Affinity precipitation of dehydrogenases.

Area of Science:

• Biochemistry
• Protein purification

Background:

• Affinity precipitation is a novel technique related to immunoprecipitation and affinity chromatography.
• Bifunctional NAD derivatives, such as Bis-NAD, can be used as ligands for affinity precipitation.

Purpose of the Study:

• To evaluate the efficacy of affinity precipitation for isolating dehydrogenases using Bis-NAD.
• To assess the suitability of affinity precipitation for preparative-scale enzyme isolation.

Main Methods:

• Enzymes (lactate dehydrogenase, glutamate dehydrogenase, yeast alcohol dehydrogenase, liver alcohol dehydrogenase) were incubated with a bifunctional NAD derivative (Bis-NAD).
• Affinity precipitation was induced, with salt added to enhance precipitation for certain enzymes.
• Preparative-scale isolation of ox heart lactate dehydrogenase from a crude extract was performed using affinity precipitation.

Main Results:

• Lactate dehydrogenase and glutamate dehydrogenase were readily precipitated.
• Yeast alcohol dehydrogenase precipitation was enhanced by the addition of salt.
• Liver alcohol dehydrogenase did not precipitate, likely due to the formation of small affinity complexes.
• Preparative-scale isolation of ox heart lactate dehydrogenase yielded satisfactory purity and yield.

Conclusions:

• Affinity precipitation is a viable method for purifying dehydrogenases, particularly lactate dehydrogenase.
• The technique's success is dependent on enzyme characteristics and reaction conditions, such as salt concentration.
• Affinity precipitation offers a promising approach for preparative-scale enzyme isolation with satisfactory results.