Multiple enzyme purifications from muscle extracts by using affinity-elution-chromatographic procedures

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Abstract
Starting with (NH4)2SO4 fractions of muscle extracts, procedures for purifying 4 to 6 separate enzymes from each fraction by using affinity-elution-chromatographic techniques are described. Schemes for purifying 12 separate enzymes from rabbit muscle, and 8 from chicken muscle extracts, are included. In nearly all cases the overall procedure involves 3 steps: the initial (NH4)2SO4 fractionation, the ion-exchange chromatography with affinity elution of the enzyme, and gel filtration. The specific activities of the enzymes so purified are comparable with the highest values in the literature. The 5 schemes described included illustrations of affinity elution of the separate enzymes at different pH values, at different ionic strengths and in combination with conventional gradient elution. They also included stepwise adsorption on columns at different pH values. Separation of 2 electrophoretically differing forms of phosphoglycerate kinase was achieved by gradient affinity elution from CM-cellulose. The lower-pI form was eluted by a lower concentration of substrate than the higher-pI form.