum for the resulting oxidation products of isolated β chains with values of g1=2.77, g2=2.27, and g3=1.68, in addition to the usual aquo-met species with g values of 5.86 and 1.99 under a magnetic field of 0–500 mT at 8.0 K in 10 mM maleate buffer (pH 6.2) and in the presence of 50% (v/v) glycerol. According to Rifkind et al. [13], such low-spin complexes characterized by the highest g values in the range 2.83-2.75 and the lowest g values in the range 1.69-1.63 have been designated as “complex B”, thereby indicating the crystal field parameters of the reversible hemichrome (i.e., a waterretained bis-histidine complex). The molar fraction of the hemichrome (complex B) in the oxidized β chains was estimated to be 85% at pH 6.2 because a low-spin species was in equilibrium with a high-spin species corresponding to the usual aquo-met species.

Interestingly, recent crystallographic and EPR spectroscopic studies concerning tetrameric Hbs isolated from the Antarctic fish species Trematomusbernacchii, Trematomusnewnesi, and Gymnodracoacuticep show that endogenous coordination at the sixth coordination site of the heme iron could be the bis-histidyl adducts in the ferric state in the solid and solution state [18,77-79]. In Antarctic fish, isolated Hbs are oxidized readily at room temperature to a partial hemichrome state in which only the iron of the β chain is bonded to the distal His. Such bis-histidyl coordination was also discovered in the crystals of horse metHb exposed to acidic pH (where the proximal His [His87(F8)α] and a water molecule are the axial heme ligands) to the hemichrome (bis-histidine) form (in which the proximal His and the distal His [His58(E7)α] are the axial heme ligands) [80]. In the case of horse metHb, the bis-histidyl coordination was seen in α chains but not in β chains. These crystal structures suggest a different binding state of α and β chains as well as a different pathway to hemichrome.

As described above, the α1–β1(and α2–β2) interface seemed to have dual faces. One is for stabilizing the HbO2 tetramer against the acidic autoxidation and the other is for controlling the fate (removal) of its own erythrocyte from the blood circulation. The α1–β1(and α2–β2) interface produces a conformational constraint in the constructed chains of the Hb molecule via tilting of the distal (E7) His residues, i.e., α58His (E7) in the α chain and β63His (E7) in the β chain. It was also shown that the α1–β1(and α2–β2) interface appears to have a pH-sensor and a temperature-sensor. By virtue of these endowments, it seemed that Hb molecules can accomplish their roles not only as suitable O2 carriers between the lungs and tissues but also as equipped “molecular sensors” within the erythrocyte to control the fate (removal) of their own erythrocytes from the blood circulation depending on internal and extraneous circumstances, including pH and temperature (Figure 13).

Regarding the latter issue, normal erythrocytes develop Heinz bodies late in their lifespan. A wide variety of biochemical changes have been reported to accompany the aging of red cells [81]. These include carboxymethylation of proteins, activation of proteases, glycosylation of proteins, loss of membrane area, decline in changes in the ratio of bands 4.1a to 4.1b, increases in oxidized lipids and proteins, changes in the rheology and fragility of cells, changes in the exposure of cell-surface sugars, and gradual accumulation of Ca2+. Low [82] demonstrated that band-3 clustering might warrant closer scrutiny as a possible transducer of distress signals from the cytoplasm to the external surface of the cell because it can be caused by hemichrome binding, ATP depletion, malondialdehyde formation, Ca2+ accumulation, oxidative crosslinking, or weakening of the skeletal membrane. It is known that hemichromes formed within erythrocytes bind to the cytoplasmic portion of band 3 in the membrane. They then rapidly copolymerize with the soluble cytoplasmic domain of membrane band 3, forming an insoluble copolymer, followed by other changes involved in the pathogenesis of red-cell destruction [83-88]. The dominant role of band-3 clustering suggests that hemichrome formation-induced band- 3 clustering could also provide a key to controlling the fate of senescent and damaged red cells in the blood circulation [84].

Discussion

Heinz bodies are intra-erythrocytic inclusions of hemichrome formed from oxidized or denatured Hb. They are commonly seen by phase contact microscopy as dark spots free in the cytoplasm or attached to the cytoplasmic surface of the erythrocyte membrane. Heinz bodies occur in red cells naturally under three distinct conditions. First, erythrocytes containing unstable Hbs, especially Hbs with mutations near the heme site in the βsubunits, show elevated levels of Heinz bodies [2,3]. Second, Heinz bodies are seen in normal cells (containing normal Hb) under conditions of oxidant stress. The oxidant stress can arise either in cells suffering from a diminished capacity to maintain intracellular reducing power, e.g., in G-6-PD and glutathione peroxidase deficiency diseases [69,70]. Third, Heinz bodies often appear in normal cells as they age [71,72]. This age-related appearance of Heinz bodies is especially pronounced in splenoectomized individuals where a major organ of senescent cell removal has been excised [73]. Heinz body formation, however, whether natural or drug induced or oxidant stress promoted or unstable Hb disease produced, has considered to be occurred by a similar mechanism.

From this point of view, in this article, we made an attempt to propose a new mode of Heinz body formation mechanisms on the basis of our experimental accomplishments and the related current interpretations and arguments, wherein the inherent tilting capability of the distal (E7) His residues (i.e., α58His (E7) in the α chain and β63His (E7) in the β chain) of the Hb molecule plays a major role as well as involvement of the α1–β1(and α2–β2) interface. As represented schematically in Figure 13, in this mechanisms, bishistidylcoordination proficiency to the heme iron of the distal (E7) His residuescan be prerequisite not only for an invocation to the end result of distal-side perturbations of the heme pocket against receipt for incoming stimuli around the molecule (i.e., pH, temperature, oxidative stress), but also for the molecular biosensing mechanisms equipped in human erythrocytes for appreciation of cell aging and determination of their lifespan. Moreover this bis-histidyl coordination of the distal (E7) His residues to the heme iron can be manifested viacollaboration of the α1–β1(and α2–β2) interface of the Hb molecule. Practically, vital hemichromes in erythrocytes can be achieved by intramolecular anioninduced nucleophilic displacement of molecular dioxygen (O2) via an intermediate ferrous heme/anion complex (a low-spin hemichrome) by relying upon intrinsic tilting capability of the distal (E7) His residues (their bis-histidylcoordination proficiency to the heme iron):

In this reaction, nucleophilic displacement can be caused within the heme pocket by the Nε-nitrogen of the distal His (E7) instead of nucleophilic incursion of the water molecule or the hydroxyl ion from outside the molecule. Hence, it was suggested that the sequential changes occurring within red corpuscles upon the removal of nonfunctional erythrocytes include: 1) receipt for internal and external stimuli (i.e., pH, temperature, oxidative stressaround the erythrocyte) via the α1–β1(and α2–β2) interface of the molecule; 2) an invocation to distal-side perturbations of the heme pocket as the end result of the receipt of incoming stimuli; 3) temporal bis-histidyl coordination of the distal (E7) His residues to the heme iron that lead to hemichrome (degradation of the Hb molecule); 4) hemichrome emergence and subsequent hemichrome precipitation and aggregation of the Hb molecules that lead to formation of Heinz bodies within the erythrocyte; and 5) hemolysis of Heinz body-containing red cell in the spleen.

Therefore, in the following, in an attempt to clarify how our hypothesis can explain a diverse status of Heinz body formation within the erythrocytes, we assess these (either in natural or drug induced or oxidant stress promoted or unstable Hb disease produced) one by one in detail.

First of all, we take up unstable Hb disease, as over 90 different unstable Hbs have been reported so far [2,3]. In establishing a diagnosis of unstable Hb disease, the heat denaturation test is considered to be the single most important procedure, in which a fresh hemolysate is diluted into 50 volumes of buffer solution and heated at 60°C; at intervals the precipitates are removed from samples by centrifugation, and the optical density of the supernatant is measured. The reason of such instability even on mild heating (i.e., inclination toward accelerated degradation of Hb molecule to hemichrome resulting hemichrome precipitation and subsequent formation of Heinz bodieswithin the erythrocytes) is quite well understood at the molecular level, but there is still a lack of knowledge of the changes occurring during precipitation and ultimate cause of hemolysis.

Addition to this, the similarity to events observed in the oxidative hemolytic anemia suggests that the precipitation of unstable Hbs is accompanied by oxidative changes [2,3]. The oxidative hemolytic anemia arise either from formation of excess oxidizing products (as with acetylphenylhydrazine administration) or from breakdown of protective mechanisms against oxidants (as in G-6-PD deficiency). In either case, the end result is the same as with unstable Hbs; the precipitation of Hb, the formation of Heinz bodies, and hemolysis.

