Direct analysis of the disulfide content of proteins: methods for monitoring the stability and refolding process of cystine-containing proteins.
So far, the cystine (disulfide) content of proteins has been routinely determined by indirect methods, i.e., cystines are first converted to more stable half-cystines prior to analysis. We present a study which demonstrates that the cystine content can be directly and accurately analyzed at low picomole to femtomole levels. The method involves (a) the employment of a vacuum during sample hydrolysis which permits quantitative recovery of cystine, and (b) the dabsyl chloride precolumn derivatization method which yields stable DABS-cystine that is subsequently analyzed by HPLC. Direct analysis of the cystine residue is important in numerous areas of protein research. It allows, by a single analysis, simultaneous determination of the sulfhydryl (cysteine, as S-carboxymethyl derivative) and disulfide (cystine) contents. The technique can be used to follow the refolding process of fully reduced, cystine-containing proteins. Direct analysis of the cystine content also serves to monitor the extent of inactivation of cystine-containing proteins caused by alkaline pH and heat treatments. In this mode of protein inactivation, cystines are selectively destroyed and converted to lanthionine and lysinoalanine. Both the decrease of cystine and the recoveries of lanthionine and lysinoalanine can be simultaneously evaluated by the proposed method. Examples of these applications are presented here.
Chang
J Y
JY
Pharmaceuticals Research Laboratories, Ciba-Geigy Ltd., Basel, Switzerland.
Knecht
R
R
D000409:Alanine / Q000031:analogs & derivatives / Q000032:analysis; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000032:analysis; D003553:Cystine / Q000032:analysis*; D003979:Diazonium Compounds; D004355:Drug Stability; D006801:Humans; D008241:Lysinoalanine / Q000032:analysis; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013425:Sulfanilic Acids; D013440:Sulfides
D000596:Amino Acids; D003979:Diazonium Compounds; D011506:Proteins; D013425:Sulfanilic Acids; D013440:Sulfides; D008241:Lysinoalanine; D003553:Cystine; C001520:lanthionine; D003545:Cysteine; D000409:Alanine; C000469:diazobenzenesulfonic acid