Determination of isoelectric point value of 3-mercaptopyruvate sulfurtransferase by isoelectric focusing using ribonuclease A-glutathione mixed disulfides as standards
✍ Scribed by Toshihiko Ubuka; Noriyoshi Masuoka; Shigeko Yoshida; Kazushi Ishino
- Publisher
- Elsevier Science
- Year
- 1987
- Tongue
- English
- Weight
- 626 KB
- Volume
- 167
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
The pI value of rat erythrocyte 3-mercaptopyruvate sulfm-transferase (EC 2.8.1.2) was determined to be 5.9 at 10°C by isoelectric focusing in a horizontal slab polyacrylamide gel containing 2% carrier ampholyte (pH 3-10). In this study, ribonuclease A-glutathione mixed disulfides (RNase-SG's) (T. Ubuka et al. (1986) J. Chromatogr., 363,43 l-437) were used as plstandards. A mixture of RNase-SC was prepared by reducing bovine pancreatic ribonuclease A (RNase) with dithiothreitol and then treating the reduced RNase with oxidized glutathione. The mixture was composed of eight species which contained 1 (RNase-SC,) to 8 (RNase-SGs) mol of glutathione per mole of RNase, and the p1 values of these species were determined under conditions minimizing the effect of carbon dioxide. The newly determined pl values of IWas.+SG, through RNm-sG8
were 8.8, 8.2, 7.7, 7.3. 6.9, 6.4, 5.8, and 5.3, respectively. The average change in pI values of these disulfides was 0.50 pH unit per mole of the bound glutathione per mole of RNase. The RNase-SC mixture was stable in acidic solutions and could be stored at 4°C as well as at -20°C with little change for at least 1 year. Thus, the mixture is shown to be an excellent standard for the determination of pZ values of proteins by isoelectric focusing in the wide range of pI value. 0 1987 Academic PRS, IIK.