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Protein Folding/refolding Analysis by Mass Spectrometry. Scrambling of Disulphide Bridges in Insulin

Overview
Journal Biochem J
Specialty Biochemistry
Date 1990 Jun 15
PMID 2194451
Citations 2
Authors
H R Morris
P Pucci
M Panico
G Marino
Affiliations
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Abstract

In this paper we present a protocol that allows a dynamic analysis of disulphide-bridge formation, based on freezing the intermediates by acid/acetone precipitation, followed by digestion with pepsin and direct fast-atom-bombardment mass-spectrometric analysis. A rapid definition of the exact nature of disulphide bridges formed can be obtained via a definitive assignment of disulphide-linked peptides according to their unique mass values. With the use of an appropriate thiol concentration, scrambling of the native disulphide bonds in bovine insulin occurs, and the process is catalysed by protein disulphide-isomerase (EC 5.3.4.1). The disruption of native and the formation of new disulphide bonds can be monitored as described above, and interestingly B-chain dimers containing Cys-B7-Cys-B7 and Cys-B7-Cys-B19 bonds are detected.

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References
1.
Creighton T . Experimental studies of protein folding and unfolding. Prog Biophys Mol Biol. 1978; 33(3):231-97. DOI: 10.1016/0079-6107(79)90030-0. View
2.
Creighton T . Intermediates in the refolding of reduced ribonuclease A. J Mol Biol. 1979; 129(3):411-31. DOI: 10.1016/0022-2836(79)90504-7. View
3.
Goldenberg D, Creighton T . Folding pathway of a circular form of bovine pancreatic trypsin inhibitor. J Mol Biol. 1984; 179(3):527-45. DOI: 10.1016/0022-2836(84)90078-0. View
4.
Givol D, DELORENZO F, GOLDBERGER R, ANFINSEN C . DISULFIDE INTERCHANGE AND THE THREE-DIMENSIONAL STRUCTURE OF PROTEINS. Proc Natl Acad Sci U S A. 1965; 53:676-84. PMC: 336997. DOI: 10.1073/pnas.53.3.676. View
5.
Marks C, Naderi H, Kosen P, Kuntz I, Anderson S . Mutants of bovine pancreatic trypsin inhibitor lacking cysteines 14 and 38 can fold properly. Science. 1987; 235(4794):1370-3. DOI: 10.1126/science.2435002. View