42-6

 

Solution conditions significantly affect sufhydryl (SH) group reactivity of bovine serum albumin (BSA). The objective of this study was to determine the effect of solution conditions on SH-reactivity. Conditions were varied in pH, temperature and the assortment of additives used. Changes in SH-group reactivity, defined in terms of a second order rate constant for SH- disulfide exchange (k, M^-1s^-1), were measured calorimetrically using 5,5’-dithiobis 2-nitrobenzoate (DTNB^2-). All results were compared to BSA under standard conditions (pH 7.0, 50mM ionic strength, 25⁰C). SH-reactivity increased from 0.49 M^-1s^-1 at pH 6, to 1.74 M^-1s^-1 at pH 7 and to 14.48 M^-1s^-1 at pH 8. This increase can be attributed to the pH microenvironment approaching the isoelectric point of cysteine. A 10⁰C increase in temperature from 25⁰C to 35⁰C doubled the rate of SH-group reactivity from 1.74 M^-1s^-1 to 3.37 M^-1s^-1. When additives were used, sodium chloride (0.1M) and urea (8M) were found to increase SH-group reactivity from 1.74 M^-1s^-1 to 23.15 M^-1s^-1 and 12.03 M^-1s^-1 respectively; whilst addition of ethanol (10%) did not have any effect on SH-reactivity (k=1.77 M^-1s^-1). Increase of reactivity upon the addition of sodium chloride (1.0M) may be due to suppression of electrostatic repulsion between BSA and DTNB^2-. The increase of SH-group reactivity upon addition of urea is due to the denaturation of BSA’s tertiary structure to expose the hidden SH-group. Findings from this study are important for better understanding of BSA structure and also characterization of SH-group location and reactivity.