Preparation of Apoprotein From Spinach Ferredoxin-NADP super(+) Reductase. Studies on the Resolution Process and Characterization of the FAD Reconstituted Holoenzyme

Ferredoxin-NADP super(+) reductase resolved into apoprotein and flavin by incubation with 2.5 M CaCl sub(2) 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 Ca super(2+) ions is a multi-step process. Binding of FAD by the apoferredoxin-NADP super(+) reductase is very rapid and it is complete in a few minutes even at 0 degree C. A K sub(d) of 3.4 x 10 super(-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.