The reactions of proteins with thiols and disulphides

Abstract

1. A method has been developed for the estimation of protein sulphydryl content. This method was used in a study of the interactions of a few biologically important sulphydryl containing proteins with non -protein thiols and disulphides.

2. No interaction was detected between GSSG or GSH and the proteins, namely, bovine and human haemoglobins bovine δ- globulin and bovine and human serum albumins, under the conditions employed.

3. CySSCy reacted with bovine haemoglobin, removing one sulphydryl group per molecule, and with bovine serum albumin. The latter reaction was studied in detail.

4. Equimolar amounts of CySSCy and BSA reacted at 37°C and pH 7.4. This reaction removed 75% of the titratable protein sulphydryl content. Increase in the CySSCy - mercaptalbumin molar ratio to 4.5:1 brought the protein sulphydryl content to zero.

The extent of the removal of CySSCy or protein sulphydryl indicated that one molecule of CySSCy was reacting with two molecules mercaptalbumin.

5. The reaction requires an intact protein sulphydryl group. Blocking of the sulphydryl group with p-chloromercuribenzoate completely abolished the reaction.

The pH dependence of the reaction suggests that it is mediated through the mercaptide ion.

Addition of catalytic amounts of heavy metal ions or non-protein thiols had no effect upon the reaction.

6. Reaction rate studies indicated second order kinetics for the reaction between mercaptalbumin and 2CySSCy, a finding explicable by postulating a rapid spontaneous oxidation to CySSCy of any CySH produced in the reaction,

7. Regeneration of the BSA sulphydryl group, removed by reaction with CySSCy, was brought about by reagents (GSH and Cyanide) which react with disulphide bonds. Some of the sulphydryl produced appeared to arise from the reduction of intramolecular disulphide bonds.

8. The findings enumerated above are compatible with the formation of a mixed disulphide between mercaptalbumin and 2CySSCy residues. No evidence for the formation of a BSA disulphide dimer was obtained.

9. BSA was found to react with other non -protein thiols and disulphides but not as readily as with CySSCy or CySH.

CySSCy also reacted with human plasma under mild conditions of temperature and pH, causing a decrease in the protein sulphydryl content.

lo. - 144 - The findings enumerated above are compatible with the formation of a mixed disulphide between mercaptalbumin and 2CySSCy residues. No evidence for the formation of a BSA disulphide dimer was obtained. BSA was found to react with other non -protein thiols and disulphides but not as readily as with CySSCy or CySH. CySSCy also reacted with human plasma under mild conditions of temperature and pH, causing a decrease in the protein sulphydryl content. The factors which affect thiol - disulphide interactions and the possible role of mixed disulphides are discussed.