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Carboxypeptidase N: colorimetric assay using a new substrate.

D Hendriks¹, S Scharpe, M van Sande

¹Faculty of Medicine, University of Antwerp, Wilrijk, Belgium.

Analytical Biochemistry

|July 1, 1987

Summary

A new colorimetric assay using hippuricase determines carboxypeptidase N activity. This method is sensitive and precise, utilizing novel substrates for accurate enzyme quantification in serum samples.

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Area of Science:

Biochemistry
Enzymology
Clinical Chemistry

Background:

Carboxypeptidase N (EC 3.4.17.3) plays a role in various physiological processes.
Accurate and sensitive methods for determining its activity are crucial for clinical diagnostics and research.

Purpose of the Study:

To develop and validate a novel hippuricase-assisted colorimetric assay for quantifying carboxypeptidase N activity.
To establish reference values for carboxypeptidase N activity in normal human serum.

Main Methods:

A colorimetric assay was developed utilizing new substrates: p-hydroxybenzoylglycine-L-Arg and p-hydroxybenzoylglycine-L-Lys.
Enzyme activity was measured by the absorbance of a quinoneimine dye at 506 nm, formed through a multi-step enzymatic and chemical reaction.
The assay's sensitivity and precision were evaluated using small serum volumes.

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Main Results:

The assay successfully determined carboxypeptidase N activity by measuring the release of p-hydroxybenzoic acid.
Mean serum carboxypeptidase N activities in 50 normal individuals were established: 30.8 (SD 5.9) nmol/mL for the L-Arg substrate and 137.8 (SD 28.1) nmol/mL for the L-Lys substrate.
The assay demonstrated high sensitivity and precision, with reliable results obtained from as little as 20 microliters of serum (RSD% 1.8-4.9).

Conclusions:

A novel, sensitive, and precise colorimetric assay for carboxypeptidase N activity has been established.
The assay provides a reliable method for enzyme quantification in biological samples.
Reference values for normal serum carboxypeptidase N activity were determined, facilitating clinical interpretation.