Adsorption on montmorillonite prevents oligomerization of Bt Cry1Aa toxin.

The adsorption of the insecticidal Cry1Aa protein from Bacillus thuringiensis (Bt-toxin) on a model clay surface was studied to understand the structural changes of the protein induced by the clay surface. We studied the adsorption of the monomeric and soluble oligomeric forms of the Cry1Aa toxin as a function of pH and ionic strength conditions on montmorillonite, which is an electronegative phyllosilicate. Cry1Aa secondary structure was determined from the amide I' FTIR absorption profiles. Accessibility to the solvent was determined by NH/ND exchange to characterize conformational flexibility of the different states of the Cry1Aa protein. The size distribution of Cry1Aa solutions was obtained by dynamic light scattering (DLS). From combined DLS and FTIR measurements, we conclude that montmorillonite traps the Cry1Aa toxin in its monomeric state, preventing the oligomerization of the protein. The oligomeric forms were adsorbed onto the clay without significant structural changes.

Fate of prions in soil: trapped conformation of full-length ovine prion protein induced by adsorption on clays.

Studying the mechanism of retention of ovine prion protein in soils will tackle the environmental aspect of potential dissemination of scrapie infectious agent. We consider the surface-induced conformational changes that the recombinant ovine prion protein (ovPrP) may undergo under different pH conditions when interacting with soil minerals of highly adsorptive capacities such as montmorillonite. The conformational states of the full-length ovine prion protein adsorbed on the electronegative clay surface are compared to its solvated state in deuterated buffer in the pD range 3.5-9, using FTIR spectroscopy. The in vitro pH-induced conversion of the alpha-helical monomer of ovPrP into oligomers of beta-like structure prone to self-aggregation does not occur when the protein is adsorbed on the clay surface. The conformation of the trapped ovPrP molecules on montmorillonite is pH-independent and looks like that of the ovPrP solvated state at pD higher than 7, suggesting the major role of Arg and Lys residues in the electrostatic origin of adsorption. The uneven distribution of positively and negatively charged residues of the ovPrP protein would promote a favored orientation of the protein towards the clay, so that not only the basic residues embedded in the N-terminal flexible part but also external basic residues in the globular part of the protein might participate to the attractive interaction. From these results, it appears unlikely that the interaction of normal prions (PrP(C)) with soil clay surfaces could induce a change of conformation leading to the pathogenic form of prions (PrP(Sc)).

Structural effects of drying and rehydration for enzymes in soils: a kinetics-FTIR analysis of alpha-chymotrypsin adsorbed on montmorillonite.

The effects of desiccation and rehydration cycles encountered by extracellular enzymes in soils are studied on -chymotrypsin adsorbed on montmorillonite. The controlled hygrometric FTIR cell used in this study enables to monitor drying and rehydration steps undergone by the -chymotrypsin-montmorillonite suspension or by the enzyme alone. Relative humidity (RH) determines the amount of deuterated water in the FTIR cell atmosphere. The molar water/protein ratio (W/P) as well as the conformational and solvation states of the enzyme have been determined using H/D exchange monitored by FTIR-transmission spectroscopy. When the W/P ratio decreases from 3500 to approximately 400, unfolding of beta-secondary structure in three different domains involves about 8% of the polypeptide backbone with respect to the most solvated states. Desiccation induces beta-unfolding, which opens channels allowing free vapor water molecules to diffuse into the enzyme at 15% RH. On drying to 0% RH, displacements of internal water (H2O) in the enzyme are demonstrated by reverse peptide isotopic exchanges (COND ==> CONH). Specific beta-structures, only formed in highly solvated states, sequester around 20 internal H2O molecules. Indeed, most of the unfolded secondary structures during the drying step are refolded at W/P approximately 1000 during rehydration. However, self-association hinders the recovery of the initial closed tertiary structure. The pD-dependent structural changes controlling inward and outward water diffusion are suppressed, whether the protein is initially in an adsorbed state or in solution. Changes in secondary structures encountered during desiccation/rehydration cycle are similar for the protein either free or in the adsorbed state. Thus domains that are unfolded by adsorption are not concerned by the desiccation/rehydration cycle.

Conformational changes of enzymes adsorbed at liquid-solid interface: relevance to enzymatic activity.

