Determination of the cysteine and cystine content of proteins by amino acid analysis: application to the characterization of disulfide-coupled folding intermediates.
A method has been developed for the simultaneous detection of cysteine and cystine in proteins by amino acid analysis. In this method, the sulfhydryl groups of the cysteine residues are first blocked with 2-aminoethyl methanethiosulfonate (AEMTS). This reagent converts all free sulfhydryl groups to mixed disulfides with 2-aminoethanethiol (AET). The isolated blocked protein is subjected to oxidation with performic acid prior to hydrolysis and amino acid analysis. This procedure quantitatively converts the 2-aminoethanethiol blocking groups into taurine, and all cysteine residues (including those involved in disulfide bonds) into cysteic acid. Both of these derivatives are stable and can be recovered quantitatively by amino acid analysis. The speed and specificity with which AEMTS reacts with thiols make this method particularly effective for the characterization of disulfide-coupled folding intermediates.
Thannhauser
T W
TW
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Sherwood
R W
RW
Scheraga
H A
HA
D003543:Cysteamine / Q000737:chemistry; D003544:Cysteic Acid / Q000737:chemistry; D003545:Cysteine / Q000032:analysis* / Q000737:chemistry; D003553:Cystine / Q000032:analysis* / Q000737:chemistry; D004220:Disulfides / Q000032:analysis; D006629:Hirudins / Q000032:analysis / Q000737:chemistry; D010084:Oxidation-Reduction; D017510:Protein Folding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013654:Taurine / Q000737:chemistry
D004220:Disulfides; D006629:Hirudins; D011506:Proteins; D013654:Taurine; D003553:Cystine; D003543:Cysteamine; D003544:Cysteic Acid; D003545:Cysteine