Comparison of Ellman's reagent with N-(1-pyrenyl)maleimide for the determination of free sulfhydryl groups in reduced cellobiohydrolase I from Trichoderma reesei.
The enzyme cellobiohydrolase I (CBH I) from Trichoderma reesei was treated with 5 mM dithiothreitol at different pH values in order to reduce some or all of its 12 disulfide bridges. A discrepancy was found in the number of free sulfhydryl (SH) groups generated upon the reduction of CBH I when they were measured using N-(1-pyrenyl)maleimide (PM) or Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid). For example, the number of SH mol generated/mol CBH I at pH 8.5 was determined to be 16 and < 1 when measured using PM or Ellman's reagent, respectively. The low value obtained with Ellman's reagent may be due to the electrostatic repulsion between the carboxylic acid groups in CBH I and those in Ellman's reagent. The fluorimetric assay used for determining SH molecules in reduced CBH I, based on their reaction with PM, is described.
Chemical Technology Division, Oak Ridge National Laboratory, TN 37831-6194.
J
J
Woodward
J
J
Tate
P C
PC
Herrmann
B R
BR
Evans
D043366:Cellulose 1,4-beta-Cellobiosidase; D003545:Cysteine / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004347:Drug Interactions; D005456:Fluorescent Dyes; D006026:Glycoside Hydrolases / Q000032:analysis* / Q000378:metabolism; D008301:Maleimides; D008623:Mercaptoethanol / Q000494:pharmacology; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000378:metabolism; D014242:Trichoderma / Q000201:enzymology*
D005456:Fluorescent Dyes; D008301:Maleimides; D013438:Sulfhydryl Compounds; D008623:Mercaptoethanol; D004228:Dithionitrobenzoic Acid; C003944:N-(3-pyrene)maleimide; D006026:Glycoside Hydrolases; D043366:Cellulose 1,4-beta-Cellobiosidase; D003545:Cysteine; D004229:Dithiothreitol