Abstract
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 CHB 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.
| Original language | English |
|---|---|
| Pages (from-to) | 121-129 |
| Number of pages | 9 |
| Journal | Journal of Biochemical and Biophysical Methods |
| Volume | 26 |
| Issue number | 2-3 |
| DOIs | |
| State | Published - May 1993 |
| Externally published | Yes |
Funding
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 Correspondence address: J. Woodward, Chemical Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6194, USA. 1 Oak Ridge Science and Engineering Research Semester (ORSERS) Student from North Dakota State University, Fargo, North Dakota. 20RSERS student from Andrews University, Berrien Springs, Michigan. 3 Managed by Martin Marietta Energy Systems, Inc., for the U.S. Department of Energy under contract DE-AC05-84OR21400. This article has been authored by a contractor of the U.S. Government under contract No. DE-AC05-84OR21400. Accordingly, the US Government retains a nonexclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for US Government purposes.
Keywords
- Cellobiohydrolase I
- Ellman's reagent
- N-(1-Pyrenyl)maleimide
- Sulfhydryl determination