可视化高通量检测天冬氨酸转氨甲酰酶活性的方法

Aspartate transcarbamylase (ATCase) is the first enzyme in the pyrimidine biosynthesis pathway, and the feedback regulation mechanism of its activity plays an important role in controlling the balance of purine and pyrimidine metabolism. Currently, the activity of ATCase is detected through a spectrophotometric method with antipyrine and 2,3-butanedione oxime as the color reagent. However, this method requires a reaction in the darkness for 16h, and then a water bath at 45℃ for 30min with uniform illumination, which is complicated to operate. In this study, p-dimethylaminobenzaldehyde (PDAB) hydrochloric acid solution was used as the color reagent to establish a method for detecting the activity of ATCase. The principle of this method is that N-carbamoyl-L-aspartic acid (the product of ATCase) reacts with PDAB at room temperature for 15min, which can produce a yellow substance and can be quantitatively detected by colorimetry. In the range of 0.1-5mmol/L, the yellow color deepened with the increase of the N-CP-DL-Asp concentration, and the absorbance at 438nm had a good linear relationship. The precision RSD was 0.87%-1.52%, and the recovery rate of standard addition was 96.6%-101.9%. This method was successfully used to determine the activity of recombinantly expressed ATCase, and the specific activity was 56.83U/mg. This method realizes the efficient, rapid and visual detection of the activity of ATCase, and can achieve high-throughput detection by using a microplate reader.