Complexation of U(VI) with highly phosphorylated protein, phosvitin: A vibrational spectroscopic approach

The complexation of uranium(VI) to variant functional groups of the highly phosphorylated protein phosvitin in aqueous solution was investigated by attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy. For the verification of the affinity of the actinyl ions to carboxyl and phosphate groups of the amino acid side chains, samples with different phosphate to uranium(VI) (P/U) ratios were investigated under denaturing conditions as well as in aqueous medium. From a comparative study with other heavy metal ions, i.e. Ba²⁺ and Pb²⁺, a strong coordination of U(VI) to carboxyl and phosphoryl groups can be derived. Furthermore, with increasing P/U ratios, a preferential binding of U(VI) to phosphoryl groups is indicated by the spectra of the batch samples. These findings are confirmed by spectra of aqueous U(VI)-phosvitin complexes reflecting an explicit coordination of the uranyl ions to phosphate groups at a high P/U ratio. Our study provides a deeper insight into the molecular interactions between actinyl ions and protein, and can be conferred to other basic biomolecules such as polysaccharides and nucleic acids.     

Histochemical observations on the role of ferric chloride in the high-iron diamine technique for localizing sulphated mucosubstances

Synopsis The binding of ferric ions to tissue sites, other than those containing sulphated mucosubstances, in sections subjected to the high iron diamine technique was followed by the Prussian blue reaction in order to throw new light on the role of ferric chloride in the high-iron diamine dye bath. From experiments involving enzyme digestions in particular, evidence was obtained that ferric ions bind to ribonucleic acid (in chief cells of the rabbit stomach), deoxyribonucleic acid (in nuclear chromatin granules), and, under certain conditions, to sialic acid residues (e.g. in mucous acini of the mouse sublingual gland) and protein carboxyl groups (in smooth muscle cells) as well.With a few exceptions, the binding of ferric ions to nucleic acids was not affected by changes in the ferric chloride concentration, pH or magnesium chloride concentration in the dye bath; the bond thus formed was very stable. It is possible that the initial linkage is not an electrostatic one. Under all the conditions investigated, it was found that the diamine complexes have a greater affinity for sulphated mucosubstances than ferric ions, but ferric chloride, by lowering the pH of the dye bath, excludes the carboxyl groups from reacting with the positively-charged diamine polymer molecules. It is possible that a high concentration of ferric ions in the high-iron diamine dye bath inhibits the binding of some diamine complexes to nuclei and gastric chief cells, i.e. sites where no sulphated mucosaccharides are present, although this conclusion needs further substantiation.     

Spectroscopic investigations of U(VI) species sorbed by the green algae Chlorella vulgaris

The green alga Chlorella vulgaris has the ability to bind high amounts of uranium(VI) in the pH range from 3 to 6. At pH 3 up to 40% of the uranium are bound by the algal cells. The uranium removal is almost complete at pH 5 and 6 under the given experimental conditions. Scanning electron microscopy and laser-induced fluorescence spectroscopy were used to characterize uranyl species formed in the selected pH range. The micrographs show a regular distribution of U(VI) on the cell surface. Fluorescence spectroscopic investigations of formed algal uranyl complexes indicate that the binding of U(VI) to carboxyl groups plays a dominating role at pH 3, whereas a minor impact of organic phosphate compounds on the U(VI) sorption cannot be excluded. In contrast, at pH 5 and 6 the phosphate groups are mainly responsible for the removal and binding of U(VI) by formation of organic and/or inorganic uranyl phosphates.     

Uranyl photoprobing of a four-way DNA junction: evidence for specific metal ion binding.

Metal ions are very important in mediating the folding of nucleic acids, as exemplified by the folding of the four-way DNA junction into the stacked X-conformation. Uranyl ion-mediated photocleavage provides a method for the localization of high-affinity ion binding sites in nucleic acids, and we have applied this to the four-way DNA junction. We have made the following observations. (i) Uranyl ions (UO2(2+)) suppressed the reactivity of junction thymine bases against attack by osmium tetroxide, indicating that the uranyl ion induces folding of the junction into a stacked conformation. (ii) DNA located immediately at the point of strand exchange on the two exchanging strands was hypersensitive to uranyl photocleavage. The relative hypersensitivity was considerably accentuated when the photocleavage was carried out in the presence of citrate ions. This suggests the presence of a tight binding site for the uranyl ion in the junction. (iii) The same positions were significantly protected from uranyl cleavage by the presence of hexamminecobalt (III) or spermidine. These ions are known to induce the folded conformation of the four-way junction with high efficiency, suggesting a direct competition between the ions. By contrast, magnesium ions failed to generate a similar protection against photocleavage. These results suggest that the uranyl, hexamminecobalt (III) and spermidine ions compete for the same high affinity binding site on the junction. This site is located at the centre of the junction, at the point where the exchanging strands pass between the stacked helices. We believe that we have observed the first known example of a metal ion 'footprint' on a folded nucleic acid structure.     

