Synthetic potential of Staphylococcus aureus V8‐protease: An approach toward semisynthesis of covalent analogs of α‐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 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 α 1–30 and α 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 α 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 α 1–47 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 α 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 α 1–30 and α 31–141 interact to form a native‐like complex, opens up an approach for the semisynthesis of covalent analogs of α‐chain of hemoglobin S.