Composition of the Cytochrome c Complex with Cardiolipin by Thermal Lens Spectrometry

Abstract

Thermal lens spectrometry along with spectrophotometric titration were used to assess the composition of the complex of oxidized cytochrome c (ferricytochrome c) with 1,1′,2,2′-tetraoleyl cardiolipin, which plays a key role in the initiation of apoptosis. Spectrophotometric titration was carried out for micromolar concentrations at which the complex is mainly insoluble, to assess the residual concentration in the solution and to estimate the solubility of the complex. Thermal lens spectrometry was used as a method of molecular absorption spectroscopy, which has two advantages over conventional optical transmission spectroscopy: the higher sensitivity of absorbance measurements and the possibility of studying the light absorption by chromophores and heat transfer in complex systems, such as living cells or tissues. Thermal lens measurements were carried out at nanomolar concentrations, where the complex is mainly in solution, i.e., under the conditions of its direct measurements. From the thermal lens measurements, the ratios of cytochrome c and cardiolipin in the complex were 50 at pH 7.4; 30 at pH 6.8; and 10 at pH 5.5, which fit well to the spectrophotometric data. The molecular solubility of the complex at pH 6.8–7.4 was estimated as 30 µmol/L.