Reversible reaction of 5,5′-dithiobis(2-nitrobenzoate) with the hemoglobins of the domestic cat: Acetylation of NH3+ terminal group of the β chain transforms the complex pH dependence of the forward apparent second order rate constant to a simple form

Reversible reaction of 5,5′-dithiobis(2-nitrobenzoate) with the CysF9[93]β sulfhydryl groups of the hemoglobins of the domestic cat: Variation of the equilibrium and reverse rate constants with pH

“…However, if the forward rate constant, k f , is known, the reverse rate constant, k r , can be determined through a knowledge of the equilibrium constant, K equ . Although our kinetic data [1] strongly indicate that the reaction of DTNB with the hemoglobins of the domestic cat is reversible, this can be confirmed only by attempting to carry out an equilibrium study. We have undertaken a comprehensive investigation of the pH dependence of K equ for the oxy, carbonmonoxy and aquomet derivatives of the major and minor hemoglobins of the domestic cat.…”

“…k r values were determined from the ratio of the k f values of Ref. [1] and the K equ values reported in Figs. 1 and 2 of the present paper.…”

“…The methods employed for preparing the cat hemolysate and for separating the major and minor hemoglobin components have been reported elsewhere [1]. Prior to its use for an experiment, each hemoglobin component was passed through a Dintzis ion exchange column [5] to remove endogenous organic phosphates and other undesired ions.…”

“…(1), HbS − þ DTNB X k f k r HbSd TNB þ TNB − , is an example of two opposing second-order processes in which two products, HbS·TNB and TNB − , result from the initial forward reaction. Under pseudo-first order conditions (with DTNB in excess), it is possible to determine the apparent second order forward rate constant, k f , by plotting the observed pseudo-first order rate constant, k obs , against the DTNB concentration [1]. It is typically difficult to directly determine the apparent reverse rate constant, k r , for such a system unless the products of the forward reaction can both be isolated.…”

“…In a recent report [1] we presented kinetic evidence indicating that the reaction of 5,5′-dithiobis (2 nitrobenzoate) -DTNBwith the CysF9[93]β sulfhydryl groups of the hemoglobins of the domestic cat might be a reversible process. If this is so, then it should be possible to determine its equilibrium constant and, hence, the apparent second order reverse rate constant, a parameter that cannot be directly determined for this system.…”