Single-Step Purification of Catalase Enzyme From Human Blood Erythrocytes Using Affinity Chromatography Technique

TL;DR

This study successfully purified catalase from human blood using a new affinity gel, achieving high purity and measuring its activity and stability.

Contribution

A newly synthesized affinity gel was used for single-step catalase purification from human erythrocytes.

Findings

Catalase was purified with a specific activity of 45.58 EU/mg and a purification fold of 529.50.

SDS-PAGE analysis showed a single 60 kDa band for catalase, indicating high purity.

The enzyme exhibited optimal activity at 30°C and thermal stability up to 60°C.

Abstract

In this study, we aimed to isolate and purify catalase from human blood erythrocytes by using a newly synthesized affinity gel. The synthesized ω-amino hexyl agarose-1,2,3-triazole-5-carboxylic acid affinity gel was analyzed by FT-IR. Then, different buffer, pH, and ionic strength parameters were optimized to determine the equilibration, washing, and elution buffer conditions. The catalase was purified from human blood erythrocytes with a specific activity of 45.58 EU/mg, purification fold of 529.50, and a yield of 0.416% using the synthesized new affinity gel. The purity and molecular weight of the enzyme were analyzed by SDS-PAGE, and a single band at 60 kDa was observed for catalase. The optimum reaction temperature of the catalase was found to be 30°C, while the thermal stability temperature was 60°C. The Km and Vmax of the enzyme for hydrogen peroxide were calculated at 0.125 mM and 2500 U mL−1, respectively.

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