The effect of solution conditions on bovine serum albumin sulfhydryl reactivity

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 (DTNB2-). 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 DTNB2-. 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.