武汉云克隆科技股份有限公司

Aspartate transaminase(AST) or aspartate aminotransferase, also known as AspAT/ASAT/AAT or glutamic oxaloacetic transaminase, is a pyridoxal phosphate-dependent transaminase enzyme AST catalyzes the reversible transfer of an α-amino group between aspartate and glutamate and, as such, is an important enzyme in amino acid metabolism. AST is found in the liver, heart, skeletal muscle, kidneys, brain, and red blood cells. In this test, an amino group is transferred of from aspartate to ⍺-ketoglutarate. The products of this reversible transamination reaction are oxaloacetate and glutamate. The oxaloacetic acid can be decomposed into pyruvate and carbon dioxide with the present of phenylamine citrate. The activity of aspartate transaminase can be measured by calculating the concentration of the pyruvate. The reaction was performed in adding 10μl different concentation recombinant AST(the blank tube add 10μl phosphate buffer) to 50μl mixture substrate contianing 2mM 2-Ketoglutaric acid, 0.1M L-aspartic acid, in 0.2M phosphate buffer,pH7.4, incubate at 37 ℃ for 1h, then add 10μl phenylamine citrate and 50μl 2,4-dinitrophenylhydrazine continue incubate at 37 ℃ for 20min, stop the action by adding 500μl 0.4M NaOH, read the OD value at 520nm. Standard curve prepare by double dilute 2μM pyruvate with phosphate bufferr then add 10μl phenylamine citrate and 50μl 2,4-dinitrophenylhydrazine, incubate at 37 ℃ for 20min and record the OD value at 520nm. One unit of AST is the amount of enzyme that will generate 1μmole of pyruvate per minute at pH7.4 at 37 ℃.