Evaluation of uranyl photocleavage as a probe to monitor ion binding and flexibility in RNAs

In order to evaluate uranyl photocleavage as a tool to identify and characterize structural and dynamic properties in RNA, we compared uranyl cleavage sites in five RNA molecules with known X-ray structures, namely the hammerhead and hepatitis delta virus ribozymes, the P4-P6 domain of the Tetrahymena group I intron, as well as tRNA(Phe) and tRNA(Asp) from yeast. Uranyl photocleavage was observed at specific positions in all molecules investigated. In order to characterize the sites, photocleavage was performed in the absence and in increasing amounts of MgCl(2). Uranyl photocleavage correlates well with sites of low calculated accessibility, suggesting that uranyl ions bind in tight RNA pockets formed by close approach of phosphate groups. RNA foldings require ion binding, usually magnesium ions. Thus, upon the adoption of the native structure, uranyl ions can no longer bind well except in flexible and open to the solvent regions that can undergo induced-fit without disrupting the native fold. Uranyl photocleavage was compared to N-ethyl-N-nitrosourea and lead-induced cleavages in the context of the three-dimensional X-ray structures. Overall, the regions protected from ENU attack are sites of uranyl cleavage, indicating sites of low accessibility which can form ion binding sites. On the contrary, lead cleavages occur at flexible and accessible sites and correlate with the unspecific cleavages prevalent in dynamic and open regions. Applied in a magnesium-dependent manner, and only in combination with other backbone probing agents such as N-ethyl-N-nitrosourea, lead and Fenton cleavage, uranyl probing has the potential to reveal high-affinity metal ion environments, as well as regions involved in conformational transitions.     

Evidence for essential carboxyls in the cation-binding domain of the Na,K-ATPase.

Treatment of isolated canine renal Na,K-ATPase with a stable diazomethane analog, 4-(diazomethyl)-7-(diethylamino)-coumarin (DEAC), results in enzyme inactivation. The inactivation rate was dramatically increased when the enzyme was treated with DEAC in the presence of ATP and Mg2+ (in imidazole buffer) or Pi and Mg2+, conditions which produce enzyme phosphorylation. Inactivation in the presence of Pi and Mg2+ could be partially prevented by Na+ and almost completely prevented by K+. The quantity of DEAC covalently bound to the Na,K-ATPase was determined spectrophotometrically. The extent of inactivation was linearly related to the amount of K-protectable DEAC incorporation. Complete inactivation of ATPase activity occurred with 2.14 +/- 0.18 nmol of DEAC covalently bound/mg of protein. This suggests that only 1 or 2 carboxyl residues/catalytic center (estimated by high affinity ADP binding) are involved in the modification leading to inactivation. The modified enzyme exhibited normal levels of high affinity [3H]ADP (and hence ATP) binding, thus, the nucleotide-binding domain of the enzyme seems unaffected by the modification. In contrast, under conditions where native enzyme was able to occlude 3.82 nmol of K+ ions/mg of protein, DEAC-modified enzyme occluded only 0.33 nmol of K+ ions. Na+ occlusion by the enzyme (in the presence of oligomycin) was also reduced (by 80%) following treatment with DEAC. Phosphorylation by [32P]inorganic phosphate and Na(+)-activated phosphorylation of the modified enzyme with [32P]ATP yielded reduced levels of phosphoenzyme (about 36%) compared to native enzyme. The DEAC-modified [32P]phosphoenzyme formed from [32P]ATP was insensitive to the addition of K+ ions, under conditions which led to the rapid hydrolysis of native phosphoenzyme. Gel electrophoresis of modified protein revealed strong fluorescence labeling of the alpha-subunit, which was substantially reduced if treatment with DEAC was performed in the presence of K+ ions. Partial tryptic digestion and electrophoretic analysis revealed normal degradation patterns in the presence of ADP (E1 form) but the typical patterns, seen with K+ ions (E2K) or Na+ ions (E1Na) in native enzyme, were absent. A typical E2-like tryptic degradation pattern was seen, however, in the presence of vanadate ions and ouabain, suggesting that the modification does not freeze the enzyme in an E1 conformation and that the enzyme is still able to undergo the E1E2 conformational transition after modification. Our results suggest that a small number of carboxyl residues in the sodium pump alpha-subunit (perhaps one) are essential for K+ and Na+ binding and stabilizing the occluded enzyme cation forms. Esterification of the carboxyl groups by DEAC inactivates the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Methanol-acetic anhydride: an efficient blocking agent for electron microscope cytochemistry. Its application to mouse testis and other tissues

