Development of a new affinity chromatography method for purification of horseradish peroxidase enzyme

In this study, benzohydroxamic acid molecules were synthesized from methyl 4-amino-2-methoxy, methyl 4-amino-3-nitro, methyl 4-amino-3-methyl, and methyl 4-amino-3-chloro benzoate molecules, and the horseradish peroxidase (HRP) enzyme was purified in one step using the affinity chromatography technique for the first time. The IC50 and Ki values for the 4-amino 3-methyl benzohydroxamic acid molecule were 0.136 and 0.132 +/- 0.054 mu M, respectively, while the IC50 and Ki values for the 4-amino-3-nitro benzohydroxamic acid molecule were 56.00 and 51.90 +/- 9.90 mu M, respectively. It was found that the IC50 and Ki values for the 4-amino-3-chloro benzohydroxamic acid molecule were 218.33 and 175.67 +/- 43.78 mu M, respectively, whereas the IC50 and Ki values for the 4-amino-2-methoxy benzohydroxamic acid molecule were 306.00 and 218.00 +/- 68.80 mu M, respectively. The HRP enzyme was synthesized from 4-amino-2-methoxy hydroxamic acid column with a 35.97% yield 601.13 times, 4-amino-3-nitro hydroxamic acid column, with a 14.00% yield 404.11 times, 4-amino-3-methyl hydroxamic acid column with an 8.70% yield 394.88 times, and 4-amino-3-chloro hydroxamic acid column with a 4.48% yield 284.85 times. Thus, the HRP enzyme was purified in a single step with hydroxamic acids, and its molecular weight was found to be 44 kDa. The optimum pH was 8.0, the optimum temperature was 15 degrees C, and the optimum ionic strength was 0.4 M for the purified HRP enzyme.