290 B, CHANCE, L. SMITH, L. CASTOR VOL. 12 (1953) 



urements of the kinetics of photodissociation and recombination of the CO compounds^*. 

 The latter method is much more direct than the manometric method^^-^^ which responds 

 too slowly to permit a direct measurement of the kinetics of photodissociation. We find the 

 molecular extinction coefficient of the a-band of cytochrome a^-CO to be £ = 12 cm~^ 

 X mM~^ for bakers' yeast cells and for heart muscle homogenates^^. 



The classical manometric method for determining the relative photochemical ac- 

 tion spectrum for the reversal of carbon monoxide inhibition of respiration that was 

 developed twenty-five years ago^' has been until now the only method so far available. 

 Although this manometric method has given excellent spectra, there seem to be large 

 changes in the heights of the major absorption bands and in the details of the subsid- 

 iary bands when the temperature is lowered^^-^^. Also, Melnick's action spectrum for 

 yeast^^ does not agree in detail with that of Kubowitz and Haas^^ nor does his 450 m^ 

 peak for heart muscle preparation agree with any of the other data on cytochrome 

 ^^i3,i9_ Xhus a new method that permits monochromatic illumination of the sample over 

 a wide range of wavelengths is highly desirable. We can report here preliminary experi- 

 ments with an apparatus for measuring photochemical action spectra in a drop of cell 

 suspension-i with the aid of the platinum microelectrode^^'^^. We have not yet perfected 

 the fourth and logical development of these techniques- — ^the plotting of the photo- 

 chemical action spectra from data on the direct measurement of the photodissociation 

 kinetics for a number of wavelengths of monochromatic photodissociating light, but 

 such an apparatus appears feasible. 



We have surveyed the respiratory pigments of various materials with these sensi- 

 tive methods^* and have recently focussed our attention upon a rather different "CO- 

 binding pigment" found in Staphylococcus albus and in other bacteria. Our absorption 

 spectrum (difference spectrum) for this CO compound shows peaks at 416 m/x^, 535 and 

 570 m/x^-', and the photodissociation spectrum shows close agreement with the absorp- 

 tion spectrum; the peak of the Soret band lies at 415 m^u.^^. We here present quantitative 

 data on the kinetics of photodissociation of this CO-binding pigment and lind that it is 

 considerably less light-sensitive at 589 m/x than the enzyme of yeast or muscle, but that 

 it has a distinctive band at 546 m/x. Our preliminary value for the molecular extinction 

 of the CO-binding pigment at this wavelength is e = 5 cm"^ X mil/~^. We have also 

 determined the relative photochemical action spectrum for this pigment and find that the 

 action spectrum in the Soret region has a peak at 418 m^i, in agreement with our ab- 

 sorption and photodissociation difference spectra and distinctly different from the 430 

 mju. peak for yeast and muscle^^ Our results suggest that this "CO-binding pigment" is 

 a new respiratory enzyme that has a prosthetic group closely allied to that of the proto- 

 hemin enzymes and distinctly different from the dichroic hemin enzymes, and should 

 therefore be classed as a completely new type of respiratory enzyme. 



Absorption difference spectra* 



One of the more useful methods for obtaining difference spectra is illustrated by 

 Fig. I. This method utilizes the light chopping and demodulating system developed 

 by Chance for a double-beam spectrophotometer^ and an automatic gain control cir- 

 cuit (age) developed by R.C.A.^^, together with a number of ingenious improvements 

 devised by Dr. C. C. Yang". The apparatus consists of a source of high intensity mono- 

 chromatic light that is split into two paths by a vibrating mirror (60 cps) and illuminates 



* Footnote see page 2g2. 

 References p. 2gyl2g8, 



