Cytochromes Cooled in Liquid Nitrogen 437 



band structure has frequently been observed. The organic solvent systems 

 (such as the ether-pentane-alcohol (EPA) mixture) generally employed by 

 chemists in the spectroscopic studies of these pigments are inadequate, 

 however, for work with proteins. Thus the introduction by Keilin and 

 Hartree (1949) of a glycerol-water-mixture as a medium and the resultant 

 five to tenfold intensification of absorption bands obtained with such a 

 mixture, has served as the basis for the identification of pigments in such low 

 amounts that they would not otherwise be discernible. 



We at the Johnson Foundation, recognizing the importance of Keilin's 

 and Hartree's (1949) work on the low temperature effect but cognizant of 

 the restriction imposed by simply describing spectra observed in a low 

 dispersion spectroscope, have adapted a wavelength scanning recording 

 spectrophotometer in order to plot automatically the spectra of haemo- 

 proteins at low temperature. These studies have confirmed in every way the 

 excellent reports of Keilin and Hartree. Indeed, we can honestly say that we 

 have not been able to add much to the basic knowledge brought forth by them 

 other than to extend the range of material studied. 



The present paper will deal with only two facets of the studies carried out 

 at the Johnson Foundation; the first is principally concerned with the 

 satelhte band structure of soluble haemoproteins, and the second with the 

 complexities observed in the spectra obtained with particulate materials. 



METHODS 



Figure 1 presents a schematic description of the instrument employed. This 

 is a more modern version of the basic instrument which has been designed 

 and developed by Chance and his colleagues (Chance, 1951; Yang and 

 Legallis, 1954). A Bausch and Lomb diffraction grating monochromator 

 serves as the source of monochromatic light; the monochromatic light from 

 the diffraction grating is divided into two equal parts by an oscillating mirror 

 and then passes through the cuvettes in the sample holder to an end-on 

 photomultiplier. As with all spectrophotometric studies of turbid material 

 the geometry of the sample to the light detector is most important, i.e. the 

 cuvettes must be placed as close to the face of the photomultiplier as possible 

 so that the greatest solid angle of transmitted and scattered light will be 

 included. For use at low temperatures (Estabrook, 1956), an unsilvered 

 Dewar flask is placed in the position normally localized for the sample. By 

 filling this Dewar with liquid nitrogen and using an appropriate sample 

 holder, one is able to cool samples and thus record the spectra of haemo- 

 proteins at the temperature of liquid nitrogen. 



Figure 2 shows in greater detail the arrangement of the sample holder and 

 Dewar flask. Two cuvettes are employed, one being essentially a reference, 

 the otlier the sample to be investigated (Note 1). The two light beams pass 

 alternately through the two cuvettes and the difference in light absorption, 



