Change in gastric-lipase adsorption on lipid layer by stigmasterols

1 Introduction

Gastric-lipase (GL) is a soluble enzyme responsible for whole lipolysis in infant and for (10–30) % in adult [1]. GL is stable at pH values between 2 and 7, and active without colipase. Although GL has a unique substrate specificity, its structure includes common characteristics of α/β-hydrolase fold and a catalytic triad as a member of a lipase family [2]. The active site of GL is hydrophobic and located inside of the enzyme structure [3]. A substrate is able to access to the active site of GL after lid opening, which occurs when the substrate contacts with the lid covering the site [4]. Furthermore, the opening induces a hydrophobic ring to interact with the substrate, and the interaction generates the interfacial adsorption of GL [5]. The adsorption leads to the hydrolysis of triacylglycerols, which produces fatty acids and diacylglycerols in an optimal way at pH 4–5.4 [6]. The hydrolysis has been found to be influenced by interfacial tension and curvature [7, 8].

Lipid layer has been not only recognized as a bio-reagent carrier but also as a bio-membrane model [9], [10], [11], [12], [13], [14], [15]. The layers have either one (called mono-) or two (bi-) layers, which have spherical and planar shapes [16], [17], [18], [19]. The spherical layers are dispersed as an emulsion in a solution. The planar monolayers are on a solid substrate or at water-oil interface, while the bilayers are usually on the substrate. The layers are composed of molecules in an arrangement that represent a specific phase – liquid-condensed or liquid-expanded phase, which affects the lateral movement of the molecules and the molecular interaction with external agents [16, 20]. These behaviors are eventually involved in the cellular process [21]. Phytosterols, components of plant cell membranes, adjust the phase of the layer by reorganizing the arrangement of the lipid headgroups [22].

Surface plasmon resonance (SPR) has been used to monitor physical or chemical adsorptions on planar surface in real-time [23]. The characteristics of the formation and the specific bindings were found for the lipid layers [24, 25]. The formation was generally performed on around 50 nm thickness film surface of zero-valent metal such as Au to generate evanescent light above the surface [26, 27]. The specific bindings between proteins and their receptors reconstituted in lipid layers have been investigated in terms of kinetics and equilibrium. In this study, we aim to investigate the GL binding to the lipid layer of specific phase according to the ratio of stigmasterol, a phytosterol, of lipid using SPR.

2 Experimental

Dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) were purchased from Sigma Aldrich (St. Louis, MO, USA). Stigmasterol, a phytosterol that maintains the structure and physiology of cell membranes in numerous plants, was also acquired from Sigma Aldrich [28]. Gastric-lipase (GL) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). All reagents were used without further purification. Either DPPC or DOPC was dissolved in 2.5 ml chloroform along with a desired ratio of stigmasterol to the lipid (0:100, 5:95. 10:90, 15:95). The evaporation of the solvent from the solution via a nitrogen stream led to the films formed on a glass vial. Then, the films were hydrated with 5 ml aqueous solution of 10 mM acetate and 100 mM NaCl at pH 5 to obtain a suspension. This suspension was changed to a solution of small unilamellar vesicles (SUVs) by extruding through 100 nm pore polycarbonate filter. The diameter of SUVs was measured using dynamic light-scattering (Otsuka Electronics Co. Ltd, Osaka, Japan).

The formation of the lipid layer on a hydrophobic surface was monitored using SPR as the procedures published previously [11]. An SPR sensor chip HPA was acquired from BIAcore SA (Little Chalfont, Buckinghamshire, UK). The HPA was inserted into an SPR equipment (BIAcore 3000, Little Chalfont, Buckinghamshire, UK), and the hydrophobic surface of the HPA was exposed to the 2 μL/min flow of the solution containing the vesicles. The change in the optical signal was monitored in real-time. After the plateau of the signal was reached, the pure buffer was injected to the surface to remove the extra vesicles. After little change in the signal was confirmed, (5, 10, 20, 30, and 40) nM GL in the buffer solution was added to the lipid layer. Therefore, the behavior of the signal after the GL addition was observed according to the ratio of stigmasterol of lipid.

3 Results and discussion

Before the addition of the vesicle solution to the HPA surface, the diameter of the vesicles was confirmed to be (100 ± 50) nm [29]. After the addition, the change in the optical signal was observed (Figure 1). Each region corresponds to a physical phenomenon. Region (1) was prior to the addition, region (2) was on-going of the addition, and region (3) was no more addition. The change in the signal can be induced by the changes in the refractive index of the solution and the adsorption on the surface. The signal of region (2) occurred due to the occupation of the vesicle solution around the surface and the adsorption of the lipid layer on the surface. The boundary between region (2) and (3) corresponded to the exchange from the vesicle solution to the buffer solution used for region (1). Therefore, the amount of the layer adsorbed on the surface was acquired from the difference between the plateaus of region (1) and (3). The value of this difference was about (1350–1800) RU (response units), which was similar with the results previously published before [30].

Figure 1:

Optical signal of monolayer formation on a hydrophobic surface; (a) DOPC and (b) DPPC; region (1) – prior to the vesicle addition, region (2) – on-going of the addition, and region (3) – no more addition.

Figure 1a shows the results with DOPC, while Figure 1b shows the results with DPPC. A clear difference between a) and b) can be found. The amplitude of the signal change is smaller in DOPC than in DPPC. For DOPC only, the change in the signal is hardly dependent on the ratio of the stigmasterol to the lipid. For DPPC, the ratio decreased to 15:85. Since the smaller amplitude is in DOPC, the addition of stigmasterol was believed to transform the phase (condensed liquid-phase) of DPPC layer closer to the phase (expanded liquid-phase) of DOPC layer. The signals between 15:85 and 20:80 of DPPC are almost identical to each other. Considering the molecular size, it was assumed that one stigmasterol was surrounded with 5–6 lipid molecules [18, 31]. This estimation corresponded to the molar ratio of 15:85. Therefore, at a ratio greater than 15:85, the effect of the stigmasterol on the DPPC was already saturated. In other words, the phase of DPPC at 15:85 was close to that of DOPC.

The adsorption behavior of GL showed a similar trend with the lipid formation. The adsorption amount was acquired from the difference between the plateaus, as described for the lipid-layer formation above. On the lipid layer made with pure DPPC, little adsorption was observed. The adsorption increased with the increase in the ratio of stigmasterol to lipid. On the DOPC layer of the expanded liquid-phase, the most adsorption seemed to occur (Figure 2a). Figure 2b shows that the characteristics of the GL adsorption depended on the ratio of stigmasterol to DPPC. Again, the curve for the ratio 20:80 is almost identical to that for the 15:85. This identical behavior was consistent with the assumption of lipid formation. The adsorption of GL was indistinguishable between DOPC and DPPC at the ratio 15:85. For proteins such as GL, 1 RU was converted to 1 pg mm⁻² [32]. Thus, the surface density of the adsorbed GL was obtained to be (1040, 700, and 350) pg mm⁻² and appears to be proportional to the decrease of the ratio 15:85. This analysis may be interpreted as the increase in the ratio leading the decrease in the GL adsorption sites. Considering the relative amount of the absorption, the relative fraction of the available sites for the adsorption was estimated. Based on DPPC entirely affected by stigmasterol at the ratio 15:85, the fraction of the stigmasterol-dependent DPPC was 0.33, 0.67, and 1.00 for the ratios 15:85, 10:90, and 5:95. Comparison of these fractions to the surface densities indicated that the fraction was proportional to the density.

Figure 2:

Optical signal of gastric-lipase adsorption on a lipid layer; (a) DOPC and (b) DPPC; Solid lines are fittings.

Using SPR data, the equilibrium constant of the GL to the lipid layer was estimated. For the estimation of the equilibrium, Langmuir adsorption isotherm was considered. To fit the isotherm to the experimental data linearly, the data were converted to the reciprocals of the coverage fraction and the GL concentration. The fitting results are shown in Figure 3. The reciprocals of the coverage fraction behave linearly to the reciprocals of the GL concentration and are distributed in the range between two curves, which means that each curve represents the upper and the lower limit. For the limit cases, the equilibrium constant was 1 × 10¹³ M⁻¹ and 2 × 10¹³ M⁻¹, respectively. In other words, the constant was in the range of these values. Since the saturation of the GL coverages were observed with the remains of the unbound GL and the available-sites, it might be argued that the reverse reaction proceeded. However, these phenomena seemed to be caused by the entropic effect of the GL distribution, and little by the reverse reaction. The high value of the equilibrium constant indicated that the reverse-reaction was almost negligible.

Figure 3:

Linear relation between reciprocal of coverage fraction and reciprocal concentration. The linear relation is located in the range of the limits.

Furthermore, kinetic analysis was possible because the optical signal for the adsorption was monitored in real-time. The data were fitted with the linear relation between the adsorption rate for the available site for the adsorption. Since the relation included the rate constant, the rate constants of the adsorptions were acquired from the fittings. The solid lines in Figure 2b correspond to the fitting of the signals. The rate constants were 0.002 s⁻¹, 0.005 s⁻¹, and 0.009 s⁻¹ for the ratios 5:95, 10:90, and 15:85 of DPPC, respectively, and the regression coefficients ranged from 0.95 to 0.98. For the layer of the ratio 0:100 of DPPC, little adsorption was required for the fitting. The interesting observation was the increase in the constant with respect to the ratio increase up to the ratio 15:85. These results were interpreted as a good proof of the affinity of GL for the lipid layer. The higher constant corresponded to the more liquid-expanded phase, which was preferred for the GL adsorption similarly to the equilibrium data suggested above.

4 Conclusions

In this study, the GL binding to the lipid layer was investigated for the phase of the layer adjusted with the ratio of stigmasterol to the lipid using SPR. While the layer was formed on the hydrophobic surface, more stigmasterol led to higher surface density only in the DPPC layer. The addition of stigmasterol was believed to transit the phase (condensed liquid-phase) of DPPC layer closer to the phase (expanded liquid-phase) of DOPC layer. At a ratio greater than 15:85, the effect of the stigmasterol on the DPPC was saturated. The adsorption behavior of GL showed the similar trend with the lipid formation. The adsorption increased with the increase in the ratio of stigmasterol to lipid up to 15:85. On the DOPC layer of the expanded liquid-phase, the most adsorption seemed to occur and was indistinguishable from that in the DPPC layer with a ratio of 15:85. The surface density of the adsorbed GL was interpreted into the fraction of the stigmasterol-dependent DPPC, 0.33, 0.67, and 1.00 for the ratios 9:95, 10:90, and 0:100 of DPPC. Furthermore, the equilibrium constant was between 1 × 10¹³ M⁻¹ and 2 × 10¹³ M⁻¹ and the kinetics of the adsorption was characterized by the increase in the adsorption rate constant with the ratio increase up to 15:85. This observation may be related to the elucidation of the lipolysis activity.