A new fluorogenic substrate method for the estimation of kallikrein in urine

Area of Science:

• Biochemistry
• Urology
• Enzymology

Background:

• Kallikrein is a key enzyme in the kinin-kallikrein system.
• Accurate measurement of urinary kallikrein is crucial for diagnosing and monitoring renal diseases.
• Existing measurement methods may lack specificity, sensitivity, or simplicity.

Purpose of the Study:

• To develop and validate a novel fluorometric assay for quantifying human urinary kallikrein.
• To assess the assay's utility in clinical samples from healthy individuals and patients with glomerulonephritis and Bartter's syndrome.
• To confirm the assay's specificity by comparing its results with radioimmunoassay and assessing enzyme elution patterns.

Main Methods:

• Fluorogenic peptide substrate, proly-phenyl-alanyl-arginine-4-methylcoumaryl-7-amide (Pro-Phe-Arg-MCA), was used for assaying kallikrein activity.
• Urine samples were incubated with Pro-Phe-Arg-MCA, and reactions were terminated using Trasylol or acetic acid.
• Arginine-Sepharose 4B chromatography was employed to analyze enzyme elution profiles.

Main Results:

• The fluorometric assay demonstrated high specificity, sensitivity, and simplicity for measuring urinary kallikrein.
• Kallikrein activity levels measured by this method were comparable to those obtained via radioimmunoassay.
• Both Pro-Phe-Arg-MCA hydrolyzing activity and kinin-releasing activity co-eluted in the same fraction during chromatography, confirming enzyme identity.

Conclusions:

• The developed fluorometric assay is a reliable and practical tool for estimating kallikrein levels in human urine.
• This method offers a valuable alternative for clinical assessment of urinary kallikrein.
• The assay's performance supports its use in research and diagnostic settings for kidney-related conditions.