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Refolding of serine proteinases
Author(s) -
Light Albert,
Duda Chester T.,
Odorzynski Thomas W.,
Moore William G. I.
Publication year - 1986
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240310104
Subject(s) - chemistry , chymotrypsinogen , cysteine , serine , cystine , protein disulfide isomerase , amino acid , glutathione , threonine , stereochemistry , cleavage (geology) , disulfide bond , biochemistry , trypsin , chymotrypsin , enzyme , geotechnical engineering , fracture (geology) , engineering
Bovine trypsinogen and chymotrypsinogen were successfully refolded as the mixed disulfide of glutathione using cysteine as the disulfide interchange catalyst. The native structures were regenerated with yields of 40%–50% at pH 8.6 and 4 °C, and the half‐time for the refolding was approximately 60–75 min. We then refolded threonine‐neochymotrypsinogen, which is a two‐chain structure held together by disulfide bonds and produced on cleavage of Tyr 146‐Thr 147 in native chymotrypsinogen [Duda CT, Light A, J Biol Chem 257 9866–9871, 1982]. Neochymotrypsinogen was denatured and fully reduced, and the thiols were converted to the mixed disulfide of glutathione. The two polypeptide fragments, representing the amino‐ and carboxyl‐terminal domains, were separated on Sephadex G‐75. Mixtures of the polypeptide fragments varying in the ratio of their concentration from 1 : 5 to 5 : 1 were refolded with yields of 21–28%. The lack of dependence on the concentration of either fragmènt and the relatively high yields suggest independent folding of the amino‐ and carboxyl‐terminal domains. When the globular structures of the domains formed, they then interacted with one another and produced the native intermolecular disulfide bridge and the proper geometry of the active site.