A mixture of absolute methanol and acetic anhydride (MA) (5:1, v/v; 24 h at 25 degrees C) which is known to methylate and acetylate--respectively--free carboxyl and amino groups in proteins, was tested for its effectiveness as a blocking agent in glutaraldehyde-fixed mouse testis and skeletal muscle. In young spermatids the staining of the acrosome with either uranyl acetate (UA) or ethanolic phosphotungstic acid (PTA) was completely prevented by prior treatment with MA. Esterification of carboxyl groups with 0.1 N HCl in methanol (24 h at 30 degrees C) eliminated the UA staining without affecting that due to PTA. It is concluded that--COOH groups are responsible for UA binding and that acetylation (of amino groups) prevented PTA binding. MA also abolishes the strong affinity of PTA to the lateral elements of the synaptonemal complex in meiotic chromosomes, the axoneme and fibrous sheath of the spermatid tail and the Z band in skeletal muscle. Reactivity was diminished in nucleoli and remained unaffected in chromatin, the outer coarse fibers of the flagellum and collagen fibrils. Different functional groups may participate in PTA staining. The ultrastructure was well preserved in all cases.     

Modification of maize phosphoenolpyruvate carboxylase by Woodward's reagentK

Maize leaf phosphoenolpyruvate carboxylase was completely and irreversibly inactivated by treatment with micromolar concentrations of Woodward's reagentK (WRK) for about 1 min. The inactivation followed pseudo-first-order reaction kinetics. The order of reaction with respect to WRK showed that the reagent causes formation of reversible enzyme inhibitor complex before resulting in irreversible inactivation. The loss of activity was correlated to the modification of a single carboxyl group per subunit, even though the reagent reacted with 2 carboxyl groups per protomer. Substrate PEP and PEP + Mg²⁺ offered substantial protection against inactivation by WRK. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification. The modified enzyme exhibited altered surface charge as seen from the elution profile on FPLC Mono Q anion exchange column. The modified enzyme was desensitized to positive and negative effectors like glucose-6-phosphate and malate. Pretreatment of PEP carboxylase with diethylpyrocarbonate prevented WRK incorporation into the enzyme, suggesting that both histidine and carboxyl groups may be closely physically related. The carboxyl groups might be involved in metal binding during catalysis by the enzyme.     

Modification of maize phosphoenolpyruvate carboxylase by Woodward's reagentK

Maize leaf phosphoenolpyruvate carboxylase was completely and irreversibly inactivated by treatment with micromolar concentrations of Woodward's reagentK (WRK) for about 1 min. The inactivation followed pseudo-first-order reaction kinetics. The order of reaction with respect to WRK showed that the reagent causes formation of reversible enzyme inhibitor complex before resulting in irreversible inactivation. The loss of activity was correlated to the modification of a single carboxyl group per subunit, even though the reagent reacted with 2 carboxyl groups per protomer. Substrate PEP and PEP + Mg²⁺ offered substantial protection against inactivation by WRK. The modified enzyme showed a characteristic absorbance at 346 nm due to carboxyl group modification. The modified enzyme exhibited altered surface charge as seen from the elution profile on FPLC Mono Q anion exchange column. The modified enzyme was desensitized to positive and negative effectors like glucose-6-phosphate and malate. Pretreatment of PEP carboxylase with diethylpyrocarbonate prevented WRK incorporation into the enzyme, suggesting that both histidine and carboxyl groups may be closely physically related. The carboxyl groups might be involved in metal binding during catalysis by the enzyme.     

