Gel tracking dye “Bromophenol blue” promote amorphous aggregation in insulin

Abstract

Protein aggregation is implicated in different human diseases. It also makes the protein less desirable candidate for industry as they exhibit reduced biological activity. Proteins tend to aggregate under different conditions which generally depends on the physico-chemical property of environment. We have examined the effects of bromophenol blue dye (BPB) on the aggregation propensity of insulin protein at pH 2.0 and 7.4. The results showed that the BPB dye is inducing amorphous aggregation in insulin protein at pH 2.0 and aggregation was not recorded at pH 7.4. To characterize the aggregation caused by BPB dye, we used various spectroscopic techniques (turbidity, light scattering, and circular dichroism (CD) techniques. Turbidity and light scattering data indicated that the insulin form aggregates in the presence of 0.05–0.5 mM BPB dye concentrations at pH 2.0. The kinetics data reveled that the BPB dye induced aggregation kinetics is very fast and aggregation reaction completed within 150 s. The kinetics data also suggest that the aggregation is dose-dependent and varies with BPB dye concentrations. The nature of BPB dye-induced insulin aggregates was characterized by far-UV CD measurements and found that the aggregated samples have lost the CD signals without the change in spectrum position which confirm that the aggregates are amorphous in nature. The transmission electron microscopy (TEM) imaging measurements were suggesting that the aggregates formed in the presence of BPB dye is amorphous in nature. The salts are not interfering in BPB dye-induced aggregates process. However, surfactants i.e., CTAB and SDS is solubilizing the BPB dye-induced amorphous aggregates and CTAB is very effective compared to SDS. Very low concentrations of CTAB solubilized dye-induced insulin aggregation.