Mechanism Of Protein Denaturation Research Articles

Role of water in the pressure-induced unfolding of Bovine Pancreatic Trypsin Inhibitor (BPTI) protein: A molecular dynamics study

The mechanism of protein denaturation under pressure is still an active area of research. Using extensive molecular dynamics simulations, for a partially unfolded structures of BPTI protein, we have studied the role of water in the unfolding of proteins at pressures of 0.001, 5, and 10 kbar at 298K. Our results show that high enough pressure leads to insertion of water into the protein’s interior, causing structural perturbation to proteins, facilitated by a decrease in the intact time of intra-protein hydrogen bond acceptor–donor distances or an increase in the residence times of shell waters or by a combination of both.

Analysis of protein denaturation, and chemical visualisation, of frozen grass carp surimi containing soluble soybean polysaccharides

SummaryThe mechanism of protein denaturation of frozen surimi enriched with soluble soybean polysaccharides (SSPS) was investigated. Near‐infrared (NIR) and hyperspectral imaging (HSI) technology were used to predict protein denaturation. The fresh grass carp surimi was divided into four groups with SSPS additions of 0%, 1%, 3% and 5%, respectively, which were frozen and stored at −18 °C. Samples were examined after 0, 1, 2, 4 and 8 weeks, for salt‐soluble protein content, total SH, NIR hyperspectral image and Raman spectrum features. The results showed that addition of SSPS decelerated protein degradation. After 8 weeks of storage, the 5% SSPS addition maintained the highest salt‐soluble protein content, while for total SH, 3% and 5% SSPS addition had similar effect. Raman spectra illustrated that SSPS had the ability of maintaining an α‐helix content, as well as decreasing the exposure of polar groups, and reducing the oxidation of SH group into disulphide bonds. NIR spectra showed that the overall reflectance of frozen surimi increased with the increase in SSPS. Two partial least squares regression (PLSR) models were established after pretreatment of HSI spectral statistics and selection of characteristic wavelengths. The distribution maps were finally generated based on the simplified PLSR models.

Free Radicals and ROS Induce Protein Denaturation by UV Photostability Assay.

Oxidative stress, photo-oxidation, and photosensitizers are activated by UV irradiation and are affecting the photo-stability of proteins. Understanding the mechanisms that govern protein photo-stability is essential for its control enabling enhancement or reduction. Currently, two major mechanisms for protein denaturation induced by UV irradiation are available: one generated by the local heating of water molecules bound to the proteins and the other by the formation of reactive free radicals. To discriminate which is the likely or dominant mechanism we have studied the effects of thermal and UV denaturation of aqueous protein solutions with and without DHR-123 as fluorogenic probe using circular dichroism (CD), synchrotron radiation circular dichroism (SRCD), and fluorescence spectroscopies. The results indicated that the mechanism of protein denaturation induced by VUV and far-UV irradiation were mediated by the formation of reactive free radicals (FR) and reactive oxygen species (ROS). The development at Diamond B23 beamline for SRCD of a novel protein UV photo-stability assay based on consecutive repeated CD measurements in the far-UV (180–250 nm) region has been successfully used to assess and characterize the photo-stability of protein formulations and ligand binding interactions, in particular for ligand molecules devoid of significant UV absorption.

The shift in urea orientation at protein surfaces at low pH is compatible with a direct mechanism of protein denaturation.

Surface-specific spectroscopic data has shown that urea undergoes a shift in orientation at protein surfaces in acidic media. Since urea denatures proteins at a wide range of pHs, the variable chemical nature of protein-urea interactions has been used to support an indirect mechanism of urea-induced denaturation. Here, we use molecular dynamics simulations, minimum-distance distribution functions (MDDFs), and hydrogen-bond analysis, to characterize the interactions of urea with proteins at neutral and low pH, as defined by the protonation state of acidic residues. We obtain the expected preferential solvation by urea and dehydration, consistently with urea-induced denaturation, while the MDDFs allow for a solvent-shell perspective of protein-urea interactions. The distribution functions are decomposed into atomic contributions to show that there is indeed a shift in the orientation of urea molecules in the vicinity of acidic side-chains, as shown by the experimental spectroscopic data. However, this effect is local, and the interactions of urea with the other side chains and with the protein backbone are essentially unaffected at low pH. Therefore, hydrophobic solvation and urea-backbone hydrogen bonds can play a role in a direct mechanism of urea-induced protein denaturation without contradicting the observed variations in the chemical nature of protein-urea interactions as a function of the acidity of the solution.

