ISSN:
0730-2312
Keywords:
semisynthesis
;
V8-protease
;
gelation
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
Notes:
Enzyme-catalyzed reformation of peptide bonds in the noncovalent fragment systems of proteins has been emerging as a convenient procedure for the semisynthesis of covalent analogs of the respective proteins. Limited proteolysis of the α-chain of hemoglobin S with Staphylococcus aureus V8-protease converts the chain into a fragment-complementing system by hydrolyzing the peptide bond Glu(30)-Arg(31) of the chain. Therefore, it is conceivable that semisynthesis of covalent analogs of α-chain could be achieved if conditions for the V8-protease catalyzed formation of peptide bonds could be established. The synthetic potential of V8-protease has been now investigated by incubating V8-protease-derived fragments of α-chain, namely α1-30 and α31-47 with the enzyme at pH 6.0 in the presence of n-propanol as the organic cosolvent. RP high performance liquid chromatography analysis showed that a new chromatographically distinct component is generated on incubation, and this has been identified as α1-47 by amino acid analysis, redigestion with V8-protease (in the absence of n-propanol), and tryptic peptide mapping. Optimal conditions for the synthesis of α1-47 is at pH 6.0, 4°C, and 24 hr of incubation with 25% n-propanol as organic cosolvent. This stereospecific condensation of the fragments proceeded to a high level of about 50% in 24 hr. Further incubation up to 72 hr did not increase the yield of α1-47, suggesting that an equilibration of synthesis and hydrolysis reactions has been attained. The demonstration of the synthetic potential of V8-protease and the fact that α1-30 and α31-141 interact to form a native-like complex, opens up an approach for the semisynthesis of covalent analogs of α-chain of hemoglobin S.
Additional Material:
5 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jcb.240300110
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