Partial Purification of 5'-Nucleotidase Enzyme in Patients with Renal Failure and Kinetic Study of the Purified Enzyme in Salah al-Din

Abstract

This study involved separating and partially purifying the 5'-Nucleotidase enzyme isoforms from the sera of patients with renal failure. The purification process consisted of several steps, beginning with precipitation using 30% ammonium sulfate, resulting in a purification fold of 1.2625 and an enzyme yield of 65.63%. This was followed by membrane filtration using a buffer solution, achieving a purification fold of 1.651 and an enzyme yield of 49%. The protein fraction separated from the dialysis bag was then passed through a gel filtration column, reaching a purification fold of 51.88 and an enzyme yield of 43.1%. The enzyme isoforms from the separated protein fraction were further separated using gel filtration, employing ion-exchange chromatography techniques. This resulted in three isoforms of the 5'-Nucleotidase enzyme with varying degrees of purity, the highest for isoform. at 2.297-fold. The study also included kinetic studies of the enzyme purified by gel filtration, which involved determining the Km value under the influence of varying concentrations of the enzyme's substrate, 5'-AMP, for the purified enzyme from the sera of patients with renal failure, which was found to be 12.4. The effects of temperature, pH, and reaction time on enzyme activity were also studied.