The Preparation and Enzymatic Hydrolysis of Reduced and S-Carboxymethylated Proteins

Arthur M Crestfield,Stanford Moore,William H Stein

doi:10.1016/s0021-9258(18)81308-4

Abstract

The Preparation and Enzymatic Hydrolysis of Reduced and S-Carboxymethylated Proteins

Highlights

The results show that a cysteic acid residue following the residue of arginine inhibits the action of the enzyme much more than does the carboxymethylcysteine group
Chromatographyof Peptides-The preparation of reduced and carboxymethyled proteins can be accomplished with a high degree of specificity, and the resulting products are readily cleaved by enzymes; further experience is required, before detailed recommendations can be made for the chromatographic separation of purified peptides containing methionine, carboxymethylcysteine, and tryptophan residues
In a comparison of reagents for the specific reduction of disulfide bonds in proteins, mercaptoethanol in 8 M urea has proved preferable to sodium borohydride

Summary

Methods

Preparation of Reduced, Carboxymethylated Protein at 50-mgScale (cj. [4, 5])-To 5 to 100 mg of protein in a 12-ml screw-cap vial (Kimble 6091OL) maintained under a nitrogen barrier [6], add 3.61 g of deionized, crystalline urea (Benesch, Lardy, and Benesch [7]), 0.30 ml of EDTA solution (50 mg of disodium EDT*4 per ml), 3.0 ml of Tris buffer, pH 8.6, (5.23 g of Tris and 9 ml of 1.0 N HCl diluted to 30 ml with water), and 0.10 ml of mercaptoethanol (Eastman Organic Chemicals). Preparation of Reduced, Carboxymethylated Protein at 50-mg. [4, 5])-To 5 to 100 mg of protein in a 12-ml screw-cap vial (Kimble 6091OL) maintained under a nitrogen barrier [6], add 3.61 g of deionized, crystalline urea (Benesch, Lardy, and Benesch [7]), 0.30 ml of EDTA solution (50 mg of disodium EDT*4 per ml), 3.0 ml of Tris buffer, pH 8.6, (5.23 g of Tris and 9 ml of 1.0 N HCl diluted to 30 ml with water), and 0.10 ml of mercaptoethanol (Eastman Organic Chemicals). A freshly prepared solution of 0.268 g of iodoacetic acid in 1.0 ml of 1.0 N NaOH is added to the reaction mixture. The iodoacetate added is slightly less on a molar basis than the amount of mercaptoethanol.

Results

Discussion

Conclusion