Isolation of phosphoglycerate kinases by affinity chromatography

Area of Science:

• Biochemistry
• Enzyme purification
• Chromatography

Background:

• Phosphoglycerate kinase is a crucial enzyme in glycolysis.
• Existing purification methods for phosphoglycerate kinase are often complex and yield-limited.

Purpose of the Study:

• To synthesize and evaluate novel Sepharose derivatives for phosphoglycerate kinase affinity purification.
• To develop a simplified, high-yield method for isolating phosphoglycerate kinase from diverse biological sources.

Main Methods:

• Synthesis of Sepharose derivatives with immobilized DL-O-phosphorylserine or adenosine nucleotides.
• Affinity chromatography using periodate-oxidized ATP or ADP bound to adipoyl-dihydrazo-Sepharose.
• Optimization of binding and elution conditions (pH, magnesium, buffer ions, ligands).
• Purification involving extract preparation, affinity chromatography, and gel filtration.

Main Results:

• The most effective affinity matrix utilized periodate-oxidized ATP or ADP linked via the ribose to adipoyl-dihydrazo-Sepharose.
• Optimized conditions facilitated high-capacity binding (5 mg/g matrix) and efficient elution.
• The developed method achieved excellent yields (85-100%) and high purity (homogeneous by SDS-PAGE).
• Phosphoglycerate kinases from spinach, erythrocytes, and various muscles showed similar affinity, molecular weight, and specific activity.

Conclusions:

• A novel and highly efficient affinity chromatography method for phosphoglycerate kinase purification has been established.
• This method is technically simple, reproducible, and provides superior yields compared to existing procedures.
• The purified enzyme from different sources exhibits conserved biochemical properties.