Partial purification of some red cell enzymes utilizing hemoglobin precipitation by zinc salts.
Carrell and Lehmann have reported a method of precipitating Hb that is attractive in terms of its rapidity, simplicity, reversibility, and lack of rigorous conditions. We have applied the methodology to the concentration and partial purification of certain red cell enzyme activities. Twenty-three glycolytic and nonglycolytic enzymes were studied (Table 1). Under conditions where 97% to 98% of Hb was precipitated, recoveries in the supernatant for 14 enzymes ranged from 40% to 100%, with nine activities virtually completely recovered as measured by routine assay. In six instances, only minimal activity was recoverable either in the supernatant or in the precipitate. In the case of those enzymes whose recovered activities in supernatant were large, more rigorous investigation to detect possible zinc-induced alterations is necessary to determine whether the procedure impairs parameters not readily evaluated by routine assay. The effect of the procedure on PK was studied more intensively. The kinetics of the enzyme with PEP and ADP were unaffected, its thermostability appeared not to differ from native PK, and ATP inhibition of activity was normal. Addition of more zinc to the supernatant precipitated PK, which could then be further partially purified by (NH4)2SO4 precipitation. Inclusion of magnesium and substrate PEP in the hemolysate prior to zinc addition increased recovery in the supernatant from 40% to 80%.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000645:Ammonium Sulfate; D011232:Chemical Precipitation; D004355:Drug Stability; D004912:Erythrocytes / Q000201:enzymology*; D006019:Glycolysis; D006454:Hemoglobins; D006358:Hot Temperature; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000097:blood*; D013431:Sulfates / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology; D019287:Zinc Sulfate
D006454:Hemoglobins; D013431:Sulfates; D015636:Magnesium Chloride; D010728:Phosphoenolpyruvate; D019287:Zinc Sulfate; D000255:Adenosine Triphosphate; D011770:Pyruvate Kinase; D008274:Magnesium; D015032:Zinc; D000645:Ammonium Sulfate