Direct Spectrophotometric Measurement of the Rate of Reduction of Disulfide Bonds: THE REACTIVITY OF THE DISULFIDE BONDS OF BOVINE α-LACTALBUMIN

Abstract

Abstract The rate of reduction of the disulfide bonds of peptides and proteins by dithiothreitol can be determined by measuring, as a function of time, the absorbance at 310 nm of the oxidized dithiothreitol formed. The method is rapid and can produce a complete kinetic curve with 0.1 µmole of disulfide. Results obtained with oxidized glutathione, ribonuclease, bovine serum albumin and α-lactalbumin show that the correct stoichiometry is obtained for the reaction. All four of the disulfide bonds of α-lactalbumin are shown to be readily available for reduction by 10-3 m dithiothreitol in aqueous buffers at room temperature. At temperatures near 0°, three of the four disulfides are no longer susceptible to reduction. Thus the conformation of the α-lactalbumin molecule is altered in a dramatic way over this temperature range.