Abstract
The relative rates of hydrolysis of the peptide bonds of the oxidized B-chain of insulin by Endothia parasitica protease have been determined at pH 3.60 and at a substrate concentration of 1.07 × 10 −3 m and an enzyme concentration of 1.95 × 10 −6 m. The Phe 24Phe 25, bond is hydrolyzed at a maximum rate followed by hydrolysis of the Tyr 16Leu 17 and Gln 4His 5 bonds. The Leu 11Val 12 and Asn 3Gln 4 bonds are hydrolyzed at slower rates. The LeunVal 12 bond appears to be considerably more resistant to hydrolysis in the peptide 5–16 than in the intact oxidized B-chain. The Leu 15-Tyr 16 bond is very slowly hydrolyzed in peptide 5–16 but there is no evidence for hydrolysis of this bond in the intact oxidized B-chain. Phe 25 is slowly hydrolyzed from the peptide 25–30 and the bond involving Gly 20Glu 21 is slowly hydrolyzed in peptide 12–24 and/or peptide 17–24. Thus the enzyme has specificity for the hydrophobic regions of the oxidized B-chain. The specificity of Endothia parasitica protease on the oxidized B-chain of insulin is similar to that of pepsin in that bonds involving Gln 4His 5, Leu 11Val 12, Leu 15Tyr 16, Tyr 16Leu 17, Phe 24Phe 25 and Phe 25Tyr 26 are hydrolyzed by both enzymes. Endothia parasitica protease hydrolyzes bonds involving Asn 3Gln 4 and Gly 20Glu 20, not attacked by pepsin, but fails to hydrolyze bonds involving Phe 1Val 2, Glu 13Ala 14, Ala 14Leu 15 and Gly 23Phe 24, hydrolyzed by pepsin. Thus, Endothia parasitica protease is more specific in its action on the oxidized B-chain of insulin than is pepsin.
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