In our hypothesis, incoming stimuli of the erythrocyte (e.g., pH, temperature, oxidative stress), whether natural or drug induced or oxidant stress promoted or unstable Hb disease produced, can be transmitted ultimately to the distal-side perturbations of residues lining the distal side of the heme pocket via the α1–β1(and α2–β2) interface, so that they let the distal (E7) His residues coordinate at the sixth coordination position of the heme iron to form a bis-histidyl complex. This temporal bis-histidyl coordination by virtue of the inherent tilting capability of the distal (E7) His residues (i.e., hemichrome emergence) in turn triggers a series of irreversible chemical reactions (including hemichrome formation-induced band-3 clustering and subsequent formation of Heinz bodies) that lead to the removal of non-functional erythrocytes from the blood circulation. Our hypothesis definitely demonstrates that there is positive correlation between the oxidative behavior of human Hb, its inseparably related instability (accelerated inclination toward degradation of Hb molecule to hemichrome), the subsequent formation of Heinz bodies within the erythrocyte and its ultimate fate (hemolysis) even on mild conditions at close-tophysiological temperatures and pH.

The defect in unstable Hbs involves important internal bonding amino acids, particularly those forming bonds with the heme group. Many of the mutants that occur at the α1–β2(and α2–β1) interface have altered oxygen affinity, but the bulk of evidence suggests that the α1–β1(and α2–β2) interface is much more important in maintaining the molecular stability of Hb than the α1–β2(and α2–β1) interface. Hemolytic anemia results from substitutions affecting the α1–β1(and α2–β2) interface or the heme pocket. If such mutations occur, the heme iron will be oxidized more readily, and a sequence of events leads to hemichrome formation and subsequent Heinz-body clustering in red cells that causes hemolytic anemia. Typical examples of such variants are summarized in Table 3. Surprisingly, almost all of these pathological mutations are found on the β chain, especially in the α1–β1(and α2–β2) contact region: Tacoma [β30(B12)Arg → Ser], Abraham Lincoln [β32(B14)Leu → Pro], Castilla [β32(B14)Leu → Arg], Peterborough [β111(G13)Val → Phe], Madrid [β115(G17)Ala → Pro], Khartoum [β124(H2)Pro → Arg], J. Guantanamo [β128(H6)Ala → Asp] and Leslie [β131(H9)Gln → deleted]. In these Hbs, the α1–β1(and α2–β2) interface would become loose or disruptive due to many different causes including: the insertion of proline (Abraham Lincoln, Madrid), the substitution with a too-small amino acid side chain (Tacoma) or a toolarge side chain (Peterborough), the introduction of a charged or very polar group (Castilla, Khartoum, J. Guantanamo), and the deletion of amino acid residue (Leslie).

• E [β26(B8)Glu → Lys] • Volga [β27(B9)Ala → Asp]
• Genova [β28(B10)Leu → Pro] • St. Louis [β28(B10)Leu → Gln]
• Tacoma [β30(B12)Arg → Ser] • Abraham Lincoln [β32(B14)Leu → Pro]
• Castilla [β32(B14)Leu → Arg] • Philly [β35(C1)Tyr → Phe]
• Rush [β101(G3)Glu → Gln] • Peterborough [β111(G13)Val → Phe]
• Madrid [β115(G17)Ala → Pro] • Khartoum [β124(H2)Pro → Arg]
• J. Guantanamo [β128(H6)Ala → Asp] • Wien [β130(H8)Tyr → Asp]
• Leslie [β131(H9)Gln → deleted] • Torino [α43(CD1)Phe → Val]
• L. Ferrara [α47(CD5)Asp → Gly] • Setif [α94(G1)Asp → Tyr]
• St. Lukes [α95(G2)Pro → Arg]  

Table 3: Unstable hemoglobin variants, showing sites of amino acid substitutions (or deletions), redrawn from Sugawara et al. [34]. Each individual row illustrates variant´s name, the residue concerned (chain; number; position), and replacement (from → to), respectively.

The situation in patients with G-6-PD deficiency [2,3] can serve as another example. Red cells are known to be vulnerable to injury by endogenous and exogenous oxidants. Oxidants can be inactivated by reduced glutathione in erythrocytes with normal G-6- PD activity because the pentose phosphate shunt supplies NADPH (which is required for glutathione recycling). In G-6-PD-deficient erythrocytes, however, the reduced glutathione cannot be restored, and the cells sustain irreversible oxidative damage. However, G-6- PD deficiency produces symptoms only if the patient is exposed to an environmental factor that results in increased oxidative stress. Such stress includes antimalarials (e.g., primaquine, pamaquine, dapsone), sulfonamides, nitrofurantoin and phenacetin. The resulting crisis can lead to hemolysis of up to 25–30% of red cells within hours. However, the crisis is self-limited, and only the older population of red cells is destroyed. The mechanisms responsible for such an acute hemolytic crisis in patients taking these drugs cannot be explained on the basis of conventional views. Figure 13 suggests that this crisis could be triggered by fluctuations in endogenous and exogenous oxidative stress under the fragile pentose phosphate shunt.

