Establishment, validation and application of a spectrophotometric method for the accurate determination of carbonic anhydrase activity

Abstract

Carbonic anhydrase (CA) has been proven to be a rather important enzyme in both medical research and industrial development, leading to great application value in accurately quantifying CA enzymatic activity. This study focuses on developing a novel spectrophotometric method for measuring the CA enzyme activity with enhanced precision, reliability, and an ultra-low limit of detection (LOD) through the transition of changing pH values into a stable UV absorbance signal by the colorimetric probe of bromothymol blue (BTB), thereby addressing the limitations of existing assay techniques. Through the establishment of a colorimetric reaction system, optimization of the CA assay reaction system, and data analysis by both least squares and two-point methods, CA conventional and trace standard curves in the range of 2–2000 U mL⁻¹ were obtained under the following conditions: a wavelength of 616 nm, a BTB concentration of 0.015 g L⁻¹, a temperature of 20 °C, and a Tris–HCl concentration of 0.028 mol L⁻¹. In addition, the results from the methodology validation revealed an LOD value of 2 U mL⁻¹, excellent repeatability, precision and recoveries within the investigated range. Finally, the application exploration, which included CA activity testing in human blood samples and pharmacokinetic research in rats, exhibited precise and stable results for clinical testing and enhanced linearity fitting of the pharmacokinetic curve, with increased resistance to interferences from hemoglobin (Hb) and other plasma proteins. The established spectrophotometric method provides a novel reliable analytical approach for CA activity measurement, pharmaceutical research and even future application prospects in environmental protection and other industrial fields.