Abstract
The kinetics of CO association to and dissociation from the two isomers of monoliganded species alpha ICO beta I(alpha II beta II) and alpha I beta I (alpha II beta COII) has been studied by double-mixing stopped-flow and microperoxidase methods. The monoliganded species were generated by hybridization between excess ferric Hb and alpha CO2 beta +2 or alpha +2 beta CO2 prepared by high-pressure liquid chromatography (HPLC). The results indicated that: 1) there were no significant differences in the reactivities of alpha and beta chains in the first step of ligation; 2) in the second step of ligation there was significant cooperativity in the reaction of deoxyhemoglobin with 0.05 or 0.1 equivalent of CO. Diliganded species were therefore formed in significant amounts. The double-mixing HPLC results suggested that in the second step of ligation alpha chains reacted faster than the beta chains, and the main diliganded species formed was alpha I beta ICO (alpha IICO beta II) or its isomer alpha ICO I(alpha II beta IICO). These results seem to indicate that the reaction of the first CO is mostly random and in the second step of ligation CO binds more to the tetramers in which one beta chain is already ligated: alpha I beta I (alpha II beta II) + CO----alpha ICO beta I (alpha II beta II) and alpha I beta ICO (alpha II beta II) + CO----alpha I beta ICO (alpha IICO beta II).
Highlights
2a2P2 two isomers of monoliganded species indicate that there are no significant differences in the reactivities of a and /3 chains
Calculations on the basis of step-wise rate constants and for the conditions maintained in HPLC experiments indicate that about 60% of CO should be in the form of diliganded species which, as argued above is most likely a?o@~(a~~I@nH~PoL)C. experiments, we should observe a:P$ = 30% if all of the monoliganded species aI@I(aI&') reacted with CO in the second step of ligation; the observed value in the HPLCexperiments was 33%.These results imply that: 1)there is significant cooperativity in the second step of ligation; and 2) the reaction of the second molecule of CO takes place more with the tetramer in which
HPLC experiments suggest that these cooperative effects are J.Biol
Summary
Vol 266,No 36, Issue of December 25, pp. 24492-24497,1991 Printed in U.S.A. Double-mixing KineticStudies of the Reactions of Monoliganded Species of Hemoglobin:c ~ & ~ and ~ ) 1 @cy~2@&CO)1*. The double-mixing HPLC results suggested that in the second step of ligation a chains reacted faster than the fi chains, and the main diliganded species formed was a&o(a~ofilI)or its isomer aFoI(aI&o)T. hese results seem to indicate that the reaction of the first CO is mostly random and in the. The type of monoliganded species formed will depend on the relative reactivities of a and /3 chains, and it will, to some extent, determine the nature of the diliganded species formed in the step of ligation. Khaleque and Sawicki (1986) and Zahroon and Sawicki (1989)have studied the kinetic properties of the monoliganded species by the flow-flash method The results of these studies and their interpretation of the dataare of direct relevance to this study and will be considered in the "Discussion." It will suffice to mention at this stage that both of these studies suggest the presence of a slow conformational change in Hb4(CO)I as a result of which the CO combination reaction following the flash, was biphasic. The rate constants for the two phases were typical of the T and R state species
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