Proteosynthetic activity of immobilized Staphylococcus aureus V8 protease: application in the semisynthesis of molecular variants of alpha-globin.
The proteosynthetic activity of Staphylococcus aureus V8 protease (endoproteinase Glu-C) immobilized onto cross-linked agarose beads by reductive alkylation procedure has been investigated. The overall substrate specificity of the enzyme, as judged by peptide mapping of performic acid oxidized RNase A, as well as the high propensity of the protease to slice selectively the alpha-chain of hemoglobin (Hb) A at the Glu(30)-Arg(31) peptide bond at pH 4.0 and 37 degrees C was essentially unperturbed by the immobilization process. This high susceptibility of Glu(30) of the alpha-chain for proteolysis appears to be a consequence of the conformational aspects of the polypeptide in this region. The proteolysis of two mutant forms of alpha-chain, namely, those of Hb I (K16E) and Hb Sealy (D47H) by immobilized V8 protease at the Glu(30)-Arg(31) peptide bond proceeds with the same selectivity. The immobilized protease also retained the proteosynthetic activity, i.e., the ability to ligate the unprotected alpha-globin fragments at the Glu(30)-Arg(31) peptide bond in the presence of 30% 1-propanol. The use of the insoluble enzyme simplifies the procedures for the construction of new semisynthetic, molecular variants of alpha-globin. The general applicability of the immobilized enzyme for protein semisynthesis has been demonstrated by the construction of a doubly mutated alpha-globin. The complementary fragments from two natural mutant forms of alpha-globin, viz., alpha 1-30 (K16E) from Hb I and alpha 31-141 (D47H) from Hb Sealy, are readily ligated to form the double mutant alpha 1-141 (K16E;D47H).
Sahni
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G
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461.
Mallia
A K
AK
Acharya
A S
AS
D002851:Chromatography, High Pressure Liquid; D004800:Enzymes, Immobilized / Q000378:metabolism*; D014644:Genetic Variation; D005914:Globins / Q000096:biosynthesis* / Q000235:genetics; D006418:Heme / Q000737:chemistry; D006801:Humans; D010446:Peptide Fragments; D012697:Serine Endopeptidases / Q000378:metabolism*; D013379:Substrate Specificity
D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D006418:Heme; D005914:Globins; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase