Chymotrypsin Adsorption on Montmorillonite: Enzymatic Activity and Kinetic FTIR Structural Analysis

Abstract

Soils have a large solid surface area and high adsorptive capacities. To determine if structural and solvation changes induced by adsorption on clays are related to changes in enzyme activity, alpha-chymotrypsin adsorbed on a phyllosilicate with an electronegative surface (montmorillonite) has been studied by transmission FTIR spectroscopy. A comparison of the pH-dependent structural changes for the solution and adsorbed states probes the electrostatic origin of the adsorption. In the pD range 4.5-10, adsorption only perturbs some peripheral domains of the protein compared to the solution. Secondary structure unfolding affects about 15-20 peptide units. Parts of these domains become hydrated and others entail some self-association. However, the inactivation of the catalytic activity of the adsorbed enzyme in the 5-7 pD range is due less to these structural changes than to steric hindrance when three essential imino/amino functions, located close to the entrance of the catalytic cavity (His-40 and -57 residues and Ala-149 end chain residue), are oriented toward the negatively charged mineral surface. When these functions lose their positive charge, the orientation of the adsorbed enzyme is changed and an activity similar to that in solution at equivalent pH is recovered. This result is of fundamental interest in all fields of research where enzymatic activity is monitored using reversible adsorption procedures. Copyright 1999 Academic Press.

Conformational Changes of Bovine Serum Albumin Induced by Adsorption on Different Clay Surfaces: FTIR Analysis

“…In order to shift the spectral absorption domain of water molecules bound to the polypeptide backbone of BSA out of the Amide I and II spectral range, all samples were prepared in 2 H 2 O medium (15,(21)(22)(23)(24) and from 6 min (minimum time for sampling) to 6 h. Spectra of BSA in solution or adsorbed on the clay surfaces were obtained from the difference spectra (BSA + buffer) − (buffer) and (BSA + clay + buffer) − (clay + buffer), respectively ( Fig. 1).…”

“…The solid phase in soils has a large specific surface area and high adsorptive capacities for proteins (2)(3)(4)(5)(6). The interaction of enzymes with soil mineral surfaces modifies their catalytic activity (7)(8)(9)(10)(11)(12)(13)(14) because of solvation and structural changes which occur upon adsorption (15). Electrostatic, hydrophobic, and hydrophilic interactions between proteins and mineral surfaces induce structural changes on the adsorbed proteins (16)(17)(18)(19)(20)(21)(22)(23)(24).…”

“…Various spectroscopic techniques (NMR, fluorescence, and circular dichroism) are currently used to study protein structural conformations in solution. FTIR-spectroscopy is best suited to compare secondary structures for proteins in solution or adsorbed on a solid support (15,(21)(22)(23)(24). In this way, we compared the conformational changes induced on BSA by adsorption, either on the hydrophobic and neutral talc, or on the electronegative montmorillonite.…”

Conformational Reorientation of Immunoglobulin G During Nonspecific Interaction with Surfaces

“…Proteins with a high internal structural stability, the so-called "hard" proteins such as a-chymotrypsin, lysozyme, ribonuclease, or blactoglobulin, in the absence of charges do not favorably adsorb onto hydrophilic surfaces, while they preferably tend to adsorb onto hydrophobic surfaces. [2][3][4][5] Proteins with a low structural stability, the so-called "soft" proteins such as bovine serum albumin, immunoglobulin G (IgG), or a-lactalbumin, in the absence of charges generally tend to adsorb onto a variety of surfaces. In these cases a conformational reorganization is discussed but the literature is not clear on this point.…”

The Influence of Surface Composition of Nanoparticles on their Interactions with Serum Albumin

“…[15] These structural changes upon adsorption to a surface may result in the loss of biological activity and hence activation of immune response. [16,17] Furthermore, the adsorption of protein molecules on the NP surface changes the surface functionality and hence may strongly influence the translocation behaviour in biological systems. [18,19] However, the mechanistic steps involved in these processes are still far from being understood.…”