Synthetic potential of Staphylococcus aureus V8-protease: an approach toward semisynthesis of covalent analogs of alpha-chain of hemoglobin S.
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 alpha-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 alpha-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 alpha-chain, namely alpha 1-30 and alpha 31-47 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 alpha 1-47 by amino acid analysis, redigestion with V8-protease (in the absence of n-propanol), and tryptic peptide mapping. Optimal conditions for the synthesis of alpha 1-47 is at pH 6.0, 4 degrees 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 alpha 1-47, 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 alpha 1-30 and alpha 31-141 interact to form a native-like complex, opens up an approach for the semisynthesis of covalent analogs of alpha-chain of hemoglobin S.
D000596:Amino Acids / Q000032:analysis; D001120:Arginine / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D005990:Glycerol / Q000494:pharmacology; D006451:Hemoglobin, Sickle / Q000096:biosynthesis*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D011433:Propranolol / Q000494:pharmacology; D012697:Serine Endopeptidases; D012997:Solvents
D000596:Amino Acids; D006451:Hemoglobin, Sickle; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012997:Solvents; D005973:Glutamine; D001120:Arginine; D011433:Propranolol; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D005990:Glycerol