ASSIGNMENT OF THE DISULFIDE BONDS IN THE SWEET PROTEIN BRAZZEIN

The thermostable sweet protein brazzein consists of 54 amino acid resi dues and has four intramolecular disulfide bonds, the location of whic h is unknown. We found that brazzein resists enzymatic hydrolysis at e nzyme/substrate ratios (w/w) of 1:100-1:10 at 35-40 degrees C for 24-4 8 h. Brazzein was hydrolyzed using thermolysin at an enzyme/substrate ratio of 1:1 (w/w) in water, pH 5.5, for 6 h and at 50 degrees C. The disulfide bonds were determined, by a combination of mass spectrometri c analysis and amino acid sequencing of cystine-containing peptides, t o be between Cys4-Cys52, Cys16-Cys37, Cys22-Cys47, and Cys26-Cys49. Th ese disulfide bonds contribute to its thermostability. (C) 1996 John W iley & Sons, Inc.