Peptide mapping of bovine pancreatic ribonuclease A by reverse-phase high-performance liquid chromatography: II. A two-dimensional technique for determination of disulfide pairings using a continuous-flow disulfide-detection system
✍ Scribed by Theodore W. Thannhauser; Charles A. McWherter; Harold A. Scheraga
- Publisher
- Elsevier Science
- Year
- 1985
- Tongue
- English
- Weight
- 754 KB
- Volume
- 149
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A procedure, developed for the cleavage and reversible blocking of disulfide bonds of proteins by S-sulfonation in preparation for peptide mapping, was applied to ribonuclease A. The complete peptide maps of sulforibonuclease A using limited Staphylococcus aureus protease digestion, tryptic digestion, and tryptic followed by chymotryptic digestion are presented. A description is given of an adaptation of the sulfonation procedure which forms the basis of a sensitive (5pmol detection limit) and quantitative (+5%) disuhidedetection system for the continuous monitoring of HPLC column effluents for disulfide-containing compounds. The sulfonation procedure, peptide maps, and disulfidedetection system are the key ingredients in a two-dimensional reverse-phase HPLC technique for the determination of disulfide pairings. The applicability of this technique is demonstrated by determining the known disuhide pairings of ribonuclease A. It is also shown that there is no disuhide interchange under the digestion conditions used. This technique is suitable for determining the distributions ofdisulfide pairings in the intermediates present in the oxidative folding of disulfide-containing proteins. o 1985 Academic ks, ~nc.