Spectrophotometric studies of the cytochrome system of heart muscle

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Abstract
A technique for studying cyto-chrome reactions in turbid suspensions of heart-muscle particles by using commercially available spectrophotometers is described. Beer''s law does not apply to measurements of the reversible change of extinction associated with the oxidation and reduction of cytochromes in heart sarcosomes, using this technique. Difference spectra (reduced minus aerobic steady-states) of heart sarcosomes and of Keilin and Hartree heart-muscle preparation were determined in the presence of succinate as reducing substrate. They are closely similar. The difference spectrum of heart sarcosomes reduced by [alpha]-oxoglutarate resembles that obtained with succinate. Difference spectra of heart sarcosomes reduced by different substrates suggest that the cytochromes contribute positive absorption bands between 320 and 340 m[mu], and that in addition endogenous diphosphopyridine nucleotide is reduced by [beta]-hydroxybutyrate and by [alpha]-oxoglutarate, but not by succinate. The kinetics and magnitude of extinction changes at 433 and at 445 m[mu] in sarcosome suspensions to which myoglobin is added suggest that the presence of myoglobin in sarcosome suspensions cannot be detected with certainty by examining the kinetics at 433 m[mu]. Carbon monoxide in low concentration does not affect the kinetics or magnitude of reversible extinction changes at 445 m[mu] or at 433 m[mu] in sarcosome suspensions. The kinetics of extinction changes at 430-435 m[mu] in sarcosome suspensions are strongly affected by the presence of 2:4-dinitrophenol and by adenosine diphosphate. This confirms observations by other workers that the reactions of cytochrome b are particularly affected by coupled phosphorylation. It is suggested that cytochrome b is reduced more rapidly in the presence of simultaneous oxidative phosphorylation than in its absence. Results presented are compared and contrasted with similar observations obtained by other workers with specially designed apparatus.