Calcium Binding Ability of Recombinant Buffalo Regucalcin: A Study Using Circular Dichroism Spectroscopy

Regucalcin is a calcium regulating multifunctional protein reported to have many important functions like calcium homeostasis, anti-oxidative, anti-apoptotic and anti-cancerous functions. Although it is demonstrated as a calcium regulating protein, the calcium binding ability of regucalcin is still a controversy. The main reason for the controversy is that it lacks a typical EF hand motif which is common to most of the calcium binding proteins. Even though many studies reported regucalcin as a calcium binding protein, there are some studies reporting regucalcin as non-calcium binding also. In the present study, we investigated the calcium binding ability of recombinant buffalo regucalcin by assessing the secondary structural changes of the protein using circular dichroism spectroscopy after adding Ca²⁺ to the protein solution. Two types of calcium binding studies were done, one with different concentration of calcium chloride (0.5 mM CaCl₂, 1 mM CaCl₂, 2 mM CaCl₂) and other at different time interval (no incubation and 10 min incubation) after addition of calcium chloride. Significant structural changes were observed in both studies which prove the calcium binding ability of recombinant regucalcin. A constant increase in the α-helix (1.1% with 0.5 mM CaCl₂, 1.4% with 1 mM CaCl₂, 3.5% with 2 mM CaCl₂) and a decrease in β-sheets (78.5% with 0.5 mM CaCl₂, 77.4% with 1 mM CaCl₂, 75.7% with 2 mM CaCl₂) were observed with the increase in calcium chloride concentration. There was a rapid increase in α-helix and decrease in β-sheets immediately after addition of calcium chloride, which subsides after 10 min incubation.