3.4. Insulin Aggregation Assay

The chaperone activities of non-gel-filtered and gel- filtered Acr samples were checked using the 1,4- dithiothreitol (DTT) induced aggregation assay using 1.0 mg/ml insulin in buffer (20 mM Tris pH 7.0, 100 mM NaCl and 5% glycerol) at 60°C at 360 nm over 20 mins. Insulin R injection (BIOCON, India) was concentrated from a stock of 0.14 mg/ml to 1.0 mg/ml using 3kDa concentrators from Amicon (Millipore). Insulin in buffer without DTT and insulin with 25 µM DTT were used as controls. All the components except DTT were added to all these samples, the absorbance values were auto zeroed, baseline adjustment was done from 350 nm to 370 nm and 25 mM DTT added to start aggregation. Acr was used at 4 different concentrations of 12, 22, 39 and 44 µM in 3 different reaction volumes of 250, 300 and 400 µl. Likewise, chaperone activity was carried out by preventing DTT induced aggregation of insulin B chain concentration at 118 μM at the wavelength of 360 nm over 30 mins at 37°C. Aggregation was started by adding 25 mM DTT and the absorbance recorded to look for inhibition of aggregation on addition of Acr. Two types of samples were used for the enzyme assays: (A) Non gel-filtered and (B) gel-filtered.

The activity of A samples was checked at concentrations of 6 and 24 μM and B samples at 1, 3 and 4 μM using the assay buffer 20 mM Tris pH 7.0, 100 mM NaCl, 5% glycerol.