Physicochemical characterization of an aspin (rBm-33) from a filarial parasite Brugia malayi against the important human aspartic proteases

Figures & data

Figure 1.  Thermal stability expressed through the CD spectrum of rBm-33 heat treated to (95°C). Thermal stability of rBm-33 on pepsin inhibition activity.*The percentage activity was calculated assuming the maximum activity at 37°C is 100% (A). CD spectrum of heat treated (95°C) rBm-33 diluted with water at final concentration of 0.1 mg/ml at pH 6.9 (B).

Figure 2.  Immunoblot to show the removal of N-terminal his-tag from rBm-33 after the incubation with pepsin. M: protein molecular-weight marker; Lane 1: flow-through of IMAC (Ni²⁺-IDA) experiment containing glutaraldehyde cross linked rBm-33-pepsin complex probed with anti Bm-33 antisera; Lane 2: same flow-through probed with anti his-tag antibody.

Figure 3.  Inhibition activity of rBm-33 against human aspartic proteases using UV spectroscopy. CA: only casein, P: pepsin, RE: renin, CE: cathepsin-E, CD: cathepsin-D, PS: pepstatin Bm: rBm-33. All the values represent the mean of six different experiments ± SD

Figure 4.  Kinetics of pepsin inhibition by rBm-33. The assay was performed with the fixed quantity of pepsin (5 mm) and varying concentrations of rBm-33 in absence and in the presence of (1 mM, 2.5 mM and 5 mM, respectively). Substrate-velocity curve (A) and Lineweaver-Burk plot (1/V vs. 1/S) (B) are shown indicating competitive inhibition of pepsin by rBm-33 (Ki = 2.5 (± 0.8) nM). P: pepsin (5 mM), P+ [B 1 mM]: pepsin+rBm-33 (1 mM), P+ [B 2.5 mM]: pepsin+rBm-33 (2.5 mM), P+ [B 5 mM]: pepsin+rBm-33 (5 mM). Assay was carried out in triplicates and the kinetic constants were determined using Graphpad Prism 2.0 (San Diego, CA, USA).

Table 1.  Association constants (Kb) and thermodynamic parameters for the binding of important human aspartic proteases to rBm-33.

Protein (human aspartic proteases)	T (K)	n	10^3 * Kb (M^−1)	−ΔGb°(kJ mol^−1)	−ΔHb°(kJ mol^−1)	−ΔSb°(J mol^−1 K^−1)
Pepsin	298	0.92 (± 0.01)	10.23 (± 0.3)	21.19	50.99 (± 0.3)	100.02(± 1.3)
Renin	298	1.12 (± 0.04)	10.01 (± 0.2)	21.17	49.02 (± 0.5)	93.45(± 2.1)
Cathepsin-E	298	1.13 (± 0.04)	8.24 (± 0.1)	20.31	47.23 (± 0.1)	90.34 (± 1.8)
Cathepsin-D	298	1.73 (± 0.04)	6.52 (± 0.4)	20.04	46.07 (± 0.7)	87.34 (± 2.7)

*Values shown are the average values from two independent titration experiments.

K_b, binding constant; n, number of binding sites; T (K), temperature; ΔG_b, Gibbs free energy; ΔH_b, enthalpy of binding; ΔS_b, entropy of binding.

Figure 5.  Iso-thermal calorimetric titration of rBm-33 (refolded) with cathepsin-E (C). Raw data obtained from 50 automatic injections of 5 µl aliquots of 0.036 mM cathepsin-E into 0.036 mM of rBm-33 (refolded) at 298 K (c). Non-linear least squares fit for the cathepsin-E and rBm-33 (refolded) (c*). Iso-thermal calorimetric titration of rBm-33 (refolded) with cathepsin-D (D). Raw data obtained from 50 automatic injections of 5 µl aliquots of 0.036 mM cathepsin-D into 0.036 mM of rBm-33 (refolded) at 298 K (d). Non-linear least squares fit for the cathepsin-D and rBm-33 (refolded) (d*).