The Expression And Purification Of Rat Acyl-CoA Oxidase And The Comparison Of Activity Determination Methods

In this study,Escherichia coli strain BL21(DE3)was used to express recombinant rat ACOX and the target protein was one-step purified by Nickel affinity chromatography.Using purified ACOX,three ACOX activity determination methods were compared in terms of the detection limit,the correlation between enzyme activity and enzyme amount,the correlation of the enzyme activity among different methods and so forth.Enoyl-CoA,the product of acyl-CoA,could be determined by the absorbance increase at 263 nm.The byproduct of peroxisomal?-oxidation hydrogen peroxide could be calculated by the fluorescent intensity decrease of scopoletin.The decrease of dissolved oxygen caused by?-oxidation could be determined by an oxygen sensor.This study could offer some method reference frame for the detection of ACOX activity in liver tissues in the theoretical research of the diseases related to fatty acid metabolism.These methods were used to determine ACOX activity in hepatic peroxisomes separated by differential centrifugation.The recombinant ACOX was purified to a single band and its formula weight was calculated to be 72 k Da at a concentration of 2.3 mg/m L.High Performance Liquid Chromatography(HPLC)was used to separate and purify the synthetic PA-CoA substrate on a small scale.The retention time of PA-CoA is 24.3 min,and its concentration was 8.6 m M.The detection limits of the acyl-CoA substrate fluorescence spectrophotometry and oxygen meter were 860 n M,lower than that acquired by UV-Vis spectrometer(1720 n M).However,compared with the correlation coefficient obtained by UV-Vis method(R²=0.98),the values obtained by the other two methods is relatively lower(0.91 and 0.69 respectively).The correlation of enzyme activity between fluorescence and UV-Vis method(R²=0.88)is higher than oxygen meter(R²=0.8).The enzyme activity determined by fluorescence method is the smallest,this could be ascribed to the instability of H₂O₂.Owing to electrode correction,constant stirring and other affecting factors,enzyme activity values obtained by oxygen meter is the largest.At the same time,the enzyme samples and expensive acyl-CoA substrate it consumed is 3 times more than the other two methods.For the determination of ACOX activity in rat liver tissue.The hepatic peroxisomes were obtained by homogenation and differential centrifugation.The values obtained by fluorescence and oxygen meter method were 3.47 and 31.2 U/m L respectively.Owing to the further oxidation of enoyl-CoA product by liver tissues,high background absorption at 263nm and some other affecting factors,the tissues ACOX activity detected by UV-Vis method u-sing PA-CoA substrate is inaccurate to some extent,which could be overcome by using IP-CoA with special structure as substrate.The ACOX in tissues was determined to be 2.4U/m L,which is equal to value obtained by fluorescence spectrophotometry.