The affinity chromatography of transketolase

Area of Science:

• Biochemistry
• Enzymology
• Protein Purification

Background:

• Transketolase (EC 2.2.1.1) is a key enzyme in the pentose phosphate pathway.
• Purification of transketolase is essential for biochemical studies.
• Contaminating enzymes, like ribulose 5-phosphate 3-epimerase (EC 5.1.3.1), can complicate purification.

Purpose of the Study:

• To develop and evaluate affinity adsorbents for transketolase purification.
• To investigate the enzyme's behavior on various chromatographic supports.
• To establish a method for removing bound cofactors like thiamine pyrophosphate.

Main Methods:

• Preparation of affinity adsorbents using inhibitors (oxythiamine, neopyrithiamine) and cofactors (thiamine pyrophosphate, D-ribose 5-phosphate).
• Chromatographic analysis of transketolase and contaminating enzymes on prepared adsorbents and Blue Sepharose CL-6B.
• Measurement of competitive inhibition constants (Ki) for oxythiamine and neopyrithiamine.

Main Results:

• Developed a procedure to remove thiamine pyrophosphate using EDTA dialysis.
• Compared enzyme affinity for various adsorbents, finding thiamine pyrophosphate adsorbents ineffective.
• Identified epoxy-linked neopyrithiamine and D-ribose 5-phosphate adsorbents as effective for transketolase binding and specific elution at pH 7.4.

Conclusions:

• Affinity chromatography using specific ligands provides an effective method for transketolase purification.
• Neopyrithiamine and D-ribose 5-phosphate are suitable ligands for developing transketolase affinity adsorbents.
• The developed methods allow for specific isolation of active transketolase from complex biological mixtures.