| Popis: |
A simple method, applicable to large amounts of starting material, has been developed for the preparation of plant ferredoxins, and applied to alfalfa. Highly purified alfalfa ferredoxin contains 2 atoms of iron and 2 moles of "labile" sulfur per molecular weight of 11,500. Spectral absorption maxima occur at 465, 422, 331, and 277 mµ. The ratios of the optical densities at 465, 422, and 331 mµ to that at 277 mµ are 0.43, 0.48, and 0.65, respectively. The amino acid composition of alfalfa ferredoxin is: Trp1, Lys5, His2, Arg1, Asp10, Thr6, Ser8, Glu17, Pro3, Gly8, Ala10, Cys6, Val9, Ile4, Leu6, Tyr4, Phe2. The aromatic residues and the spectral contribution of the 2 iron atoms account for the entire absorption at 277 mµ. The spectrum provides no evidence for the existence of any nonprotein organic constituent, such as a prosthetic group. The activity of the purest preparations in the photoreduction of TPN in the presence of chloroplasts is over 40 units per mg of protein. It was shown that the labile sulfur does not arise from cysteinyl residues by β elimination. The native protein deteriorates rapidly in the presence of O2 but can be protected indefinitely under N2. During deterioration there is a parallel loss of enzymic activity, of the spectral bands at 465, 422, and 331 mµ and of the labile sulfur. There is also a concomitant polymerization, the weight average molecular weight rising to about 30,000. The polymers are colorless and separate readily from the intact protein by electrophoresis. No disulfide bonds occur either in native or deteriorated ferredoxin. However, following deterioration the cysteinyl sulfhydryl groups become less reactive and the iron more strongly bound than in the native protein. Mercurial titrations and carboxymethylation experiments indicate that all 6 cysteinyl residues have identical reactivities, and it appears that the active site of alfalfa ferredoxin does not conform to the model proposed for the bacterial proteins. |
