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.
Woodward
J
J
Chemical Technology Division, Oak Ridge National Laboratory, TN 37831-6194.
Tate
J
J
Herrmann
P C
PC
Evans
B R
BR
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