Preparation of Apoprotein from Spinach Ferredoxin‐NADP+ Reductase

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Abstract
Ferredoxin-NADP reductase resolved into apoprotein and flavin by incubation with 2.5 M CaCl2 at pH 7.5 and 2.degree. C. Essential factors to recover a reconstitutable apoprotein are dithiothreitol, glycerol and guanidine/HCl. The apoprotein is stable for at least a week at -20.degree. C. The release of the prosthetic group from the protein by the Ca2+ ions is a multi-step process. Three different effects of these ions are identifiable: a rapid 20-25% inhibition of catalytic activity probably caused by an increase in the ionic strength of the medium (other cations can produce it as well); a slower induction of a conformational change in the protein causing complete loss of activity and exposure to solvent of the flavin moiety, the FAD being finally released from the protein; and complete conversion of FAD to FMN (which blocks reconstitution to holoenzyme) caused by the well-known hydrolytic action of Ca2+ ions on the pyrophosphate bridge of FAD. Binding of FAD by the apoferredoxin-NADP+ reductase is very rapid and it is complete in a few minutes even at 0.degree. C. A Kd of 3.4 .times. 10-9 M was determined by fluorescence titration. The reconstituted holoenzyme has catalytic activity, spectral and fluorescence properties nearly identical to the native enzyme. The gel electrophoresis and isoelectrofocusing patterns of the 2 enzymes are very similar. Removal of factors from the apoprotein solution, such as dithiothreitol and glycerol, promotes the appearance of protein aggregates.