A new modified micromethod for quantitative estimation of cytochrome c.

A new convenient micromethod is presented for the quantitative determination of cytochrome c by measurement in the α-region of a low-temperature difference spectrum (reduced minus oxidized) using a commercially available split-beam spectrophotometer and its low-temperature spectrometry attachment. The method shows excellent reproducibility and precision with high sensitivity. Interference by other pigments such as cytochrome b and b₅ was avoided by the extraction of cytochrome c from the material in 0.5% aluminium sulfate solution, pH 4.5. Direct extraction from the homogenate minimized the loss of cytochrome c. Distortion of the spectrum induced by low-temperature treatments was compensated for by calculations. With the present method, one can well estimate quantitatively 0.2 nmol of cytochrome c in a minimum of 0.8 ml (0.4 ml × 2) of sample, e.g., the supernatant obtained from five adults of Tenebrio molitor (500 mg: 2 ml of homogenate).