The molecular mechanism of protein denaturation in supercritical CO2: The role of exposed lysine residues is explored

Although there are many interests to use proteins in supercritical CO2, protein destabilization in this solvent is the major drawback. This is because of the non-natural CO2 environment which imposes essential instability on the protein structure. Due to the limitations of the experimental techniques to obtain in situ molecular levels information about protein microstructure at high pressures of the supercritical state, the molecular level reasons of the protein destabilization remains controversial and relatively unknown in supercritical CO2. While there is a consensus about the probable roles of surface lysine residues in destabilization of proteins in supercritical CO2, no clear mechanism is presented about the way that these amino acids impose instabilities on the protein conformation. Here, using molecular dynamics simulations, we extensively examined the structural behavior of the surface lysine residues of chymotrypsin inhibitor 2 as a model protein in supercritical CO2. In this way, a new molecular mechanism is proposed which states that the inappropriate interactions of polar side chain of lysine residues with the non-polar nature of supercritical CO2 enforce these exposed residues to return from the solvent bulk to the protein exterior and also to form new non-native hydrogen bonds with the acidic residues such as glutamic acid. This induces some surface regions to be very labile and dynamics. These regions are the starting sequence in the process of protein denaturation in supercritical CO2. Denaturation is extended by collapsing the structure due to the formation of multiple non-native interactions between different residues. To evaluate the proposed mechanism, surface exposed lysine residues were replaced to leucine amino acids with the hydrophobic side chains. Interestingly, the protein remains intact by this replacement even at 200 bar in supercritical CO2. However, replacing of lysine residues by negatively charged glutamic acid and also by glycine residues cannot prevent protein denaturation process. These molecular levels explorations not only address some unknown aspects of protein denaturation mechanism in supercritical CO2, but it also can essentially help to design new CO2-tolerant proteins which can be used in popular processes such as biocatalysis in supercritical CO2.

A detailed analysis of the influence of β-cyclodextrin derivates on the thermal denaturation of lysozyme

The influence of HPβCD on the thermal denaturation of lysozyme was analyzed mainly from microcalorimetry and Raman investigations carried out in the molecular fingerprint and the low-frequency regions. It was shown that Raman spectroscopy investigations performed on a wide spectral range give the opportunity to describe the influence of HPβCD on the mechanism of protein denaturation. Using D2O as solvent allowed us to show that HPβCD mainly destabilizes the tertiary structure of lysozyme by enhancing the protein flexibility and thus inducing the destabilization of the secondary structure. Principal components analysis (PCA) was used for spectra treatment, providing important information about inclusion complex formation between protein hydrophobic residues and CDs molecules. Combining PCA and classical technics (curve fitting) of data analysis allowed a better understanding of the influence of HPβCD on the protein denaturation that seems to be related to the CDs capacity to form inclusion complex. It was observed that these interactions prevent the formation of new strong H-bonds between β-sheet structures thereby inhibiting protein aggregation. This study reveals that CDs are promising systems for inhibiting protein aggregation without protein denaturation, only if designing derivative CDs is carefully controlled.

Atomic scale insights into urea-peptide interactions in solution.

The mechanism by which proteins are denatured by urea is still not well understood, especially on the atomic scale where these interactions occur in vivo. In this study, the structure of the peptide GPG has been investigated in aqueous urea solutions in order to understand the combination of roles that both urea and water play in protein unfolding. Using a combination of neutron diffraction enhanced by isotopic substitution and computer simulations, it was found, in opposition with previous simulations studies, that urea is preferred over water around polar and charged portions of the peptides. Further, it appears that while urea directly replaces water around the nitrogen groups on GPG that urea and water occupy different positions around the peptide bond carbonyl groups. This suggests that urea may in fact weaken the peptide bond, disrupting the peptide backbone, thus ultimately causing denaturation.

