Beta-thiopropionyl cytochromes c modified at lysyl residues: preparation and characterization of the monosubstituted horse cytochromes c.

The study focuses on the preparation and characterization of modified forms of horse cytochrome c, specifically beta-thiopropionyl derivatives. Through various analyses, it was found that these modifications did not significantly change the structure of the protein, except in certain cases. The modifications did, however, affect the protein’s electron transfer activity depending upon the position of the modification. It was also shown that these effects originate solely from electrostatic effects.

Preparation and characterization of beta-thiopropionyl cytochromes c

• The researchers prepared beta-thiopropionyl derivatives of horse cytochrome c, with a single modification at each of 18 different lysine epsilon-amino groups.
• These derivatives were prepared using sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate and then purified by high-pressure liquid chromatography, ensuring a homogenous sample.
• Characteristics of these derivatives were studied, including the location of the modification, reduction potentials, visible and NMR spectra, and the measurement of electron transfer activity with cytochrome-c oxidase.

Effects of the modifications on structure and activity

• The researchers found that, in general, the structure of the derivatives did not show significant changes upon modification, with the exception of the ferric forms of the derivatives modified at lysines 72, 73, and 79.
• It was observed that the electron transfer activity of the modified cytochromes c with bovine heart cytochrome-c oxidase was decreased. The extent of this decrease was dependent on the position of the modification.
• The researchers conducted aminoethylation, a secondary modification process that reverses the charge change. This process restored the electron transfer rate to what was observed with the unmodified cytochrome c, irrespective of the primary modification’s location.

Implications and future directions

• The results provide experimental evidence that changes in kinetics with physiological electron transfer partners resulting from modifications that cause a change of surface side chain charge are primarily due to electrostatic effects.
• Of the many chemically modified cytochromes c prepared till date, the singly substituted beta-thiopropionyl cytochromes c are deemed particularly useful. Their thiol group allows the attachment of any sulfhydryl-reactive reagent to a specific location on the protein surface, providing a crucial advantage for potential applications.