Journal of Analytical & Bioanalytical Techniques
Open Access

Abstract

Human adult hemoglobin (HbA) has four subunits, namely, two- a subunits and two b -subunits. X-ray crystallographic analysis have indicated that there are two distinct quaternary structures, namely, the deoxy state, represented by the tense (T), low-affinity structure, and oxy state, represented by the relaxed (R), high-affinity structure. A characteristic spectral change of HbA in the near-UV CD occurs: from a small positive band in the oxy-R form to a negative CD band with a distinct peak at 287nm in the deoxy-T form. This negative CD band of deoxyHbA known as T-state marker has been supposed to derive from the changes of Tyr and Trp residues at the �?±1�?²2 subunit interface. To identify the aromatic residue responsible for the CD band, we have synthesized five recombinant Hbs in E. coli in which non aromatic residue is substituted for Tyr or Trp residue; rHb (�?±14Trpâ�?�?Leu), rHb (�?²15Trpâ�?�?Leu), rHb (�?²37Trpâ�?�?His), rHb (�?±42Tyrâ�?�?Ser), and rHb (�?²145Tyrâ�?�?Thr). We examined the near-UV CD spectra of these rHbs and a natural mutant, Hb Rouen (�?±140Tyrâ�?�?His). The CD spectra of individual aromatic residue were extracted from the difference between Hb A and each mutant. We concluded that changes in CD bands arising fromï�? �?²37Trp,ï�?  �?±140Tyr, �?²145Tyr and �?±42Tyr residues contributed to the appearance of the negative CD bands at 287nm. To examine different signals of CD band among aromatic residues, the effects of environments on CD spectra were examined using model compounds of Tyr and Trp dissolved in various solvants.

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