Direct Spectrophotometric Measurement of the Rate of Reduction of Disulfide Bonds

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.