The application of chromatographic techniques for the purification of aconitate hydratase isolated from the hearts of rats with pathology, and the investigation of certain catalytic properties of this enzyme

Abstract

In this study, we evaluated the catalytic and regulatory properties of an enzyme preparation derived from the cytoplasmic fraction of rat aconitate hydratase (AH, EC 4.2.1.3), which was purified using gel filtration and ion exchange chromatography. The enzyme preparation was derived from the hearts of rats (Rattus norvegicus, Wistar strain) that had undergone myocardial injury induced by diclofenac and treated with dipicolinic acid (DPA). Three groups of animals were used in the experiment. From the 15th day of the experiment, a saline solution was administered intraperitoneally to the rats in the control group for a period of 7 days. On the first day, animals in the second group received 100 μl of Freund’s adjuvant subcutaneously into the left plantar surface of their hind paw. Thereafter, from the 15th day onwards, diclofenac sodium was given intraperitoneally at a dosage of 10 mg/kg body weight for a duration of 7 days. A third group of animals concurrently received intragastric administration of DPA at 10 mg/kg, along with diclofenac sodium. Heart and blood serum samples were collected 24 hours following the final injection. Commercial kits were used to analyze marker enzymes of cardiomyocyte cytolysis in the blood serum. The level of protein oxidative modification was determined through reaction with 2,4-dinitrophenylhydrazine. The activity of AH was measured spectrophotometrically at 235 nm. Separation of the cytoplasmic and mitochondrial fractions was performed using differential centrifugation. Purification of the enzyme from rat heart tissue was achieved through techniques such as ammonium sulfate precipitation, gel filtration using G-25 and G-150 sephadex columns, and ion exchange chromatography on KM-cellulose. During the course of this work, a cytoplasmic fraction with a 120-fold purification factor was isolated from the hearts of rats with myocardial damage induced by diclofenac, and a fraction with a purification factor of 122.1 was isolated from hearts of rats with pathology treated for DPA. Using the Lineweaver-Burke double inverse coordinate method, it has been demonstrated that the administration of DPA to rats resulted in a restoration of the enzyme's affinity for citrate and isocitrate compared to pathological indicators. In addition, there were changes in the control of parameters such as temperature, pH-optimum, and activation energy of the enzymatic reaction. For AH in the heart of pathologically treated rats, there was a more significant increase in enzyme activity when reduced glutathione was added to the reaction medium, compared to animals treated with diclofenac alone. The observed alterations in the properties of AH may be linked to changes in the structural and functional characteristics of the enzyme molecule. Such alterations could be a consequence of the antioxidant activity of DPA in response to oxidative stress induced by diclofenac treatment.