cytochrome-c-t and nile-red

Cytochrome C (Cyt c) is an electron transporting protein that resides within the inter-membrane space of the mitochondria. It plays a critical role as an electron carrier in the process of oxidative phosphorylation and production of cellular ATP. Cyt c is also involved in the apoptosis process and functions as a death messenger. On the other hand, it is well known that the metallo-pharmaceuticals such as palladium complex offer potential as anti-tumor agents to fight cancer. In order to identify the role of anticancer Pd complex in release of Cyt c from the biological membrane, an artificial monolayer was assembled which is able to adsorb Cyt c. A monolayer containing a mixture of two long chain thiols (mercapto-undecanoic acid and mercapto-undecanol) was self-assembled on the surface of a gold electrode. Due to the existence of both hydrophobic and electrostatic interactions between Cyt c and the assembled monolayer, this membrane could be considered as a rough analogue of the biological membrane to study the release of Cyt c by Pd complex. The electrochemical and spectroscopic studies showed that bounding of Pd complex to Cyt c causes a conformational change which leads to the release of Cyt c from the model membrane.

Topics: Adsorption; Animals; Antineoplastic Agents; Circular Dichroism; Cytochromes c; Electrochemical Techniques; Electrodes; Horses; Immobilized Proteins; Membranes, Artificial; Oxazines; Palladium; Protein Structure, Secondary; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Time Factors

The highly solvatochromic dye Nile red is used in conjunction with synchronous scan fluorescence spectroscopy to elucidate changes in the internal environment of cytochrome c, upon incorporation into differently modified sol-gel derived media. Nile red was first studied in a variety of solvents in order to quantify changes in polarity. Matrix modifications involved the addition of several silanes, intended to interact with any unreacted hydroxyl entities left from the matrix forming reaction, while polymers were used to help reduce shrinkage and modify the internal pore environment. Slight unfolding of the protein was observed on incorporation into the sol-gel derived media. During the aging process further changes were monitored by using difference synchronous scan fluorescence spectra and complementary measurements of catalytic activity, expressed as the initial velocity. Combining Nile red synchronous scan fluorescence with cytochrome c activity data lead to a method to elucidate effects linked to protein conformation and those related to the sol-gel derived host.

Topics: Catalysis; Cytochromes c; Fluorescent Dyes; Gels; Oxazines; Peroxidase; Phase Transition; Protein Conformation; Silanes; Spectrometry, Fluorescence