Determination of serum and urinary urate with reusable uricase strip

Abstract

<span style="color:black;mso-bidi-language:HI">Commercial uricase has been immobilized covalently onto alkylamine glass beads affixed on a plastic strip with a conjugation yield of 87mg/g and 11.5% retention of initial activity of free enzyme. Maximum activity of immobilized enzyme was attained at pH 8<span style="color:#292929;mso-bidi-language:HI">.<span style="color:black; mso-bidi-language:HI">8, when incubated at 40°C for 20 min. <i>K</i>_m<i> </i>for uric acid and <i>V</i>_{<span style="mso-bidi-font-family: Arial;color:black;mso-bidi-language:HI">max</span>}<span style="mso-bidi-font-family: Arial;color:black;mso-bidi-language:HI"> <span style="color:black; mso-bidi-language:HI">of immobilized enzyme were 0<span style="color:#292929;mso-bidi-language:HI">.<span style="color:black; mso-bidi-language:HI">15mM and 0.68µmoles H₂O₂/min respectively. A method for uric acid determination in serum and urine was developed using the strip bound enzyme. The method is based on the measurement of H₂O₂<span style="mso-bidi-font-family:Arial; color:black;mso-bidi-language:HI"> <span style="color:black;mso-bidi-language: HI">generated from uric acid by using a colour reaction consisting of 4-aminophenazone, <i>p</i>-hydroxybenzoic acid and horseradish peroxidase as chromogenic system. The minimum detection limit of the method was 1.68mg/dl and recovery of added uric acid in urine was 90%. The coefficient of variation (CV) were <5 and <2% for within batch and between batch respectively. The immobilized enzyme lost 50% of its initial activity after its regular uses for over 3 weeks, when stored in 0<span style="color:#424242;mso-bidi-language: HI">.<span style="color:black;mso-bidi-language:HI">05M glycine NaOH buffer, pH 8.8 at 4°C.

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