A New Functional Model for Prediction of Chaperone Activity of the Recombinant M. tb Acr (α-Crystallin) Using Insulin as Substrate

Figure 3

Chaperone activity of (H) and (G) samples at 37°C and 60°C. (a) Chaperone activity of (H) at 60°C: insulin (1 mg/ml) was used for the assay and aggregation measured at 360 nm over 20 mins by adding 25 (mM DTT). Acr was used at 4 different concentrations: 5.5 μM, 11 μM, 18.75 μM, and 37.5 μM. (b) Chaperone activity of (H) at 37°C: insulin (1 mg/ml) was used for the assay, and aggregation was measured at 360 nm over 30 mins by adding 25 mM DTT. Acr was used at 4 different concentrations of 5.5, 11, 18, and 30 μΜ. (c) Chaperone activity of (G) pool 1: run 1: insulin 1 (mg/ml) was used for the assay and aggregation measured at 360 nm over 30 mins by adding 25 mM DTT. Acr was used at concentrations of 6, 9, and 12 μM. (d) Chaperone activity of (G) pool 2, run 1: insulin (1 mg/ml) was used for the assay and aggregation measured at 360 nm over 30 mins by adding 25 mM DTT. Acr was used at concentrations of 2, 4, and 6 μM. (e) Chaperone activity of (G) run 2: insulin (1 mg/ml) was used for the assay and aggregation measured at 360 nm over 30 mins by adding 25 mM DTT. Acr was used at concentrations of 2, 3, 4, and 8 μM. (f) Consolidated curve of Figures 3(a) to 3(e): the mole ratios of Acr to insulin obtained from Figures 3(a) to 3(e) (Table 2) were replotted versus % inhibition and numerical best fits with polynomials plotted. (g) Chaperone activity of (H) at 37°C after pre-heat treatment at 37°C and 60°C: insulin (1 mg/ml) was used for the assay and aggregation measured at 360 nm over 30 mins by adding 25 mM DTT. Acr was used at a concentration of 11 μM, with no pre-heat treatment and with pre-heat treatment at 37°C and 60°C. (h) Pre-heat treatment studies and chaperone activity of (G): insulin (1 mg/ml) was used to induce aggregation with 25 mM DTT and aggregation measured at 360 nm over 30 mins. The concentration of Acr used was 1 μM, and pre-heat treatment was carried out at 37°C, 60°C, and 70°C for 15 minutes.

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