Mechanism of Protein Denaturation: Partial Unfolding of the P22 Coat Protein I-Domain by Urea Binding

The I-domain is an insertion domain of the bacteriophage P22 coat protein that drives rapid folding and accounts for over half of the stability of the full-length protein. We sought to determine the role of hydrogen bonds (H-bonds) in the unfolding of the I-domain by examining 3JNC’ couplings transmitted through H-bonds, the temperature and urea-concentration dependence of 1HN and 15N chemical shifts, and native-state hydrogen exchange at urea concentrations where the domain is predominantly folded. The native-state hydrogen-exchange data suggest that the six-stranded β-barrel core of the I-domain is more stable against unfolding than a smaller subdomain comprised of a short α-helix and three-stranded β-sheet. H-bonds, separately determined from solvent protection and 3JNC’ H-bond couplings, are identified with an accuracy of 90% by 1HN temperature coefficients. The accuracy is improved to 95% when 15N temperature coefficients are also included. In contrast, the urea dependence of 1HN and 15N chemical shifts is unrelated to H-bonding. The protein segments with the largest chemical-shift changes in the presence of urea show curved or sigmoidal titration curves suggestive of direct urea binding. Nuclear Overhauser effects to urea for these segments are also consistent with specific urea-binding sites in the I-domain. Taken together, the results support a mechanism of urea unfolding in which denaturant binds to distinct sites in the I-domain. Disordered segments bind urea more readily than regions in stable secondary structure. The locations of the putative urea-binding sites correlate with the lower stability of the structure against solvent exchange, suggesting that partial unfolding of the structure is related to urea accessibility.

Effects of Concentration on Like-Charge Pairing of Guanidinium Ions and on the Structure of Water: An All-Atom Molecular Dynamics Simulation Study.

Like-charge ion-pair formation in an aqueous solution of guanidinium chloride (GdmCl) has two important facets. On one hand, it describes the role of the arginine (ARG) side chain in aggregation and dimer formation in proteins, and on the other hand, it lends support for the direct mechanism of protein denaturation by GdmCl. We employ all-atom molecular dynamics simulations to investigate the effect of GdmCl concentration on the like-charge ion-pair formation of guanidinium ions (Gdm(+)). From analyses of the radial distribution function (RDF) between the carbon atoms of two guanidinium moieties, the existence of both contact pairs and solvent-separated pairs has been observed. Although the peak height corresponding to the contact-pair state decreases, the number of Gdm(+) ions in the contact-pair state actually increases with increasing GdmCl concentration. We have also investigated the effect of the concentration of Gdm(+) on the structure of water. The effect of GdmCl concentration on the radial and tetrahedral structures of water is found to be negligibly small; however, GdmCl concentration has a considerable effect on the hydrogen-bonding structure of water. It is demonstrated that the presence of chloride ions, not Gdm(+), in the first solvation shell of water causes the distortion in the hydrogen-bonding network of water. In order to establish that Gdm(+) not only stacks against another Gdm(+) but also directly attacks the ARG residue of a protein or peptide, simulation of an ARG-rich peptide in 6 M aqueous solution of GdmCl has been performed. The analyses of RDFs and orientation distributions reveal that the Gdm(+) moiety of the GdmCl attacks the same moiety in the ARG side chain with a parallel stacking orientation.

Influence of xanthan gum on the structural characteristics of myofibrillar proteins treated by high pressure.

The effects of xanthan gum on the structural modifications of myofibrillar proteins (0.3 M NaCl, pH 6) induced by high pressure (200, 400, and 600 MPa, 6 min) were investigated. The changes in the secondary and tertiary structures of myofibrillar proteins were analyzed by circular dichroism. The protein denaturation was also evaluated by differential scanning calorimetry. Likewise, the protein surface hydrophobicity and the solubility of myofibrillar proteins were measured. High pressure (600 MPa) induced the loss of α-helix structures and an increase of β-sheet structures. However, the presence of xanthan gum hindered the former mechanism of protein denaturation by high pressure. In fact, changes in the secondary (600 MPa) and the tertiary structure fingerprint of high-pressure-treated myofibrillar proteins (400 to 600 MPa) were observed in the presence of xanthan gum. These modifications were confirmed by the thermal analysis, the thermal transitions of high-pressure (400 to 600 MPa)-treated myofibrillar proteins were modified in systems containing xanthan gum. As consequence, the high-pressure-treated myofibrillar proteins with xanthan gum showed increased solubility from 400 MPa, in contrast to high-pressure treatment (600 MPa) without xanthan gum. Moreover, the surface hydrophobicity of high-pressure-treated myofibrillar proteins was enhanced in the presence of xanthan gum. These effects could be due to the unfolding of myofibrillar proteins at high-pressure levels, which exposed sites that most likely interacted with the anionic polysaccharide. This study suggests that the role of food additives could be considered for the development of meat products produced by high-pressure processing.

