Interactions in globular proteins with polyampholyte: coacervation route for protein separation

• Published in: RSC advances 2015, Vol.5 (18), p.13579-13589
• Main Authors: Pathak, Jyotsana, Rawat, Kamla, Aswal, V. K., Bohidar, H. B.
• Format: Article
• Language: English
• Subjects: Arrays; Binding energy; Fluorescence; Fretting; Polypeptides; Proteins; Separation; Spectroscopy
• ISSN: 2046-2069; 2046-2069
• DOI: 10.1039/C4RA13133A

In this work, we report exclusive separation of Bovine Serum Albumin (BSA) from a solution where this protein was present with β-lactoglobulin (β-Lg) in 1 : 0.75 (w/v) ratio at their common isoelectric pH (5 ± 0.02). A polyampholytic polypeptide Gelatin B (GB) also having the same pI was used to extract protein (BSA or β-Lg) molecules selectively from this solution through a process called complex coacervation. In our study, the protein-rich condensate, called coacervate, comprised of GB–BSA complexes while the supernatant mostly contained β-Lg molecules. For the separation of BSA from BSA–GB coacervate, we used ethyl alcohol, which removed the BSA to the supernatant. The differential binding affinity of BSA versus β-Lg to GB chains was established through fluorescence quenching and fluorescence resonance energy transfer (FRET) studies. The BSA–GB binding protocol followed a surface selective patch binding mechanism and these results were obtained from an array of experimental methods such as UV-vis and fluorescence spectroscopy, small angle neutron scattering (SANS), FTIR and circular dichroism spectroscopy. Herein, it is clearly established that selective coacervation at pI can be used as a method for protein separation.