Preparation of Apoprotein from Spinach Ferredoxin‐NADP + Reductase

1 Ferredoxin‐NADP + reductase resolved into apoprotein and flavin by incubation with 2.5 M CaCl 2 at pH 7.5 and 2°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°C. 2 The release of the prosthetic group from the protein by the Ca 2+ ions is a multi‐step process. Three different effects of these ions are identifiable: (a) 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; (b) a slower induction of a conformational change in the protein which causes complete loss of activity and exposure to solvent of the flavin moiety; the FAD is finally released from the protein; (c) complete conversion of FAD to FMN, which blocks reconstitution to holoenzyme, caused by the well‐known hydrolytic action of Ca 2+ ions on the pyrophosphate bridge of FAD 3 Binding of FAD by the apoferredoxin‐NADP + reductase is very rapid and it is complete in a few minutes even at 0°C. A K d of 3.4 × 10 −9 M has been 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 two enzymes are very similar. Removal of factors from the apoprotein solution such as dithiothreitol and glycerol promotes the appearance of protein aggregates.