M. Perrella et al., FUNCTIONAL-HETEROGENEITY OF THE ALPHA-SUBUNIT AND BETA-SUBUNIT IN THEASSOCIATION REACTION BETWEEN HEMOGLOBIN AND CARBON-MONOXIDE, Biophysical chemistry, 61(2-3), 1996, pp. 169-176
A technique is described for the rapid inactivation and removal of exc
ess ferricyanide used for the non-cryogenic oxidation of the unligande
d subunits of the intermediates in the association reaction between he
moglobin and carbon monoxide. Under these conditions the asymmetric ox
idized intermediates, which dissociate into non-identical dimers, disp
roportionate into their parent tetramers and four species, Hb(+), HbCO
, alpha(2)(+)beta(2)(CO), alpha(2)(CO)beta(2)(+), are isolated by non-
cryogenic isoelectric focusing. The relative concentrations of species
alpha(2)(CO)beta(2)(+) and alpha(2)(+)beta(2)(CO) measure the overall
distribution of the ligand between the alpha and beta subunits in the
association reaction. At 20 degrees C in 0.1 M KCI, pH 7, preferentia
l CO binding to the beta subunits was observed, in agreement with obse
rvations made by the cryogenic technique for the isolation of the inte
rmediates [M. Perrella, N. Davids and L. Rossi-Bernardi, J. Biol. Chem
. 267 (1992) 8744].