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
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.