442 



R. W. ESTABROOK 



Effect of Glycerol 



When samples of reduced cytochrome c are diluted in various concentra- 

 tions of glycerol and then cooled in liquid nitrogen, it is observed that glycerol 

 is required in order to resolve clearly the c^^ and c^o absorption bands. This is 

 illustrated in Fig. 5 where three representative spectra are presented. When 

 the concentration of the glycerol is less than 5 % (v/v) the absorption band 

 attributed to c^2 appears merely as a shoulder on c^^. In addition, some of the 

 /?-bands as well as the Cg^g-band are less distinct. This failure to obtain maximal 

 resolution in the absence of glycerol may be due to the type of crystal struc- 

 ture formed when the sample is cooled to — 190''C, or it may represent a 

 hitherto unrecognized reaction of reduced cytochrome c with glycerol forming 

 a derivative with distinctive absorption bands discernible only at hquid 

 nitrogen temperatures. In the subsequent discussion most of the measure- 

 ments have been carried out in the presence of about 50% (v/v) of glycerol 

 in order to measure the maximal effect of low temperature on the absorption 

 bands of reduced haemoproteins. 



Fig. 6. A comparison of the spectral properties of reduced cytochrome c of 

 heart muscle, the alkaline haemochrome of cytochrome c, and the peptide core of 

 cytochrome c. Curve A — Samples of peptide core of cytochrome c were dissolved 

 in 01 M phosphate buffer of pH 7-4 and a few crystals of Na^S.^Oj added. This 

 was then mixed with an equal volume of glycerol. Curve B — Samples of horse 

 heart cytochrome c were diluted with 1 n NaOH, permitted to incubate lOmin 

 at room temperature, reduced with Na2S204, and then mixed with an equal 

 volume of glycerol. Curve C — Samples of heart muscle cytochrome c were 

 treated as described in Fig. 4. Optical depth of cuvette was 1 mm. 



