Horse-liver glutathione reductase: purification and characterization.

This research paper describes the process and results of purifying horse-liver glutathione reductase, a type of enzyme. Multiple techniques were used in this process and their effectiveness evaluated. In addition, the characteristics of the enzyme under different conditions were also assessed.

Purification of Horse-liver Glutathione Reductase

• The researchers purified the enzyme from horse liver through a series of techniques including affinity chromatography on N6-(6-aminohexyl)-adenosine-1’5′-bisphosphate Sepharose (N6-2’5′-ADP-Sepharose) and Reactive Red-120-Agarose, as well as chromatography on DEAE-Sephadex and Sephacryl S-300. These complex chemical compounds and procedures are used to isolate the enzyme from other components present in the horse liver.
• The outcome was a high-degree of purification, as denoted by a specific activity of 248 units per milligram, indicating a high concentration of active enzyme in the preparation. The enzyme was purified 11,174 times and there was a 47% final recovery.
• SDS-electrophoresis, a laboratory technique used for studying proteins, showed that the final enzyme preparation was homogeneous, meaning that it had a uniform composition and was pure.

Characteristics of the Enzyme

• The enzyme demonstrated varied charge properties shown through non-denaturing electrophoresis and chromatofocusing. There were several peaks of isoelectric point (pI), a measure of the pH at which a particular molecule or surface carries no net electrical charge, between 5.7 and 6.7.
• The enzyme was found to be homodimeric, meaning it is made up of two identical subunits. Its native molecular weight was 107,000.
• The researchers found that the enzyme had peaks of absorption at 270, 370 and 462 nm, indicating that it absorbs light at these specific wavelengths due to its characteristic as a flavoprotein.

Enzyme Activity

• When NADPH, the normal cofactor of the enzyme, was substituted with deamino-NADPH or NADH, the enzyme still showed 69% and 8.5% activity, respectively. When mixed with glutathione-CoA disulfide, the enzyme demonstrated 23% of the activity compared to GSSG, the standard substrate of the enzyme.
• The apparent Km values, which measure the affinity of the enzyme for its substrates, were calculated for NADPH, NADH, GSSG and ferricyanide, providing insight into the enzyme’s performance and its possible roles in various biological reactions.