Development of on-line HPLC-carbonic anhydrase enzyme determination method and its application in protein mixture

The purification and separation of active proteins from complex samples is a very laborious and time-consuming process. On-line HPLC applications becoming icreasingly widespread offer significant advantages for the detection of natural bioactive compounds. In this study, an on-line HPLC-CA enzyme determination method was developed, which enables the on-line determination of proteins with esterase activity in protein mixtures and the simultaneous determination of carbonic anhydrase enzyme. The developed method was optimized over the parameters of flow rates, buffer solution selection, concentration and pH, reaction temperature, tubing length and substrate concentration. The optimum working conditions were obtained by pumping 0.15 M phosphate buffer with a pH of 8.2 to the system at a flow rate of 0.5 mL/min, and injecting 5 mM p-nitrophenyl acetate with a flow rate of 0.3 mL/min, to achieve protein separation detected at 210 nm and esterase activity at 400 nm at 25 °C with 3 m tubing length. The optimized on-line HPLC-CA enzyme determination method has been validated on linearity, linear range, LOD, LOQ, precision, and robustness. It was determined that the method was linear up to 5 mg/mL BCA levels, the LOD was 0.03 mg/mL, and the LOQ was 0.07 mg/mL. The relative standard deviation below 1% revealed the precision of the method, the relative error of less than 5% as a result of small changes in the flow rate, reaction temperature, pH and concentration of the mobile phase demonstrated the robustness of the method. Studies have shown that the developed method can be easily applied to all solutions with known and unknown protein content.