FTIR with attenuated total reflectance spectroscopy was used to study in situ adsorption of enzymes at water-solid interfaces to better understand how conformational changes may monitor enzymatic activity. Because the adsorption process depends on hydrophobic and electrostatic interactions, conformational changes were studied as a function of the nature of the adsorbing substrates, which are hydrophobic or hydrophilic in character. The adsorption kinetics of two examples of serine enzymes, alpha-chymotrypsin (alpha-chym) and Humicola lanuginosa lipase (HLL), were studied. The secondary structure and solvation of the adsorbed enzymes were both compared to the dissolved enzymes. The positively charged alpha-chym was adsorbed on a negatively charged hydrophilic support with minor structural changes, but the negatively charged lipase had no affinity for a similar support. Both enzymes were strongly retained on the hydrophobic support. The secondary and tertiary structures of the alpha-chym adsorbed on the hydrophobic support were strongly altered, which correlates to the inhibition of enzymatic hydrolysis. The specific solvation obtained for the adsorbed HLL is consistent with the existence of the open conformer in relation to the enhanced enzymatic activity at the water-hydrophobic interface.

Structural changes of human serum albumin immobilized on chromatographic supports: a high-performance liquid chromatography and Fourier-transform infrared spectroscopy study.

Chiral stationary phases obtained by immobilization of HSA on [C8] and [C18] reversed-phases and on poly(1-vinylimidazole)-coated silica were tested to resolve DL-tryptophan, N-benzoyl-DL-phenylalanine, RS-oxazepam and RS-warfarin racemic mixtures. Parameters of enantioselectivity measured in HPLC are correlated to structural and solvation states for adsorbed HSA, evaluated by FTIR spectroscopy. HSA immobilized on [PVI]-anion-exchangers is highly selective. HSA molecules are not self-associated, only unfolded for a small hydrophobic helix. The HSA-coated reversed-phases have a lower selectivity. Unfolding is larger but the indole-benzodiazepine chiral site is preserved and remains accessible.

Structural changes of cytochrome c(552) from Thermus thermophilus adsorbed on anionic and hydrophobic surfaces probed by FTIR and 2D-FTIR spectroscopy.

The structural changes of cytochrome c(552) bound to anionic and hydrophobic clay surfaces have been investigated by Fourier transform infrared spectroscopy. Binding to the anionic surface of montmorillonite is controlled by electrostatic interactions since addition of electrolyte (0.5 mol L(-1) KCl) causes desorption of more than 2/3 of the protein molecules. Electrostatic binding occurs through the back side of the protein (i.e., remote from the heme site) and is associated only with subtle changes of the secondary structure. In contrast, adsorption to the hydrophobic surface of talc leads to a decrease in alpha-helical structure by ca. 5% and an increase in beta-sheet structure by ca. 6%. These structural changes are attributed to a hydrophobic region on the front surface of cytochrome c(552) close to the partially exposed heme edge. This part on the protein surface is identified as the interaction domain for talc and most likely also serves for binding to the natural reaction partner, a ba(3)-oxidase. Fourier transform infrared spectra of cytochrome c(552) and the clay-cytochrome c(552) complexes have been measured as a function of time following dissolution and suspension in deuterated buffer, respectively. A two-dimensional correlation analysis was applied to these spectra to investigate the dynamics of the structural changes in the protein. For both complexes, adsorption and subsequent unfolding processes in the binding domains are faster than the time resolution of the spectroscopic experiments. Thus, the processes that could be monitored are refolding of peptide segments and side chain rearrangements following the adsorption-induced perturbation of the protein structure and the solvation of the adsorbed protein. In each case, side chain alterations of solvent-exposed tyrosine, aspartate, and glutamate residues were observed. For the cytochrome c(552)-talc complex, these changes are followed by a slow refolding of the peptide chain in the binding domain and, subsequently, a further H/D exchange of amide group protons.

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

Interactions between proteins and clays perturb biological activity in ecosystems, particularly soil extracellular enzyme activity. The pH dependence of hydrophobic, hydrophilic, and electrostatic interactions on the adsorption of bovine serum albumin (BSA) is studied. BSA secondary structures and hydration are revealed from computation of the Amide I and II FTIR absorption profiles. The influence of ionization of Asp, Glu, and His side chains on the adsorption processes is deduced from correlation between p(2)H dependent carboxylic/carboxylate ratio and Amide band profiles. We quantify p(2)H dependent internal and external structural unfolding for BSA adsorbed on montmorillonite, which is an electronegative phyllosilicate. Adsorption on talc, a hydrophobic surface, is less denaturing. The results emphasize the importance of electrostatic interactions in both adsorption processes. In the first case, charged side chains directly influence BSA adsorption that generate the structural transition. In the second case, the forces that attract hydrophobic side chains toward the protein-clay interface are large enough to distort peripheral amphiphilic helical domains. The resulting local unfolding displaces enough internal ionized side chains to prevent them from establishing salt bridges as for BSA native structure in solution. On montmorillonite, a particular feature is a higher protonation of the Asp and Glu side chains of the adsorbed BSA than in solution, which decreases coulombic repulsion. Copyright 2000 Academic Press.