Protein denaturants at aqueous-hydrophobic interfaces: self-consistent correlation between induced interfacial fluctuations and denaturant stability at the interface.

The notion of direct interaction between denaturing cosolvent and protein residues has been proposed in dialogue relevant to molecular mechanisms of protein denaturation. Here we consider the correlation between free energetic stability and induced fluctuations of an aqueous–hydrophobic interface between a model hydrophobically associating protein, HFBII, and two common protein denaturants, guanidinium cation (Gdm+) and urea. We compute potentials of mean force along an order parameter that brings the solute molecule close to the known hydrophobic region of the protein. We assess potentials of mean force for different relative orientations between the protein and denaturant molecule. We find that in both cases of guanidinium cation and urea relative orientations of the denaturant molecule that are parallel to the local protein–water interface exhibit greater stability compared to edge-on or perpendicular orientations. This behavior has been observed for guanidinium/methylguanidinium cations at the liquid–vapor interface of water, and thus the present results further corroborate earlier findings. Further analysis of the induced fluctuations of the aqueous–hydrophobic interface upon approach of the denaturant molecule indicates that the parallel orientation, displaying a greater stability at the interface, also induces larger fluctuations of the interface compared to the perpendicular orientations. The correlation of interfacial stability and induced interface fluctuation is a recurring theme for interface-stable solutes at hydrophobic interfaces. Moreover, observed correlations between interface stability and induced fluctuations recapitulate connections to local hydration structure and patterns around solutes as evidenced by experiment (Cooper et al., J. Phys. Chem. A2014, 118, 5657.) and high-level ab initio/DFT calculations (Baer et al., Faraday Discuss2013, 160, 89).

Modelling of pepsin digestibility of myofibrillar proteins and of variations due to heating

Digestibility of myofibrillar proteins by pepsin was determined by in vitro trials and mathematical modelling. A primary model was developed to predict in vitro digestion kinetics, and a secondary model based on the mechanisms of protein denaturation was then added to take into account the effect of meat heating. Model predictions agreed with measurements in the pH and pepsin concentration ranges 1.8–3.8 and 6–50Umg−1 respectively. The utility of the model is illustrated by a simple example where meat is assumed to be heated homogeneously, and myofibrillar proteins to be directly in contact with pepsin. The combined effects of heating time, temperature, enzyme concentration and pH modified the digestibility value, which also depends on residence time in the stomach.

Protein denaturation kinetic processes of a simple and a complex reaction mechanism analyzed by an iso-conversional method

The protein denaturation kinetic processes of a simple and a complex reaction mechanism represented by bovine serum albumin and hen egg-white lysozyme were analyzed by an iso-conversional method using differential scanning calorimetry. After differential scanning calorimetry using the iso-conversional method, the results were found to pose distinct contrasts between the two proteins. Bovine serum albumin showed an increasing peak temperature of the transition as the scan rate and protein concentration increased, whereas hen egg-white lysozyme exhibited almost constant peak temperature. The differential scanning calorimetry transition of bovine serum albumin was calorimetrically irreversible, while one part of hen egg-white lysozyme denaturation process was irreversible during which aggregation occurred and the other part was reversible. The iso-conversional method indicated that the value of bovine serum albumin apparent activation energy hardly varied with the degree of conversion, which showed that the denaturation kinetic process should conform to single reaction model. Using the master plots method, the most possible kinetic model for bovine serum albumin denaturation might be described by F n kinetic model. On the contrary, the hen egg-white lysozyme value of apparent activation energy decreased with the increase of degree of conversion. It was not a process involving the two standard reversible states, and can be described by the simple Lumry–Eyring model. The iso-conversional method provides new opportunities in exploring a simple and a complex reaction mechanism of protein denaturation.

Kinetic evidence for a two-stage mechanism of protein denaturation by guanidinium chloride.

Dry molten globular (DMG) intermediates, an expanded form of the native protein with a dry core, have been observed during denaturant-induced unfolding of many proteins. These observations are counterintuitive because traditional models of chemical denaturation rely on changes in solvent-accessible surface area, and there is no notable change in solvent-accessible surface area during the formation of the DMG. Here we show, using multisite fluorescence resonance energy transfer, far-UV CD, and kinetic thiol-labeling experiments, that the guanidinium chloride (GdmCl)-induced unfolding of RNase H also begins with the formation of the DMG. Population of the DMG occurs within the 5-ms dead time of our measurements. We observe that the size and/or population of the DMG is linearly dependent on [GdmCl], although not as strongly as the second and major step of unfolding, which is accompanied by core solvation and global unfolding. This rapid GdmCl-dependent population of the DMG indicates that GdmCl can interact with the protein before disrupting the hydrophobic core. These results imply that the effect of chemical denaturants cannot be interpreted solely as a disruption of the hydrophobic effect and strongly support recent computational studies, which hypothesize that chemical denaturants first interact directly with the protein surface before completely unfolding the protein in the second step (direct interaction mechanism).

