VOL. 12 (1953) BIOCHIMICA ET BIOPHYSICA ACTA 289 



NEW METHODS FOR THE STUDY OF THE 

 CARBON MONOXIDE COMPOUNDS OF RESPIRATORY ENZYMES* 



BRIXTON CHANCE, LUCILE SMITH and LAROY CASTOR** Ao /i <-<^ <■ 



Johnson Research Foundation, University of Pennsylvania, IllJ / •»'«' '"'--i> ■'^'' 



Philadelphia, Pennsylvania (U.S.A.) I^C \ L' t^ ■'"'^ '■*- * 



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MASS. ys 



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In the past few years it has been possible to improve considerably and to extend 

 range of three basic methods for the study of respiratory enzymes, especially their carbon 

 monoxide compounds. Visual spectroscopy- — ^used first by MacMunn in 1885^ to dis- 

 cover the histohemins, by Keilin in 1925^ to identify and to study the cytochromes in 

 detail, and by Warburg and his co-workers to identify the CO compound of the respira- 

 tory enzyme in Acetohader pasteurianum^ — until recently has had no successful compet- 

 itor for the study of the absorption bands of the respiratory enzymes in suspensions of 

 intact cells or in muscle tissue. But improved photoelectric surfaces and electronic tech- 

 niques have in our hands and more recently in the hands of others^ brought sharply 

 into focus the absorption spectra that could at best be only dimly perceived by earlier 

 photoelectric techniques such as were used by Warburg and Christian to show the 

 presence of flavoprotein in Bacterium delbrilckii^ and by Haas to measure the speed of 

 reduction of cytochrome c m Torula utilis^. It is now possible to observe the reduction of 

 respiratory enzymes in the range 320 to 660 m/x in many types of respiring cell suspen- 

 sions. In bakers' yeast, for example, the reaction kinetics and spectra of the pyridine 

 nucleotides, flavoproteins and cytochromes of types a, a^, b and c can be separately 

 studied by rapid and sensitive spectroscopic methods''^. In some cases the sensitivity 

 exceeds that achieved by highly skilful visual observers since we can regularly record 

 the 590 mju, band of cytochrome a^ in cultures oi Azotobacter chroococcum. More recently 

 these spectroscopic methods have been improved so that they are suitable for measuring 

 the changes in optical density caused by the formation of the carbon monoxide com- 

 pounds^ of the respiratory enzymes^" or cytochrome oxidases^^ of the cell suspensions. 



About ten years ago Bucher and Negelein developed an "optical method" for 

 the study of the kinetics of photodissociation of the CO compounds of the soluble pig- 

 ments myoglobin and hemoglobin^^. By introducing new electronic techniques, we now 

 have developed a more sensitive method for use with turbid cell suspensions, first, for 

 demonstrating that the photodissociation of the cytochrome a^-CO compound actually 

 occurs^^, secondly, for obtaining "photodissociation spectra" of the CO compounds of 

 respiratory enzymes^^, and thirdly, for obtaining accurate values for the molecular ex- 

 tinction of the a-bands of the CO compounds of the respiratory enzymes by direct meas- 



This research was supported in part by the National Institutes of Health, United States Pubhc 

 Health Service, by the Office of Naval Research, and by the National Science Foundation. 

 ** Lalor Foundation predoctoral fellow. 



References p. 2gyl2g8. 



