Conformation change of trypsin induced by acteoside as studied using multiple spectroscopic and molecular docking methods

Figures & data

Figure 1. Molecular structure and ball-and-stick model of acteoside.

Figure 2. Ultraviolet visible absorption spectra of trypsin in absence and presence of acteoside at different concentrations. c(trypsin) = 2.50 × 10^–7 mol L⁻¹. Concentrations of acteoside from a to k are 0, 0.67, 1.33, 2.00, 2.67, 3.33, 4.00, 4.67, 5.33, 6.00, 6.67 (×10^–6 mol L⁻¹), respectively.

Figure 3. Positions of tryptophan (a) and tyrosine (b) residues in three-dimensional structure of trypsin.

Figure 4. Fluorescence spectra of 2.50 × 10^–7 mol L⁻¹ trypsin in absence and presence of acteoside in phosphate buffer (pH 8.0) at 310 K, c(acteoside) (a–k): 0, 0.67, 1.33, 2.00, 2.67, 3.33, 4.00, 4.67, 5.33, 6.00, 6.67 (×10^–6 mol L⁻¹), respectively.

Table 1. Quenching constants for acteoside-trypsin-system at different temperatures.

T (K)	Ksv (L mol^−1)	Kq (L mol^−1 s^−1)	R^2 a
288	1.96 × 10^5	7.00 × 10^13	0.992
298	2.38 × 10^5	8.50 × 10^13	0.996
310	2.56 × 10^5	9.14 × 10^13	0.998

^aThe correlation coefficient.

Table 2. Binding parameters of acteoside to trypsin at different temperatures.

T (K)	Ka (L mol^−1)	n	R^2 a
288	2.11 × 10^5	1.13	0.997
298	2.50 × 10^5	1.08	0.998
310	2.74 × 10^5	1.05	0.998

^aThe correlation coefficient.

Figure 5. (a) Van’t Hoff plots of lnK_a versus 1/T; (b) bar diagram showing thermodynamic parameters (T = 298 K) for binding of acteoside to trypsin.

Figure 6. Synchronous fluorescence spectra of trypsin with different concentrations of acteoside, (a) Δλ = 15 nm, (b) Δλ = 60 nm, pH = 8.0, c(trypsin) = 2.50 × 10^–7 mol L⁻¹, c(acteoside) (a–k): 0, 0.67, 1.33, 2.00, 2.67, 3.33, 4.00, 4.67, 5.33, 6.00, 6.67 (×10^–6 mol L⁻¹); (c) Quenching of trypsin synchronous fluorescence by acteoside, pH = 8.0, c(trypsin) = 2.50 × 10^–7 mol L⁻¹, x-axis represents the concentration of acteoside.

Table 3. Effect of acteoside on secondary structure content of trypsin.

Molar ratio of acteoside to trypsin	Secondary structural elements of trypsin
	α-Helix (%)	β-Sheet (%)	β-Turn (%)	Unorder (%)
0:1	4.80	39.60	21.60	34.00
3:1	5.60	38.50	22.10	33.60
7:1	6.90	36.30	23.30	33.40

Figure 7. Circular dichroism spectra of trypsin and trypsin–acteoside complexes. c(trypsin) = 2.50 × 10^–7 mol L⁻¹.

Figure 8. (a) Best docked conformation of trypsin–acteoside complex, acteoside is shown as stick model; (b) two-dimensional illustration (generated by LIGPLOT) of interaction of amino acid residues with acteoside.