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Title: Assessment of roles of surface histidyl residues in the molecular basis of the Bohr effect and b143 histidine in the binding of 2,3-bisphosphoglycerate in human normal adult hemoglobin
Authors: Tsuei-Yun Fang
Ming Zou
Virgil Simplaceanu
Nancy T. Ho
Chien Ho
Contributors: 國立臺灣海洋大學:食品科學系
Date: 1999-09
Issue Date: 2017-07-24T02:25:23Z
Publisher: Biochemistry
Abstract: Abstract:Site-directed mutagenesis has been used to construct two mutant recombinant hemoglobins (rHbs), rHb(βH116Q) and rHb(βH143S). Purified rHbs were used to assign the C2 proton resonances of β116His and β143His and to resolve the ambiguous assignments made over the past years. In the present work, we have identified the C2 proton resonances of two surface histidyl residues of the β chain, β116His and β143His, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance (NMR) spectra of human normal adult hemoglobin (Hb A) with those of rHbs. Current assignments plus other previous assignments complete the assignments for all 24 surface histidyl residues of human normal adult hemoglobin. The individual pK values of 24 histidyl residues of Hb A were also measured in deuterium oxide (D2O) in 0.1 M N-(2-hydroxyethyl)piperazine-N‘-2-ethanesulfonic acid (HEPES) buffer in the presence of 0.1 M chloride at 29 °C by monitoring the shifts of the C2 proton resonances of the histidyl residues as a function of pH. Among those surface histidyl residues, β146His has the biggest contribution to the alkaline Bohr effect (63% at pH 7.4), and β143His has the biggest contribution to the acid Bohr effect (71% at pH 5.1). α20His, α112His, and β117His have essentially no contribution; α50His, α72His, α89His, β97His, and β116His have moderate positive contributions; and β2His and β77His have a moderate negative contribution to the Bohr effect. The sum of the contributions from 24 surface histidyl residues accounted for 86% of the alkaline Bohr effect at pH 7.4 and about 55% of the acid Bohr effect at pH 5.1. Although β143His is located in the binding site for 2,3-bisphosphoglycerate (2,3-BPG) according to the crystal structure of deoxy-Hb A complexed with 2,3-BPG, β143His is not essential for the binding of 2,3-BPG in the neutral pH range according to the proton NMR and oxygen affinity studies presented here. With the accurately measured and assigned individual pK values for all surface histidyl residues, it is now possible to evaluate the Bohr effect microscopically for novel recombinant Hbs with important functional properties, such as low oxygen affinity and high cooperativity. The present study further confirms the importance of a global electrostatic network in regulating the Bohr effect of the hemoglobin molecule.
Relation: 38(40), pp.13423-13432
URI: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/43383
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