Abstract
Serum uric acid (SUA) is an important biomarker for prognosis and management of gout and other diseases. The development of a low-cost, simple, rapid and reliable assay for SUA detection is of great importance. In the present study, to save the cost of enzyme production and to shorten the reaction time for uric acid quantification, bifunctional proteins with uricase and peroxidase activities were engineered. In-frame fusion of Candida utilis uricase (CUOX) and Vitreoscilla hemoglobin (VHb) resulted in two versions of the bifunctional protein, CUOX-VHb (CV) and VHb-CUOX (VC). To our knowledge, this is the first report to describe the production of proteins with uricase and peroxidase activities. Based on the measurement of the initial rates of the coupled reaction (between uricase and peroxidase), CV was proven to be the most efficient enzyme followed by VC and native enzymes (CUOX+VHb), respectively. CV was further applied for the development of an assay for colorimetric detection of SUA, which was based on VHb-catalyzed oxidation of Amplex Red in the presence of hydrogen peroxide (H2O2). Under the optimized conditions, the assay exhibited a linear relationship between the absorbance and UA concentration over the range of 2.5 to 50 μM, with a detection limit of 1 μM. In addition, the assay can be performed at a single pH (8.0) so adjustment of the pH for peroxidase activity was not required. This advantage helped to further reduce costs and time. The developed assay was also successfully applied to detect UA in pooled human serum with the recoveries over 94.8%. These results suggest that the proposed assay holds great potential for clinical application.
Highlights
Uric acid (UA) is the end product of purine nucleotide catabolism in the human body
Recombinant CUOX, Vitreoscilla hemoglobin (VHb), CUOX-VHb (CV) fusion protein and VHb-CUOX-VHb (VC) fusion proteins were successfully expressed in E. coli BL21 (DE3) and purified to homogeneity
This result indicated that VC was susceptible to protein degradation, which could be due to steric effects presented within the engineered polypeptide chain
Summary
The normal ranges of serum uric acid (SUA) in females and males are 2.4–6.0 mg/dL (143–357 μM) and 3.4–7.0 mg/dL (202–416 μM), respectively. Monitoring of SUA levels is essential for the prevention, diagnosis and treatment of these diseases. Various modern techniques have been proposed, the most widely employed technique for UA determination by automated systems in clinical laboratories is classical spectrophotometry [3]. This is because the technique is designed with an automation-friendly protocol and the cost is more favorable. The principle of this technique is based on the cascade reactions of uricase and peroxidase enzymes [4,5]
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