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

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.

Characterization of Human Serum Albumin Adsorbed on a Porous Anion-Exchange Support.

Fourier transform infrared (FTIR) spectroscopy and high-performance liquid chromatography (HPLC) were used to study the adsorption behavior of human serum albumin (HSA) onto a polymer-layer type anion exchanger, poly(vinylimidazole) (PVI) adsorbed and cross-linked on a porous silica support. The data are compared with previous results for HSA adsorbed on C6 alkyl chains grafted on the same silica matrix. For the adsorption onto the PVI support from a phosphate buffer solution of low ionic strength (pD 7.4), the FTIR experiments reveal only very weak structural and solvation changes. A large fraction of the protein remains irreversibly adsorbed and the amount retained at equilibrium is close to that observed for the adsorption on the reversed-phase support, although the structural effect of the stationary phase was much larger with the grafted C6 alkyl chains. Comparing to the solution state, only 2% of the HSA backbone is modified by adsorption on PVI, whereas 12% alterations are involved for the protein adsorbed on the reversed-phase support. When adsorbed from an eluent containing 20% acetonitrile, the amount of HSA retained by the PVI ion exchanger is about twice that measured with the buffer alone. This result is explained by a more compact structure of the protein when dissolved in the organoaqueous solvent. The presence of acetonitrile does not markedly affect the elution front of HSA adsorbed on the charged hydrophilic adsorbent. This result contrasts with the much lower apparent adsorption rate observed when HSA is adsorbed on the grafted alkyl chain support in the presence of acetonitrile in the buffer. Copyright 1998 Academic Press.

Composition of uterine flushings from Large White and prolific Chinese Meishan gilts.

This study examined differences in selected components of uterine secretions from Large White and prolific Chinese Meishan gilts during the oestrous cycle or early pregnancy. Total recoverable protein, uteroferrin (measured as acid phosphatase activity), acyl aminopeptidase, calcium, sodium, potassium, immunoglobulins A and G, glucose, fructose, oestradiol-17 beta, and prostaglandins F2 alpha (PGF2 alpha) and E2 (PGE2) in uterine flushings were measured. During the oestrous cycle, breed effects were detected only for total protein (P = 0.07), which tended to be higher for Large White gilts. However, for pregnant gilts, total recoverable glucose (P less than 0.05), fructose (P less than 0.05) sodium (P less than 0.05), immunoglobulin A (P less than 0.01), PGF (P less than 0.01), PGE (P less than 0.01), and acyl aminopeptidase (P less than 0.05) were greater in uterine flushings from Meishan gilts. Only uteroferrin was higher (P = 0.06) in uterine flushings from Large White gilts. Concentrations of prolactin were higher (P less than 0.05) in plasma from cyclic or pregnant Meishan gilts, but concentrations of total oestrogens and progesterone were not affected by pregnancy status or breed. These results suggest that Meishan conceptuses, individually or collectively, are more stimulatory to endometrial secretion and/or transport of the components of histotroph into the uterine lumen, or that the endometrium of Meishan gilts is more sensitive to conceptus signals responsible for the accumulation of histotroph in the uterine lumen.

[Not Available]. / Applications de la colorimetrie de precision a la microanalyse elementaire Dosages du rhenium, du rhodium, du tellure et du zirconium.

The sample (< 1 mg) is burnt in an oxygen flask for the determination of rhenium. It is destroyed by a wet process in a Kjeldahl flask for the determination of rhodium or tellurium (sample <4-5 mg) or zirconium (sample <1.5 mg). These elements are determined spectrophotometrically. The following reactions are used: reduction of perrhenate by tin(II) chloride in the presence of 2,2'-furildioxime or thiourea, complexation of rhodium(III) by sodium hypochlorite, formation of iodotellurite, complexation of zirconium(IV) with Xylenol Orange.

[Remarques sur le microdosage du bore et du germanium dans les composes organiques et mineraux]. / Remarques sur le microdosage du bore et du germanium dans les composes organiques et mineraux.

A wet destruction in a Kjeldahl flask followed by spectrophotometric measurement with azomethine H as reagent, combustion in oxygen in a Parr bomb, and destruction by heating with sodium peroxide followed by acidimetric titration of the mannitol-boric acid complex have been tested for the microdetermination of boron in organic and inorganic compounds and compared with the Schöniger-flask method, which fails to give good results in the case of inorganic compounds; the wet-combustion method is the most useful. Similar techniques, combined with the acidimetric titration of the mannitol-germanic acid complex, or spectrophotometric measurement using phenylfluorone as a complexing reagent, or gravimetric determination as GeO(2), have been tested for the microdetermination of germanium, but none of them is entirely satisfactory, for reasons of lack of either universality or precision.