204 RESPIRATION 



which depends on the reduction of methylene blue coupled to oxida- 

 tion of substrate: 



Substrate + oxidized dye — > oxidized substrate + reduced dye (2) 



The Thunberg method has been used to demonstrate the presence of 

 respiratory enzymes in cell-free extracts from fungi (182, 226). Methyl- 

 ene blue is often inhibitory (271) and other dyes, e.g., 2,6-dichloro- 

 phenolindophenol, are in general to be preferred (30, 207, 216, 225). 



Spectrophotometric methods are both more powerful and more 

 quantitative. The most generally useful of these relies upon the 

 fact that the reduced forms of the coenzymes diphosphopyridine 

 nucleotide (DPN) and triphosphopyridine nucleotide (TPN) absorb 

 strongly at 340 m^; the change in density can be followed easily and 

 rapidly after addition of substrate to a suitable system (312). Exam- 

 ples of the use of this method in studies of fungi and actinomycetes 

 include work on the enzymes of Neurospora crassa (81, 301), Aspergil- 

 lus niger (148), Fusarium lini (62), and Streptomyces coelicolor (66). 



Spectrophotometric measurements can, of course, be used whenever 

 a component of the system changes its light absorption as a conse- 

 quence of an enzymatic reaction. Oxidation and reduction of cyto- 

 chrome c can be followed at 550 imx (46, 47, 71, 280), or reducible 

 dyes may be used (50). 



Like all other sensitive measurements, the spectrophotometric re- 

 quires a certain amount of judgment. Too often, a crude enzyme 

 preparation contains unsuspected activities which complicate a given 

 determination. For example, an extract may reduce both DPN and 

 TPN when fructose- 1,6-diphosphate is added. The DPN reduction 

 is straightforward: aldolase forms triose phosphate, which is then 

 oxidized with concurrent reduction of the coenzyme. The reduction 

 of TPN is not, however, as might first be thought, the same process; 

 instead, fructose-l,6-diphosphate is first converted to glucoses-phos- 

 phate, which is oxidized by glucose-6-phosphate dehydrogenase and 

 TPN to 6-phosphogluconate. The crude experiment falsely suggests 

 that triose phosphate dehydrogenase can react with either DPN or 

 TPN. 



These manometric and spectrophotometric methods are general 

 in their application and therefore stand in contrast to the problem 

 of preparing cells and extracts of fungi for respirometric study. In a 

 certain sense, each organism is a problem in itself, but it may be 

 useful here to mention some of the methods which have been used 

 in particular instances. Discs of growing mycelium from a surface 



