HELMU r JiE/NERT 165 



(22). These changes, as well as the quenching of fluorescence, have 

 been attributed to formation of an intramolecular con;plex between 

 the isoalloxa/ine moiety and the adenine moiety of FAD (22, 21) . 

 Similar spectral changes are foiuid in a variety of flavin complexes. 

 As examples we may mention those with phenol (23) , benzoate, or 

 nai)htiioate (9) . In addition to a shift toward longer wavelengths 

 and a decrease in absorption, such complexes often show a pronounced 

 shoidder in the 460-490 m^ region. It is of interest here that a num- 

 ber of pure flavoproteins show a shoulder in this region of the spec- 

 trum (16, 8, 18, 15) or even an additional peak (19, 5), which 

 may indicate an intramolecular complex of the protein moiety with 

 the prosthetic flavin. Other flavoproteins, which show a smooth 

 ilavin spectnuii initially, may develop a shoulder in this region when 

 substrate or an inhibitor is bound to them (17, 24). The spectral 

 changes demonstrated by Yagi (24) , which occur in going from FAD 

 to D-amino acid apooxidase-FAD and then to the D-amino acid apo- 

 oxidase-FAD-benzoate complex are typical here. Recently Harbury 

 et al. succeeded in j^roducing a shoulder and even an additional peak 

 in the 460-490 nijx region of the spectrum of 3-methyl lumiflavin when 

 they dissolved this compound in solvents of decreasing polarity (10) . 

 They observed the simultaneous emergence of a shoulder at 400-430 

 m/x, which has also been found to be associated with the 460-490 

 nifi shoulder in flavoproteins (17) . 



It is often assumed that a shoulder at 400-430 niyu, in a pure flavo- 

 protein indicates contamination with heme compounds. The fact, 

 however, that this shoulder can be produced by changing solvents (10) 

 or adding substrate (17) shows that this interpretation is not always 

 correct. Observation of the spectral changes on reduction may permit 

 one to reach a decision here, as heme compounds shovdd then show 

 a distinct peak. 



It is not clear whether the complex formation of the old yellow 

 enzyme with TPNH (7, 6) bears more relation to those interactions 

 which lead to a band in the 460-490 mjx region or to those which 

 produce spectral changes at longer wavelengths, such as oxidoreduc- 

 tive interactions. It appears from the spectrum (7, 4) that both com- 

 ponents may be represented. This complex represents one of the first 

 instances in which a flavoprotein substrate complex was clearly demon- 

 strated by a spectral shift (7) . Interpretation is, however, complicated 

 by the fact that a certain oxidoreductive interaction between the 

 complexing partners is bound to oc(ur. The actual nature of the 

 complex is still unknown (6, 4) . 



