Synthesis and hydrolysis by arginyl-hydrolases of p-nitroanilide chromogenic substrates containing polyethylene glycol and D-gluconyl moieties.
D-Gluconic acid and alpha-carboxymethyl-polyethylene-glycol-omega-methyl ether (PEG) (mol wt 550) were covalently bound at N alpha-amino group of H-Phe-Arg-pNa to study the effect on hydrolysis by arginyl-hydrolases of chromogenic substrates containing high hydrophilic and amphiphilic groups. For comparison, epsilon-aminocaproyl-, sarcosyl- and succinyl-Phe-Arg-pNa were also synthesized. The obtained compounds were assayed as substrates for porcine pancreatic kallikrein, horse urinary kallikrein, tonin and beta-trypsin. Both PEG- and gluconyl-Phe-Arg-pNa had kcat values of hydrolysis 2-4 times higher than the N-acetyl derivative for all the studied enzymes. epsilon-NH2caproyl-Phe-Arg-pNa resulted in the best chromogenic substrate described for the two tissue kallikreins. The PEG-derivative and D-gluconyl groups were also introduced in the N alpha-amino group of H-Arg-pNa and assayed as beta-trypsin substrates. In comparison with benzoyl-Arg-pNa, the D-gluconyl group had no effect on Km but reduced the kcat value more than 15 times; however, PEG-Arg-pNa was hydrolyzed with similar Km but with kcat 5 times higher. The presence of D-gluconyl and PEG groups in the chromogenic substrate molecules increased their water solubility significantly.
Juliano
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MA
Escola Paulista de Medicina, Brazil.
Juliano
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L
Biondi
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Rocchi
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R
D000595:Amino Acid Sequence; D000813:Anilides / Q000378:metabolism; D000818:Animals; D002863:Chromogenic Compounds / Q000737:chemistry / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D011092:Polyethylene Glycols / Q000378:metabolism; D013379:Substrate Specificity
D000813:Anilides; D002863:Chromogenic Compounds; D009842:Oligopeptides; D011092:Polyethylene Glycols; D010450:Endopeptidases