COMPARISON OF ELLMANS 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 tre ated with 5 mM dithiothreitol at different pH values in order to reduc e 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, t he 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. T he low value obtained with Ellman's reagent may be due to the electros tatic repulsion between the carboxylic acid groups in CBH I and those in Ellman's reagent. The fluorimetric assay used for determining SH mo lecules in reduced CBH I, based on their reaction with PM, is describe d.