A novel method has been proposed to develop a simple, rapid, sensitive and affordable chromogenic attempt for the quantification of catalase (CAT) activity in blood samples. The method is based on the oxidation of pyrocatechol (PC) to give quinone form which by oxidative coupling with aminyl radical of 4-aminoantipyrine (4-AAP) resulting from H2O2/CAT to produce a pink colored quinone-imine product with λmax=530 nm in a 100 mmol/L of tris buffer of pH 9.8 at room temperature (30 ºC). The linearity of CAT assay was between 0.316 and 10 U/mL. The accuracy ranges for CAT having concentrations of 1.25, 5 and 7.5 μmol/L were 89-105.52, 90–107%, and 91–104.58 % respectively. Within-run and between-run precision studies showed CV’s of 1.98 – 3.02 % (n=7) and 2.97 – 4.40 % (n=7), respectively. The detection and quantification limits of CAT were 0.12 and 0.225 μmol/L, respectively. The Michaelis-Menten constant and maximum velocity of the reaction was = 1.052 mM and = 0.168 μmol/min, respectively. The present method provides a convenient means for investigating the usefulness of CAT measurements in biological sample assessing the potential for free radical-induced pathology.
Keywords: Somatic oxidant defense; catalase assay; serum catalase; erythrocytes; oxidative coupling.
Dr. Honnur Krishna received his PhD degree in enzyme assays from the University of Mysore, Mysore under the guidance of Prof. P. Nagaraja, Professor of Analytical Chemistry, Department of Studies in Chemistry, University of Mysore. He currently works as an Assistant Professor of Chemistry, SDVS Sangh’s, S. S. Arts College and T. P. Science Institute, at Sankeshwar affiliated to Rani Channamma University, Belagavi district, Karnataka state, from India. His research focuses on analytical method development and its validation for the clinically important biomarkers for better understanding of the biochemical pathways and also for different environmental analytes. He is also interested in DFT studies for prediction of chemical reactions insight through computational chemical approach; aptamers based sensors, nano technological applications and its sensing applications.