Journal of Chromatography & Separation Techniques
Open Access
ISSN: 2157-7064
ISSN: 2157-7064
Natalia Y Marchenko, Victor V Marchenkov, Ivan A Kashparov and Gennady V Semisotnov
Natalia Y Marchenko, Victor V Marchenkov, Ivan A Kashparov and Gennady V Semisotnov
Posters & Accepted Abstracts: J Chromatogr Sep Tech
Statement of the Problem: Molecular chaperones are a large group of proteins (presented in both prokaryotes and eukaryotes) that assist other proteins in their folding. The chaperones play important roles under normal conditions as well as in stress response and in disease development. Thus, detection of changes in chaperone content in cells under different conditions is important. Methodology & Theoretical Orientation: Tight interaction with denatured proteins is a general property of molecular chaperones. That is why affinity chromatography on a base of denatured proteins is a perspective method for chaperone analysis. Affinity sorbents were made on the base of Sepharose and a small target protein (e.g. lysozyme) covalently attached to it. The experiments were carried out in the conditions which are native for chaperones and denaturing for the target protein (pH 7.5 and presence of dithiothreitol). Findings: Cell lysates of bacteria (Escherichia coli, Thermus thermophilus, and Yersinia pseudotuberculosis), archaea (Halorubrum lacusprofundi) as well as lysate of rat liver mitochondria (in normal conditions and after oxidative stress) and human blood serum were analyzed. It is shown that Hsp60 chaperonins were the main proteins that bind to the affinity sorbent (except for the case of blood serum where albumin was the main bound protein). It is demonstrated that, apart from Hsp60, other proteins with a molecular weight of about 100, 50, 40, and 20 kDa are able to interact with denatured lysozyme. Conclusion & Significance: We are able to evaluate the quantity of bound proteins and to detect stress changes in protein content. So, this approach can be useful for studying various diseases connected with changes in chaperone content.
Natalia Y Marchenko has completed her PhD from Kharkiv National University (Ukraine) and Institute of Protein Research, RAS (Russia). Her research is focused on protein-protein interactions, specifically on studying chaperone-protein interaction (especially, chaperonin GroEL from Escherichia coli) in various conditions using different physical and chemical methods, in particular, affinity chromatography. The method of affinity chromatography on a base of denatured proteins was developed and validated with her direct participation, and the application of this method for research and medical purposes is her main goal. She has published 10 papers in peer-reviewed journals.
Email: lita@phys.protres.ru