The Study And Application Of A Static Injection Chemiluminescence Method In Assay Of Oxidase Activities

Objective: Oxidase is a kind of enzyme which reacts with molecular oxygen to catalyze the oxidation of substrate and the producton of H₂O₂, such as glucoseoxidase, acyl CoA oxidase and so on. Therefore, the assay of oxidase avtivity can measure the formation of product, H₂O₂. Because of rapid analysis, simple apparatus, high sensitivity, and wide linear range, the chemiluminescence has been often used as a means of determining H₂O₂ to reflect the oxidase's activity. At present, mostly used chemiluminescence method is flowing injection method which needs larger volum of reagent. The big volum prevents this method from being used to assay enzyme activity of small sample. Conversely, static injection chemiluminescence just requires less volum of reagent. So the static injection chemiluminescence was chosen at present experiment to assay oxidase activity in small sample.Luminol is a widely used chemiluminescence reagent for its simple structure, water solubility and high light quantum efficiency. It has been reported that trace amount of horseradish peroxidase activity was determined in a 4-Iodophenol enhanced luminol-H₂O₂-horseradish-peroxidase chemiluminescence system.In this study,we will couple the reaction catalyzed by oxidase with the system of 4-Iodophenol enhanced luminol-H₂O₂-horseradish-peroxidase chemiluminescence to detect H₂O₂ with static injection chemiluminescence, and establish a new method on assay the activities of glucoseoxidase and acyl CoA oxidase. The method will be used in the glucoseoxidase activity assay of honey and acylCoA oxidase activity assay of liver, heart and brain of normal, type-2 diabetes and insulin resistance rat.Methods:Two steps were used to determine the oxidase avtivity, namely the enzyme-catalyzed reaction and the chemiluminescence reaction.1 Assay of glucoseoxidase avtivity1.1 glucoseoxidase catalyzes the oxidation of glucose to produce H₂O₂ After starting the reaction by addition of 10μl of sample, the enzymatic reactions were performed under 35℃in total 30μl of 0.1mol/L potassium phosphate, pH 5.5 containing glucose.1.2 The H₂O₂ amount was determined by luminol chemiluminescence with static injectionAfter a period of time, put the container into the cassette of IFFS-A Multi chemiluminescence analyzer, inject 400μl of luminol solution(luminol, HRP, PIP, potassium phosphate buffer) into the container to stop the oxidase reaction and start chemiluminescence under 40℃. Record the chemiluminescence signal(CL) at 400V of PMT voltage. Use the peak as relative CL intensity. Calculated the H₂O₂ amount corresponded to the CL intensity according to the H₂O₂ standard curve. One enzyme unit was defined as the amount of the enzyme producing 1.0μmol of H₂O₂ per min under assay conditions.2 Assay of acyl CoA oxidase activity2.1 Acyl CoA oxidase catalyzes the oxidation of acyl CoA to produce H₂O₂ After starting the reaction by addition of 10μl of sample, the enzymatic reactions were performed under 35℃in total 30μl of potassium phosphate buffer containing palmitoyl-CoA, FAD, 200μmol/L NAD,170μmol/L CoA, 4mmol/L NaN3, 5mmol/L EDTANa3.2.2 The H₂O₂ amount was determined by luminol chemiluminescence with static injectionThe H₂O₂ amount was determined as described in 1.2 above. One enzyme unit was defined as the amount of the enzyme producing 1.0μmol of H₂O₂ per min under assay conditions.Results:1 Optimization of conditions of glucoseoxidase activity assay1.1 The reaction catalyzed by glucoseoxidase After starting the reaction by addition of 10μl of sample, the enzymatic reactions were performed for 60 min under 35℃in total 30μl of 0.1mol/L potassium phosphate, pH 5.5 containing 0.2mol/L glucose. 1.2 The reaction of luminol chemiluminescence with static injectionBy statically injecting 400μl of luminol solution containing 0.1mol/L potassium phosphate buffer, pH8.5, 40μmol/L luminol, 50μg/ml HRP, 20μmol/L PIP into the enzymatic reaction to stop the oxidase reaction and start chemiluminescence under 40℃.The leaner rage of the static injection chemiluminescence method for glucoseoxidase activity assay is 1.67×10-4-3.0×10-3U/ml. The relatively standard deviation of sample is 5.239%.2 Optimization of conditions of acyl CoA oxidase activity2.1 The reaction catalyzed by acyl CoA oxidase activityThe enzymatic reactions were performed for 10 min under 35℃in total 30μl of 0.1mol/L potassium phosphate, pH 8.5 containing 30μmol/L palmitoyl-CoA, 10μmol/L FAD, 200μmol/L NAD,170μmol/L CoA, 4mmol/LNaN3, 5mmol/L EDTANa3.2.2 The reaction of luminol chemiluminescence with static injectionThe Assay conditions are the same as that described in 1.2 above. One enzyme unit was defined as the amount of the enzyme producing 1.0μmol of H₂O₂ per min under assay conditions.Using the static injection chemiluminescence method to assay acyl CoA oxidase activity, relatively standard deviation of sample is 7.616%.3 Activities of samples3.1 Glucoseoxidase activity of honeyThe activity in honey is 5.379U/mg.3.2 Acyl CoA oxidase activities of tissues in rats3.2.1 In liver of ratsCompared with control groups(65.57±9.16 mU/mg), the activity of Acyl CoA oxidase increased in diabetes groups (78.33±12.18 mU/mg, P <0.05 ) and did not changed in insulin-resistant groups ( 60.75±10.15 mU/mg,P >0.05).3.2.2 In heart of ratsCompared with control groups (31.81±5.54 mU/mg ), the activity of Acyl CoA oxidase increased in diabetes groups (37.82±6.69 mU/mg, P <0.05 ) and did not changed in insulin-resistant groups (30.69±3.74 mU/mg,P >0.05).3.2.3 In brain of ratsCompared with control groups (19.86±4.40 mU/mg), the activity of Acyl CoA oxidase decreased both in diabetes groups (14.51±2.80 mU/mg P <0.01 ) and in insulin-resistant groups (15.74±2.90 mU/mg , P <0.05).Conclusion:1 The novel static injection chemiluminescence method can be applied for the determination of glucoseoxidase activity and acyl CoA oxidase activity. The method is simple, fast, sensitive and reliable.2 In type-2 Diabetes rats,the acyl CoA oxidase activities increase in liver and heart,but reduce significantly in brain;In insulin resistant rats,the acyl CoA oxidase activities do not significantly change in liver and heart,but reduce in brain.