Mechanical Protein Unfolding and Translocation by AAA+ Proteases

In all cells, protein degradation regulates key processes and maintains protein quality control. The AAA+ protease ClpXP is one of the best-studied examples of the ATP-dependent machinery responsible for intracellular proteolysis. A ring-shaped hexamer of the ClpX ATPase first recognizes and denatures folded protein substrate, then translocates the unfolded polypeptide into the barrel-shaped ClpP peptidase for degradation. In recent studies, dual-laser optical trapping was used to dissect the molecular events resulting in protein degradation by single molecules of ClpXP (Aubin-Tam et al. 2011, Maillard et al. 2011). This technique allows for the detection of small events (nm scale) with high temporal resolution (sub-ms scale). Here, we adapt our single-molecule assay to study a related AAA+ protease ClpAP and to address whether the similar architecture shared by all AAA+ proteases indicates a common mechanism of protein denaturation and degradation. Unlike ClpX, ClpA contains two ATPase modules per hexamer subunit and represents a major subclass of double-ring protein remodeling enzymes. We find similarities but also differences in mechanochemical activity between single- and double-ring ATPases, which contribute to their distinct activities in protein unfolding and degradation. We anticipate that the methods used to study ClpXP and ClpAP at the single-molecule level can be readily applied to examine the activity of other AAA+ proteases and protein remodeling enzymes.Aubin-Tam ME, Olivares AO, Sauer RT, Baker TA, Lang MJ (2011). Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine. Cell 145: 257-67.Maillard RA, Chistol G, Sen M, Righini M, Tan J, Kaiser CM, Hodges C, Martin A, Bustamante C (2011). ClpX(P) generates mechanical force to unfold and translocate its protein substrates. Cell 145: 459-69.

Analysis of Bulk and Hydration Water During Thermal Lysozyme Denaturation Using Raman Scattering

We describe a method for analyzing protein hydration by Raman spectroscopy on the model protein lysozyme. The analysis of the protein hydration shell is made possible by dissolving the protein in D2O, providing via isotopic exchange the uncoupled O – H stretching spectrum of water molecules early bound to the protein, which are thereafter spread into the solvent. The spectrum of the hydration water can be obtained by subtracting the spectrum of the contribution of D2O from that of the aqueous lysozyme solution in the intramolecular O – D stretching vibrations region (2,200–2,800 cm−1). Raman investigations were simultaneously carried out in the amide I region (1,500–1,800 cm−1) and in the O – D/H stretching spectrum (3,200–3,800 cm−1) during thermal denaturation of lysozyme, to analyze structural changes of the protein in relation to the physical properties of hydration water. It was found that the H-bond network of hydration water is slightly distorted compared to the bulk water at room temperature, with a loss of the tetrahedral local order. The difference between hydration and bulk water is significantly enhanced at T = 90 °C in the denaturated state of the protein. The quantification of water molecules in direct interaction with the protein provides the temperature dependence of the solvent-accessible surface area during the denaturation process. Both kinds of information on hydration water and protein structure lead to a detailed description and overall understanding of the mechanism of protein denaturation.

Investigation of the Mechanism of Protein Denaturation by Guanidine Hydrochloride-Induced Dissociation of Inhibitor-Protease Complexes

In this communication we describe an approach in which guanidine hydrochloride-induced dissociation of a protein inhibitor-serine protease complex is used to explore the molecular basis of protein denaturation. The rationale behind this approach is that the inhibitor-protease complex is stabilized by the same types of non-covalent interactions that stabilize the native state of a protein. The dissociation of inhibitor-protease complex can be performed at concentrations of guanidine hydrochloride at which the inhibitor and the protease retain their native conformations. Here, we present our results on the effect of 0.1 M to 0.4 M guanidine hydrochloride concentrations on the association equilibrium constants (reciprocal of dissociation constant) of P₁G, P₁A, P₁V, P₁N, and P₁S variants of turkey ovomucoid third domain with bovine α-chymotrypsin. We use these results to calculate the free energy change in the dissociation of inhibitor-protease complexes (the m value) per mol of guanidine hydrochloride concentration. Our results agree with the general consensus that the denaturing effect of guanidine hydrochloride is due to its favorable interaction with the polar parts of proteins and that the non-polar side chains have no or little favorable interaction with guanidine hydrochloride.

