PREPARATION AND KINETIC CHARACTERIZATION OF A SERIES OF BETA-W37 VARIANTS OF HUMAN HEMOGLOBIN-A - EVIDENCE FOR HIGH-AFFINITY T-QUATERNARY STRUCTURES

Citation
Ld. Kwiatkowski et al., PREPARATION AND KINETIC CHARACTERIZATION OF A SERIES OF BETA-W37 VARIANTS OF HUMAN HEMOGLOBIN-A - EVIDENCE FOR HIGH-AFFINITY T-QUATERNARY STRUCTURES, Biochemistry, 37(13), 1998, pp. 4325-4335
Citations number
38
Categorie Soggetti
Biology
Journal title
ISSN journal
00062960
Volume
37
Issue
13
Year of publication
1998
Pages
4325 - 4335
Database
ISI
SICI code
0006-2960(1998)37:13<4325:PAKCOA>2.0.ZU;2-U
Abstract
Four variants of human beta globin in which the Trp at position 37 has been replaced with a Tyr Ala, Gly; or Glu have been expressed in Esch erichia coli. These globins have been combined with normal human alpha chains and heme to form tetrameric hemoglobin molecules. A technique for the preparation of alpha chain dimers, which are cross-linked betw een their (alpha 99 lysine residues, has been developed, and these alp ha dimers were combined with two of the beta globins, beta W37G and be ta W37E, to form the corresponding cross-linked variants. The kinetics of CO binding to the deoxygenated derivatives following rapid mixing and of CO rebinding following flash photolysis have been examined as f unctions of pH in the presence and absence of the organic phosphate in ositol hexaphosphate, IHP. The kinetic measurements indicate that repl acement of the tryptophan with other residues destabilizes the hemoglo bin tetramer, resulting in considerable dissociation of even the deoxy genated hemoglobins into alpha beta dimers at micromolar protein conce ntrations. Substitutions at beta 37 also alter the properties of the d eoxygenated hemoglobin tetramer. The alteration of the functional prop erties of the T states of these variants as well as the tendency of th e deoxygenated derivatives to dissociate into alpha beta dimers increa ses in the order HbA < beta W37Y < beta W37A < beta W37G < beta W37E. Stabilizing the beta W37G or beta W37E tetramers by addition of MP or by crosslinking does not restore the normal functional properties of t he T state. Measurements of the geminate rebinding of CO establish a k inetic difference between the normal R state tetramer and the alpha be ta dimer consistent with quaternary enhancement, the greater affinity of oxygen for the R state tetramer than for the alpha beta dimer. Kine tics of geminate rebinding also suggest that quaternary enhancement ma y be altered by substitutions at the beta 37 position.