The reaction of nitro-blue tetrazolium with d,l-L-tetrahydrofolate: The effect of pH,oxygen, formaldehyde and palladium(II)

d,l-L-Tetrahydrofolate (d,l-L-FH₄) transfers two electrons to nitro-blue tetrazolium (NBT) in oxygen-free buffers to form the highly coloured nitro-blue formazan and oxidized folate. Both the rate and extent of this reaction are affected by the pH, the nature of the buffer and the oxygen concentration. Inhibition of both the rate and extent of this reaction in air-saturated solutions by superoxide dismutase (SOD) indicates that the superoxide anion is an intermediate in the reaction so that formazan can be produced by both superoxide independent and superoxide dependent routes in air-saturated solutions.In oxygen-free solutions several lines of evidence can be interpreted to mean that the reduced pteridine ring of tetrahydrofolate is the electron donor in the reaction with NBT. Ionization of the amide hydrogen of the pteridine ring and subsequent increase in electron density of that ring might explain the large increases observed in the rate and extent of the reaction of tetrahydrofolate with NBT as the pH increases. Formaldehyde reacts non-enzymatically with tetrahydrofolate to form a methylene bridge between nitrogens 5 and 10 of methylenetetrahydrofolate. This molecule is much less reactive with nitro-blue tetrazolium than tetrahydrofolate. Complexes formed between tetrahydrofolate and palladium(II) ions are also less reactive with NBT than the tetrahydrofolate alone. This result provides added evidence that palladium(II) ions interact with tetrahydrofolate at the nitrogen 5, nitrogen 10 site of the molecule.     

Activation of latent phenolase from spinach chloroplasts by ageing and by frost

Activation of latent phenolase by freezing and thawing occurs in both thylakoid sediments and membrane washings from spinach chloroplasts, while ageing and digitonin treatment activates membrane-bound enzyme only. Disc clectrophoresis reveals that frost converts a soluble, latent phenolase to an active form after its release from the thylakoid membrane. Ageing of membranes containing latent phenolase results in direct liberation of other active forms. There are further active, soluble forms, which are exclusively found in the chloroplast stroma fraction.     

Relation between photosynthetic and phenolase activities in spinach chloroplasts

During O°-storage of class I chloroplasts from spinach leaves, activation of phenolase strongly correlates with the inactivation of photosynthetic re