Purification and characterization of a new arginine carboxypeptidase in human serum.

A carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates is present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its unstability at room temperature and at 37 degrees C, we named it unstable carboxypeptidase (carboxypeptidase U). Carboxypeptidase U was partially purified from fresh human serum by chromatography on DEAE-cellulose and Mono-Q sepharose and was found to be a 435 kDa protein. We compared this enzyme with carboxypeptidase N, purified from human serum by a two-step affinity chromatography on arginine-Sepharose 4B, followed by ion-exchange chromatography on Mono-Q sepharose. Carboxypeptidase U cleaves hippuryl-L-arginine and hippuryl-L-lysine, but at a different relative rate than carboxypeptidase N, and has no esterase activity on hippuryl-L-argininic acid. Its activity was inhibited by o-phenanthroline, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, CoCl2, 2-mercaptoethanol, dithiothreitol and 4-chloromercuribenzoic acid. These characteristics differentiate carboxypeptidase U from carboxypeptidase N and other known carboxypeptidases.

Characterisation of a carboxypeptidase in human serum distinct from carboxypeptidase N.

Arginine carboxypeptidase activity in human serum, measured with the hippuryl-L-arginine substrate, is about three times higher than in human plasma. This difference is much smaller when hippuryl-L-lysine is used as the substrate. When fresh serum is incubated at 30 degrees C, the arginine and lysine carboxypeptidase activity decreases until a stable activity, close to the plasma activity, is reached. This stable carboxypeptidase activity is attributed to carboxypeptidase N. The unstable carboxypeptidase differs from carboxypeptidase N in pH-optimum, esterase activity, substrate specificity, Co2+-activation and dithiotreitol activation. Blood cells are not responsible for the release of this enzyme during coagulation. No activator of carboxypeptidase N was detectable in human serum. Ion-exchange chromatography on DEAE-cellulose confirms the presence of two different molecular forms of arginine carboxypeptidase activity.

Carboxypeptidase N: colorimetric assay using a new substrate.

A method has been developed for determining carboxypeptidase N (EC 3.4.17.3) activity by a hippuricase (EC 3.5.1.14)-assisted colorimetric assay. The method is based on the absorbance at 506 nm of a quinoneimine dye, produced by the action of carboxypeptidase N on the new substrates p-hydroxybenzoylglycine-L-Arg and p-hydroxybenzoylglycine-L-Lys. The enzyme acts on the substrates producing p-hydroxybenzoylglycine and L-Arg or L-Lys. The former is then hydrolyzed by hippuricase into p-hydroxybenzoic acid and Gly. Subsequently, oxidative coupling of p-hydroxybenzoic acid with 4-aminoantipyrine by sodium periodate forms a quinoneimine dye. The mean value of carboxypeptidase N activities in sera of 50 normal individuals was 30.8 (SD 5.9) nmol of p-hydroxybenzoylglycine released per milliliter of serum for the p-hydroxybenzoylglycine-L-Arg substrate and 137.8 (SD 28.1) for the p-hydroxybenzoylglycine-L-Lys substrate. The sensitivity of the assay is such that as little as 20 microliters of serum provides reliable and precise results (RSD% ranging from 1.8 to 4.9).