A More Sensitive Hummel Assay for Chymotrypsin

Rafiq, Amjad; Bailey, Graham

doi:10.1006/abio.1997.2570

Cited by 4 publications

(1 citation statement)

References 8 publications

(16 reference statements)

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“…The assays were carried out using the method described by Rafiq & Bailey [35]. The enzyme was freshly prepared and active chymotrypsin was assessed by active site titration using p-nitrophenyl acetate [36].…”

Section: Kinetic Studiesmentioning

confidence: 99%

Detection of native peptides as potent inhibitors of enzymes

et al. 2004

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Chymotrypsin is a prominent member of the family of serine proteases. The present studies demonstrate the presence of a native fragment containing 14 residues from Ile16 to Trp29 in a-chymotrypsin that binds to chymotrypsin at the active site with an exceptionally high affinity of 2.7 ± 0.3 · 10 )11 m and thus works as a highly potent competitive inhibitor. The commercially available a-chymotrypsin was processed through a three phase partitioning system (TPP). The treated enzyme showed considerably enhanced activity. The 14 residue fragment was produced by autodigestion of a TPP-treated a-chymotrypsin during a long crystallization process that lasted more than four months. The treated enzyme was purified and kept for crystallization using vapour the diffusion method at 295 K. Twenty milligrams of lyophilized protein were dissolved in 1 mL of 25 mm sodium acetate buffer, pH 4.8. It was equilibrated against the same buffer containing 1.2 m ammonium sulfate. The rectangular crystals of small dimensions of 0.24 · 0.15 · 0.10 mm 3 were obtained. The X-ray intensity data were collected at 2.2 Å resolution and the structure was refined to an R-factor of 0.192. An extra electron density was observed at the binding site of a-chymotrypsin, which was readily interpreted as a 14 residue fragment of a-chymotrypsin corresponding to Ile-Val-Asn-Gly-GluGlu-Ala-Val-Pro-Gly-Ser-Trp-Pro-Trp(16-29). The electron density for the eight residues of the C-terminus, i.e. Ala22-Trp29, which were completely buried in the binding cleft of the enzyme, was of excellent quality and all the side chains of these eight residues were clearly modeled into it. However, the remaining six residues from the N-terminus, Ile16-Glu21 were poorly defined although the backbone density was good. There was a continuous electron density at 3.0 r between the active site Ser195 Oc and the carbonyl carbon atom of Trp29 of the fragment. The final refined coordinates showed a distance of 1.35 Å between Ser195 Oc and Trp29 C Abbreviations OW, water molecule;

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Section: Kinetic Studiesmentioning

confidence: 99%

Detection of native peptides as potent inhibitors of enzymes

et al. 2004

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Al-Ajlan

Bailey

1999

Molecular and Cellular Biochemistry

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A carboxypeptidase B-like enzyme was purified 116-fold with a recovery of activity of 29% from a crude extract of camel pancreas by a four-step procedure consisting of two anion exchange chromatographies in succession, gel filtration and hydrophobic interaction chromatography. The enzyme was homogeneous on SDS and non-denaturing gel electrophoresis and on gel isoelectric focusing. Its molecular mass was found to be 31.5 kDa and its isoelectric point was estimated as 6.1. It was active towards a number of substrates that are cleaved by carboxypeptidases B from other species and was also susceptible to inhibition by inhibitors of such enzymes. The camel enzyme showed a pH optimum of 8.0 and it was seen to be a relatively potent kininase in vitro. The enzyme purified in this study was very similar to carboxypeptidases B isolated from other species in size, charge, substrate specificity and susceptibility to inhibition and thus it can be identified as camel carboxypeptidase B.

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Al-Ajlan

2000

Molecular and Cellular Biochemistry

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This study reports on the purification and characterization of a cationic enzyme with chymotryptic activity from camel pancreas. The enzyme was purified 52-fold in a 48% yield by a three-step chromatographic procedure consisting of anion-exchange, cation-exchange and affinity chromatographies. The purified enzyme was homogeneous on gel isoelectric focusing and on SDS gel electrophoresis. Its isoelectric point was estimated to be 7.3 and its molecular mass was found to be 23,600 Da. The enzyme was identified as a cationic chymotrypsin according to its physiochemical properties, substrate specificity and susceptibility to inhibition. It was active towards esters of aromatic amino acids but much less active towards a leucine ester. In all cases, the k(cat) values of the camel enzyme were less than the corresponding values of bovine chymotrypsin A. It also showed a lower level of kininase activity. Camel chymotrypsin was more susceptible than its bovine equivalent to inhibition by soybean trypsin inhibitor and aprotinin. It showed the same pH optimum as bovine chymotrypsin A for its esterolytic activity, but was more dependent on CaCl2 for long-term stability.

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A More Sensitive Hummel Assay for Chymotrypsin

Cited by 4 publications

References 8 publications

Detection of native peptides as potent inhibitors of enzymes

Detection of native peptides as potent inhibitors of enzymes

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