The acceptor specificity of flavins and flavoproteins. I. Techniques for anaerobic spectrophotometry

1. Easily constructed apparatus is described for spectrophotometry under strictly anaerobic conditions without requiring special cuvettes. It permits the addition of several reagents successively without opening the system to the air.

2. The absorption spectrum of dithionite shows a strong peak at 314 nm, the molar absorbance of which has been determined. This gives a convenient method for the titration of acceptors with dithionite.

3. One molecule of dithionite reacts very rapidly with one molecule of O₂ in solution. The O₂ is reduced quantitatively to H₂O₂. With excess of dithionite another, much slower, reaction follows, in which a second molecule of dithionite is oxidized by the peroxide.

4. A study has been made of the reduction by dithionite of a variety of acceptors commonly used in the study of flavoproteins. The majority react very rapidly, but a few are reduced relatively slowly or not at all.

5. The majority of acceptors do not react significantly with sulphite, the oxidation product of dithionite. One molecule of dithionite then provides two reduction equivalents. A few acceptors, however, react with the sulphite formed, giving a second reaction involving two more equivalents.