Femtosecond study of the effects of ions and hydrophobes on the dynamics of water

We study the effects of ions and hydrophobic molecular groups on the orientational dynamics of water using THz dielectric relaxation (THz-DR) and polarization-resolved femtosecond infrared (fs-IR) pump-probe spectroscopy. We measure the dynamics of water in solutions of NaI, NaCl, CsCl, guanidinium chloride (GndCl) and tetramethyl guanidinium chloride (TMGndCl) of different the static dipoles of their surrounding water molecules. With fs-IR we find that concentrations. With THz-DR we observe that strongly hydrated cations align the OD groups that form hydrogen bonds to halide anions reorient with two distinct time constants of 2 +/- 0.3 ps and 9 +/- 1 ps. The fast process is assigned to a wobbling motion of the OD group that keeps the hydrogen bond with the anion intact. The amplitude of this wobbling motion depends on the nature of both the anion and the counter cation. The replacement of four of the six hydrogen atoms of the weakly hydrated cation guanidinium by hydrophobic methyl groups leads to an exceptionally strong slowing down of the water dynamics. Hydrophobic groups thus appear to have a much stronger effect on the dynamics of water than ions. These findings give new insights in the mechanism of protein denaturation by GndCl and TMGndCl.

Investigation of the Mechanism of Protein Denaturation by Guanidine Hydrochloride-Induced Dissociation of Inhibitor-Protease Complexes

In this communication we describe an approach in which guanidine hydrochloride-induced dissociation of a protein inhibitor-serine protease complex is used to explore the molecular basis of protein denaturation. The rationale behind this approach is that the inhibitor-protease complex is stabilized by the same types of non-covalent interactions that stabilize the native state of a protein. The dissociation of inhibitor-protease complex can be performed at concentrations of guanidine hydrochloride at which the inhibitor and the protease retain their native conformations. Here, we present our results on the effect of 0.1M to 0.4M guanidine hydrochloride concentrations on the association equilibrium constants (reciprocal of dissociation constant) of P1G, P1A, P1V, P1N, and P1S variants of turkey ovomucoid third domain with bovine α-chymotrypsin. We use these results to calculate the free energy change in the dissociation of inhibitor-protease complexes (the m value) per mol of guanidine hydrochloride concentration. Our results agree with the general consensus that the denaturing effect of guanidine hydrochloride is due to its favorable interaction with the polar parts of proteins and that the non-polar side chains have no or little favorable interaction with guanidine hydrochloride. Keywords: Chymotrypsin, denaturation, osmolytes, protease inhibitor, serine proteases, turkey ovomucoid third domain

Adsorption induced enzyme denaturation: The role of protein surface in adsorption induced protein denaturation on allyl glycidyl ether (AGE)–ethylene glycol dimethacrylate (EGDM) copolymers

The effects of protein size on adsorption and adsorption-induced denaturation of proteins on copolymers of allyl glycidyl ether (AGE)–ethylene glycol dimethacrylate (EGDM) have been studied. Different responses were observed for the amount of protein adsorbed and denatured on the polymer surface for different proteins (trypsin, alchol dehydrogenase from baker's yeast (YADH), glucose dehydrogenase (GDH) from Gluconobacter cerinus, and alkaline phosphates from calf intestinal mucosa (CIAP). Protein adsorption on the copolymer with 25% crosslink density (AGE-25) was dependent not only on the size of the protein but also on the presence of glycoside residues on the protein surface. Adsorption and denaturation of proteins follows the order YADH > trypsin > GDH ≫ CIAP although the molecular weights of the proteins follow the order YADH > CIAP > GDH > trypsin. The lack of correlation between amount of adsorbed protein and its molecular weight was due to the presence of glycoside residues on CIAP and GDH which protect the enzyme surface from denaturation. Enzyme stabilities in aqueous solutions of 1-cyclohexyl-2-pyrrolidinone (CHP) correlate well with the trend in denaturation by the copolymer, strongly suggesting that hydrophobic interactions play a major role in protein binding and the mechanism of protein denaturation is similar to that for water–miscible organic solvents.