Mono- (Ag, Hg) and di- (Cu, Hg) valent metal ions effects on the activity of jack bean urease. Probing the modes of metal binding to the enzyme

The inhibition of urease by heavy metal ions has been habitually ascribed to the reaction of the ions with enzyme thiol groups, resulting in the formation of mercaptides. To probe the modes of metal binding to the enzyme, in this work the reaction of mono- (Ag, Hg) and di- (Cu, Hg) valent metal ions with jack bean urease was studied. The enzyme was reacted with different concentrations of the metal ions for different periods of times, when its residual activity was assayed and thiol content titrated. The titration carried out with DTNB was done to examine the involvement of urease thiol groups in metal ion binding. The binding was further probed by reactivation of the metal ion-enzyme complexes with DTT, EDTA and dilution. The results are discussed in terms of the HSAB concept. In inhibiting urease the metal ions showed a common feature in that they inhibited the enzyme within a comparable micromolar range, and also in that their inhibition was multisite. By contrast, the main distinguishing feature in their action consisted of the involvement of enzyme thiol groups in the reaction. Hg (2+) and Hg2(2+) inhibition was found thoroughly governed by the reaction with the enzyme thiols, and the complete loss of enzyme activity involved all thiols available in the enzyme under non-denaturating conditions. In contrast, Ag+ and Cu2+ ions for the complete inactivation of the enzyme required 53 and 60% of thiols, respectively. Accordingly, Ag+ and Cu2+ binding to functional groups in urease other than thiols, i.e. N- and O-containing groups, cannot be excluded. Based on the reactivation experiments this seems particularly likely for Cu2+, whose concurrent binding to thiols and other groups might distort the architecture of the active site (the mechanism of which remains to be elucidated) resulting in the observed inhibitory effects.     

谷胱甘肽S-转移酶活性中心的研究—羧基、氨基和胍基的化学修饰

用1-乙基-3-(3-二甲基氨基丙基)-碳二亚胺(EDC),2.4.6.三硝基苯磺酸(TNBS)和丁二酮(DIC)分别修饰人胎盘型谷胱甘肽S-转移酶(GST-π)的羧基、氨基和胍基,研究了酶的失活动力学,发现引起一分子酶亚基全部失活所需抑制剂的分子数分别为1.0、1.08和0.98,提示每亚基只有一个羧基、氨基和胍基参与酶的活性中心。底物及其类似物谷胱甘肽,S-己烷或S-辛烷谷胱甘肽可保护GST-π免受上述抑制剂的修饰,使假一级反应速度常数k_1明显降低,说明羧基、氨基和胍基是GST-π和GSH结合部位的组成基团。作者曾证明GST-π中的一个快反应巯基也参与酶与GSH的结合,故至少有四个不同的基团是酶亚基的结合基团,本文还对TNBS对氨基修饰的特异性作了验证,并讨论了GST-π与GSH结合时形成离子键的情况。     

Effects of di-isopropyl phosphorofluoridate on rat liver microsomal and lysosomal beta-glucuronidase

N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.     

Methanol-Acetic anhydride: an efficient blocking agent for electron microscope cytochemistry. Its application to mouse testis and other tissues

Summary A mixture of absolute methanol and acetic anhydride (MA) (5:1, v/v; 24 h at 25° C) which is known to methylate and acetylate — respectively — free carboxyl and amino groups in proteins, was tested for its effectiveness as a blocking agent in glutaraldehyde-fixed mouse testis and skeletal muscle. In young spermatids the staining of the acrosome with either uranyl acetate (UA) or ethanolic phosphotungstic acid (PTA) was completely prevented by prior treatment with MA. Esterification of carboxyl groups with 0.1 N HCl in methanol (24 h at 30° C) eliminated the UA staining without affecting that due to PTA. It is concluded that — COOH groups are responsible for UA binding and that acetylation (of amino groups) prevented PTA binding. MA also abolishes the strong affinity of PTA to the lateral elements of the synaptonemal complex in meiotic chromosomes, the axoneme and fibrous sheath of the spermatid tail and the Z band in skeletal muscle. Reactivity was diminished in nucleoli and remained unaffected in chromatin, the outer coarse fibers of the flagellum and collagen fibrils. Different functional groups may participate in PTA staining. The ultrastructure was well preserved in all cases.     

The stimulation of Na+ uptake in frog skin by uranyl ions.

1. The Na+ uptake in the isolated from skin of Rana esculenta was measured by the short-circuit current (Isc). Uranyl ions increase at pH 5.5 the Isc up to 200% at concentrations of 10 mM. The half-maximal value for this effect is at about 1 mM uranyl salt. 2. The effect is (a) specific for the Na+-selective membrane, (b) fully reversible. No stimulation can be seen in presence of 1 mM H+ or 0.1 mM amiloride. 3. The decrease of the sodium permeability of the apical membrane (PNa), normally induced by increasing concentrations of Na+ in the mucosal solution, %Na]o, is partially prevented by uranyl ions. The apparent Michaelis constant of the saturable Na+ uptake is shifted to much higher values. 4. A comparison between the uranyl effect and similar effects of the other drugs leads to the conclusion that uranyl ions might act in a polar hydrophobic environment, possibly by combining with phosphate groups (of phospholipids), and, thus, enhancing Na+ permeability by changes in tertiary structure near each Na channel. The interaction of mucosal Na+ with their receptor, normally triggering the [Na]o-dependent decrease of PNa, is thought to be diminished by uranyl association in a neighbouring region, causing a noncompetitive stimulation of the Na+ translocation though the apical frog skin membrane.     

