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).
D000328:Adult; D000581:Amidohydrolases / Q000378:metabolism; D002268:Carboxypeptidases / Q000097:blood*; D003124:Colorimetry; D004151:Dipeptides / Q000378:metabolism*; D004492:Edetic Acid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D001121:Lysine Carboxypeptidase / Q000097:blood*; D008875:Middle Aged; D011786:Quality Control; D012016:Reference Values
D004151:Dipeptides; C053950:4-hydroxybenzoylglycylarginine; C053951:4-hydroxybenzoylglycyllysine; D004492:Edetic Acid; D002268:Carboxypeptidases; D001121:Lysine Carboxypeptidase; D000581:Amidohydrolases; C019672:aminoacylase I