Malaria provides the other example. This is a protozoal disease transmitted by the bite of female Anopheles mosquitoes (Plasmodium falciparum, P. malariae, P. vivax, and P. ovale). Blackwater fever syndrome is caused by infection with P. falciparum, and is characterized by repeated bouts of chills and fevers, severe intravascular hemolysis and anemia, jaundice, hemoglobinuria (black urine) and splenic enlargement [89-91]. Patients often develop fevers above 40°C. A feature of this syndrome is that symptoms can be exacerbated in patients with G-6-PD deficiency as a result of taking quinine. In these patients, massive destruction of red cells infected by the parasite occurs, but similar numbers of normal erythrocytes are also ruptured, resulting in characteristic hemoglobinuria. The reason for the massive hemolysis of non-infected red cells is unclear. However, the possibility of a relationship between quinine ingestion and the associated massive hemolysis, as well as the relevance of G-6-PD deficiency, is addressed in Figure 13. Fevers above 40°C, endogenous G-6-PD deficiency, and exogenous quinine ingestion represent conditions that induce acute hemolysis, thereby leading to massive blood loss and hemoglobinuria.

Conclusion

Cellular life is reliant upon rapid and efficient responses to internal and external conditions whereby basic molecular events associated with these processes are the structural transitions of the proteins (structural protein allostery) involved. The human Hb molecule (α2β2) holds a special position in these structural transitions. Hb has two types of α–β interface (i.e., α1–β1[and α2–β2] and α1–β2[and α2–β1]). The latter α1–β2(and α2–β1) interface is associated with cooperative O2 binding, and exhibits principal roles if the molecule goes from its deoxygenated to oxygenated quaternary structure. However, the role of the former α1–β1(and α2–β2) interface has been unclear for a long time.

In this regard, important and intriguing observations have been accumulating, so that a new gaze can be focused on the α1–β1(and α2–β2) interface. In this article, on the basis of our experimental accomplishments and the related current interpretations and arguments, we first proposed a new mode of Heinz body formation mechanisms, wherein the inherent tilting capability of the distal (E7) His residues(i.e., α58His (E7) in the α chain and β63His (E7) in the β chain) of the Hb molecule plays a major role as well as involvement of the α1–β1(and α2–β2) interface. Specifically, the suggested sequential changes occurring within the erythrocyte upon the removal of nonfunctional erythrocytes, whether natural or drug induced or oxidant stress promoted or unstable Hb disease produced, include: 1) receipt for internal and external stimuli around the erythrocyte (e.g., pH, temperature, oxidative stress) via the α1–β1(and α2–β2) interface of the molecule; 2) an invocation to distal-side perturbations of the heme pocket as the end result of the receipt of incoming stimuli; 3) temporal bis-histidylcoordination of the distal (E7) His residuesto the heme iron that lead to hemichrome (degradation of the Hb molecule); 4) hemichrome emergence and subsequent hemichrome precipitation and aggregation of the Hb molecules that lead to formation of Heinz bodies within the erythrocyte; and 5) hemolysis of Heinz bodycontaining red cell in the spleen.

Granting this new mode of Heinz body formation mechanisms to be basic premise, we then made an attempt to examine how this new mode of mechanisms can serve as a basis for deeper understanding of clinical aspects of drag-induced hemolytic anemia, defects in the intraerythrocytic reducing system and unstable Hb disease, in which the mechanisms for acute hemolytic crisis cannot be explained on the basis of conventional views. The followings were shown:

(1) Almost all of pathological mutations of unstable Hbs are found on the β chain, especially in the α1–β1(and α2–β2) contact region: Tacoma [β30(B12)Arg → Ser], Abraham Lincoln [β32(B14)Leu → Pro], Castilla [β32(B14)Leu → Arg], Peterborough [β111(G13)Val → Phe], Madrid [β115(G17) Ala → Pro], Khartoum [β124(H2)Pro → Arg],J. Guantanamo [β128(H6)Ala → Asp] and Leslie [β131(H9)Gln → deleted].

(2) Acute hemolytic crisis observed in patients of G-6-PD deficiency while taking drugs such as antimalarials (e.g., primaquine, pamaquine, dapsone), sulfonamides, nitrofurantoin and phenacetin can be explained by increased oxidative stress against inadequate reduced glutathione levels in erythrocytes of the patients.

(3) Acute hemolytic crisis associated with black water fever syndrome of malarial patients caused by infection with Plasmodium falciparum can be also explained by fevers above 40°C, endogenous G-6-PD deficiency, and exogenous quinine ingestion.

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