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

• Published in: Biophysical chemistry 2006-04, Vol.121 (1), p.65-73
• Main Authors: Okonjo, Kehinde Onwochei, Fodeke, Adedayo A., Kehinde, Abisola Temilade
• Format: Article
• Language: English
• Subjects: 5,5′-dithiobis(2-nitrobenzoate); Animals; Cat hemoglobins; Cats; CysF9β sulfhydryl group; Dithionitrobenzoic Acid - chemistry; Hemoglobins - chemistry; Hydrogen-Ion Concentration; Kinetics; Oxidation-Reduction; pH dependence of equilibrium constant; Reverse rate constant; Sulfhydryl Reagents - chemistry
• ISSN: 0301-4622; 1873-4200
• DOI: 10.1016/j.bpc.2005.12.012

We have determined for the first time the equilibrium constant, K eq, for the reaction of Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoate), with the CysF9[93]β sulfhydryl groups of the hemoglobins of the domestic cat. In the pH range 5.6 to 9.0 K equ varies over four orders of magnitude — between ca 10 and 10 − 3 — for all hemoglobin derivatives. Using these K equ values and published data on the dependence of the apparent second order forward rate constant, k f, on pH we have calculated the apparent second order reverse rate constant, k r, as a function of pH. This parameter increases strongly with pH, particularly above pH 7.5. Quantitative analyses of the pH dependence profiles of log 10 k r indicate that the reverse reaction is coupled to the ionization of two groups on the protein with p K as of 7.2 ± 0.2 and 9.4 ± 0.1 in the major hemoglobin and 6.7 ± 0.3 and 8.4 ± 0.1 in the minor hemoglobin.