Evaluation of methods for the quantitation of cysteines in proteins.
Anal Biochem
; 265(1): 8-14, 1998 Dec 01.
Article
em En
| MEDLINE
| ID: mdl-9866701
ABSTRACT
Several methods for the quantitation of cysteines in proteins have been evaluated and compared. Titration of protein sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) under carefully controlled conditions has extended the detection limits of this method with high accuracy and reproducibility. Results are reported for a variety of enzymes containing a range of total cysteines with different degrees of solvent accessibility and reactivity. A papain amplification assay has also been examined, in which reactivation of the disulfide-blocked active site cysteine of papain can be achieved by a coupled reaction with protein sulfhydryl groups. Detection of sulfhydryls by this amplification assay can be extended, by increasing the enzyme assay times, to achieve over a 40-fold increase in sensitivity over the improved DTNB titration method. Alternatively, titration of enzyme cysteinyl residues with either bromobimane or a maleimide derivative of naphthopyranones has the advantage that a fluorescent product results upon modification of the sulfhydryl group. Reaction of bromobimane with several different enzymes results in nonspecific background fluorescence that limits the detection range of this method unless the products are separated. In contrast, low background fluorescence and high quantum yields with maleimide naphthopyranoses has allowed detection of protein cysteinyl residues with very high sensitivities.
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Eixos temáticos:
Pesquisa_clinica
Base de dados:
MEDLINE
Assunto principal:
Proteínas
/
Cisteína
Tipo de estudo:
Diagnostic_studies
/
Evaluation_studies
Idioma:
En
Ano de publicação:
1998
Tipo de documento:
Article