Contribution of Surface Histidyl Residues in the -Chain to the Bohr Effect of Human Normal Adult Hemoglobin: Roles of Global Electro
详细信息 查看全文
- 作者：Dazhen Philip Sun ; Ming Zou ; Nancy T. Ho ; and Chien Ho
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：June 3, 1997
- 年：1997
- 卷：36
- 期：22
- 页码：6663 - 6673
- 全文大小：297K
- 年卷期：v.36,no.22(June 3, 1997)
- ISSN：1520-4995
文摘
We have applied site-directed mutagenesis to our Escherichiacoli hemoglobin expressionplasmid and constructed five recombinant mutant hemoglobins (r Hbs):r Hb(
20His
Gln or :H20Q);r Hb(:H50Q); r Hb(:H72Q); r Hb(:H89Q); and rHb(:H112Q). We have constructed these r Hbstohelp us assess the contribution of the surface histidyl residues in the-chain to the alkaline Bohr effectof human normal adult hemoglobin (Hb A). In our laboratory, wehave monitored the variation of protonnuclear magnetic resonances arising from the C2 protons of the histidylresidues of Hb A as a functionof pH and buffer conditions. Several of these resonances have beenassigned to the individual histidylresidues on the surface of the hemoglobin molecule using naturallyoccurring mutant hemoglobins andchemically modified hemoglobins. In the present work, we haveidentified the C2 proton resonances offive surface histidyl residues of the -chain, 20, 50, 72,89, and 112, in both the carbonmonoxyand deoxy forms, by comparing the proton nuclear magnetic resonancespectra of Hb A with those of ther Hbs. For the assignment of the C2 proton resonances of 20Hisand 112His, we have used combinationsof mutations to compensate for the spectral perturbations resultingfrom the single mutations, which obscurethe resonance assignment. On the basis of the new findings, insolvent containing 0.1 M chloride, theoverall contributions from surface histidyl residues of both the -and 
-chain and from other previouslyidentified alkaline Bohr groups account for approximately 75% of theobserved Bohr effect at pH 7.3 (themaximum Bohr effect under the prescribed solvent conditions). Ourresults show that some histidyl residuescontribute to the Bohr effect and some oppose the net Bohr effect.In some cases, the addition of anionscan diminish or reverse the contributions of specific histidyl residuesto the overall Bohr effect. Thus,the Bohr effect, a heterotropic effect, depends on the intricatearrangement and interactions of all hydrogenand anion binding sites in the hemoglobin molecule. It is anexcellent example of global electrostaticeffects in proteins.
20HisGln or :H20Q);r Hb(:H50Q); r Hb(:H72Q); r Hb(:H89Q); and rHb(:H112Q). We have constructed these r Hbstohelp us assess the contribution of the surface histidyl residues in the-chain to the alkaline Bohr effectof human normal adult hemoglobin (Hb A). In our laboratory, wehave monitored the variation of protonnuclear magnetic resonances arising from the C2 protons of the histidylresidues of Hb A as a functionof pH and buffer conditions. Several of these resonances have beenassigned to the individual histidylresidues on the surface of the hemoglobin molecule using naturallyoccurring mutant hemoglobins andchemically modified hemoglobins. In the present work, we haveidentified the C2 proton resonances offive surface histidyl residues of the -chain, 20, 50, 72,89, and 112, in both the carbonmonoxyand deoxy forms, by comparing the proton nuclear magnetic resonancespectra of Hb A with those of ther Hbs. For the assignment of the C2 proton resonances of 20Hisand 112His, we have used combinationsof mutations to compensate for the spectral perturbations resultingfrom the single mutations, which obscurethe resonance assignment. On the basis of the new findings, insolvent containing 0.1 M chloride, theoverall contributions from surface histidyl residues of both the -and -chain and from other previouslyidentified alkaline Bohr groups account for approximately 75% of theobserved Bohr effect at pH 7.3 (themaximum Bohr effect under the prescribed solvent conditions). Ourresults show that some histidyl residuescontribute to the Bohr effect and some oppose the net Bohr effect.In some cases, the addition of anionscan diminish or reverse the contributions of specific histidyl residuesto the overall Bohr effect. Thus,the Bohr effect, a heterotropic effect, depends on the intricatearrangement and interactions of all hydrogenand anion binding sites in the hemoglobin molecule. It is anexcellent example of global electrostaticeffects in proteins.