Quantification of cysteine residues following oxidation to cysteic acid in the presence of sodium azide.
Quantification of cysteines by amino acid composition analysis is inaccurate because of decomposition of these residues during protein hydrolysis. Cysteine (and cystine) residues are oxidized to cysteic acid following hydrochloric acid hydrolysis in the presence of sodium azide. Using selected native and recombinant proteins, containing different numbers of cysteine residues, we investigated the conditions for the quantitative oxidation of cysteines to cysteic acid in the presence of sodium azide. Protein hydrolysis with hydrochloric acid in the presence of 0.20% sodium azide resulted in 87-100% oxidation of the cysteines to cysteic acid which was easily quantified. The results were highly reproducible so that the azide-induced oxidation can be used as a general method to determine cysteine residues in a given protein. The sodium azide-dependent oxidation is superior to oxidation with performic acid because (i) it can be performed in solution not requiring protein lyophilization and in approximately half of the time; (ii) it delivers slightly higher yields of cysteic acid; and (iii) it does not affect tyrosine residues, which can be modified during the performic acid treatment.
Manneberg
M
M
F. Hoffmann-LaRoche Ltd., Pharmaceutical Research-Gene Technologies, Basel, Switzerland.
Lahm
H W
HW
Fountoulakis
M
M
D000596:Amino Acids / Q000032:analysis; D001386:Azides; D003544:Cysteic Acid / Q000737:chemistry*; D003545:Cysteine / Q000032:analysis*; D006851:Hydrochloric Acid; D006868:Hydrolysis; D007202:Indicators and Reagents; D016898:Interferon-alpha / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry; D010084:Oxidation-Reduction; D012710:Serum Albumin, Bovine / Q000737:chemistry; D019810:Sodium Azide; D013879:Thioredoxins / Q000737:chemistry
D000596:Amino Acids; D001386:Azides; D007202:Indicators and Reagents; D016898:Interferon-alpha; D012710:Serum Albumin, Bovine; D013879:Thioredoxins; D019810:Sodium Azide; D003544:Cysteic Acid; D009113:Muramidase; D003545:Cysteine; D006851:Hydrochloric Acid