VOL. 12 (1953) 



CO COMPOUNDS OF RESPIRATORY ENZYMES 



295 



In order to demonstrate that our method for measuring the photodissociation spec- 

 trum gives a result that agrees accurately with the actual absorption spectrum, we 

 compare in Fig. 7 the spectrum obtained by subtracting the ferromyoglobin-CO spec- 

 trum from that of ferromyoglobin (Beznak^^) with a photodissociation spectrum ob- 

 tained with this apparatus. The agreement of the data shows that the photodissociation 

 method gives nearly as accurate results as the direct measurement of the absorption 

 spectra. 



It should be noted that exact coincidence of the peaks of the absorption photodisso- 

 ciation difference spectra with those of the relative photochemical action spectra is not 

 to be expected. Fig. 7 (Curve A) clearly shows that for protohemin pigments the peak of 

 the difference spectrum lies 2.5 rufx below that of the absolute spectrum and, in the case 

 of the dichroic hemin enzyme lactoperoxidase, the displacement is 1.5 m/i,^^. Thus the 

 displacement is small, but significant. 



240 n 



180 



1 160 



c 

 a> 

 E 

 <u 



g 120 



a. 



O 



80 



40 



Reduced 

 + 

 CO 



Oxidized 



Reduced 



400 



420 440 



A 



460 



420 



440 460 



B 



480 



Fig 7. (A), the oxidized, reduced, and reduced-CO spectra for myoglobin (from Beznak^^) and (B), 

 (open circles), the difference spectrum of the CO compound. Solid circles of (B) show experimental 

 data on the photodissociation spectrum of myoglobin-CO obtained by the method of Fig. 4. In 

 order to facilitate the comparison, the ordinates of the photodissociation curve were multiplied by 

 a constant factor to cause the two sets of data to match at the peak of the curve (i fiM Mgb, pH = 



7.0, 2.g fiM CO) (Expt. 143a). 



Calculation of the molecular extinction coefficients 



By measuring the kinetics of photodissociation and recombination of the CO com- 

 pound on a faster time scale as in Fig. 8, the molecular extinction coefficient may be cal- 

 culated in a manner similar to that used by Bucher and Kaspers^^ provided the inten- 

 sity of the photodissociating light is known. But instead of a bolometer we use myo- 

 globin-CO as a standard and thereby avoid the need for an accurate measurement of the 

 light intensity as well as the distribution of intensities in the cuvette. To simulate light 

 scattering when myoglobin is used, Escherichia coli are added to give the same scattering 

 effects as the yeast cells (see reference 14 for details). On this basis, we have computed 

 the values of the molecular extinction coefficients for the CO compounds in heart muscle 



References p. 2gyl2g8. 



