Presenting B-DNA as macromolecular crowding agent to improve efficacy of cytochrome c under various stresses

Abstract

Existence of numerous biomolecules results in biological fluids to be extremely crowded. Thus, Macromolecular crowding is an essential phenomenon to sustain active conformation of proteins in biological systems. Herein, double helical deoxyribonucleic acid (B-DNA) is presented for the first time as a biomacromolecular crowding system for sustainable packaging of cytochrome c (Cyt C). The peroxidase activity of Cyt C was investigated in the presence of various concentrations of B-DNA (from salmon milt). At an optimized concentration of 0.125 mg/mL B-DNA, an 11-fold higher catalytic activity was found than in native Cyt C with improved stability. Molecular docking and spectroscopic analyses revealed that electrostatic and H-bonding are the main interactions between DNA and Cyt C that affect the structural stability and activity of the protein. Moreover, the catalytic activity and stability of the protein were further investigated in the presence of severe process conditions by UV-visible, circular dichroism, and Fourier-transform infrared spectroscopies. Molecularly crowded Cyt C showed significantly higher activity and stability under severe environments such as high temperature (110 °C), oxidative stress, high pH (pH 10) and biological (trypsin) and chemical denaturants (urea) compared to bare Cyt C. The observed results support the suitability of DNA-based macromolecular crowding media as a viable and effective stabilizer of proteins against multiple stresses.

Keywords: B-DNA; Biocatalysis; Cytochrome c; Macromolecular crowding; Multiple stresses; Protein packaging.