Carboxyl groups at the two active centers of sucrase-isomaltase from rabbit small intestine.

1. Seveal selective reagents were employed to identify the amino acid residues essential for the catalytic activity of sucrase-isomaltase. 2. Modification of histidine, lysine and carboxyl residues resulted in a partial inactivation of the enzyme. Substrates or competitive inhibitors provided protection against inactivation only in the reaction of carboxyl groups with carbodiimide (+lycine ethyl ester) or with diazoacetic ethyl ester. This indicated the occurrence of carboxyl groups at the two active centers of the enzyme complex. 3. Protection against inactivation of the enzyme by carbodiimide was provided also by the presence of alkali and alkaline earth metal ions, which are non-essential activators of sucrase-isomaltase. The presence of Na+ and Ba2+ protected approximately one carboxyl group per active center from reacting with carbodiimide plus glycine ethyl ester. 4. The carbodiimide-reactive groups were not identical with the two carboxylate groups recently found to react with conduritol-B-epoxide, an active-site-directed inhibitor of sucrase-isomaltase (Quaroni, A. and Semenza, G., 1976, J. Biol. Chem 251,3250--3253). A possible role for the carbodiimide-reactive carboxyl groups at the active centers of sucrase-isomaltase is discussed.     

Site specificity of metallic ion binding in Escherichia coli K-12 lipopolysaccharide

The site specificity of metallic ion binding in Escherichia coli K-12 lipopolysaccharide was assessed by collecting high-resolution phosphorus nuclear magnetic resonance spectra in the presence of manganese, a paramagnetic divalent cation. This technique revealed high-affinity interactions between the cation and all of the lipopolysaccharide phosphoryl groups. To ascertain whether the carboxyl groups of 2-keto-3-deoxyoctonate contributed to the metal cation binding, lipopolysaccharide was chemically modified using a glycine ethyl ester - carbodiimide reaction. Of the three available carboxyl groups, only one was neutralized by the exogenously added ligand; the others appeared to be cross-linked within the molecule. By analogy, only one carboxyl group should be freely available for binding metallic ions, while the others are probably neutralized by the close proximity of endogenous amino substituents. Although high-resolution phosphorus nuclear magnetic resonance showed that an intermolecular conformational change had occurred after the carboxyl groups were neutralized, titration with manganese revealed no differences in the apparent strength of the interactions between the cation and the phosphoryl groups. Together, these data suggest that the high affinity of lipopolysaccharide for divalent metallic ions can be attributed primarily to the phosphoryl substituents and not free carboxyl groups.     

The Effects of the Porosity of a Support and of Attachment on the Mode of Action of Immobilized Endopolygalacturonase

Endopolygalacturonase of Aspergillus sp. was immobilized by three different methods; viz. (a) via amino groups, (b) via carboxyl groups and (c) by means of epoxy groups to a nonporous microparticular silicon dioxide (Cabosil), functionalized by 3-(amino)-propyl groups and 3-(2',3'-epoxypropoxy)-propyl groups, respectively. The conjugates were compared in their mode of action with corresponding immobilized preparations based on microporous ceramics. The binding via amino groups and via carboxyl groups lead, by itself, to changes in the mode of action of the enzyme, consisting of a decrease in randomness of glycosidic linkage splitting. The changes were greater in microporous support conjugates due to additional size-exclusion effects. The action pattern of endopolygalacturonase bound by means of epoxy groups was modulated exclusively by the porosity of the support, whereas the binding alone did not play any role.     

The Effects of the Porosity of a Support and of Attachment on the Mode of Action of Immobilized Endopolygalacturonase

Endopolygalacturonase of Aspergillus sp. was immobilized by three different methods; viz. (a) via amino groups, (b) via carboxyl groups and (c) by means of epoxy groups to a nonporous microparticular silicon dioxide (Cabosil), functionalized by 3-(amino)-propyl groups and 3-(2′,3′-epoxypropoxy)-propyl groups, respectively. The conjugates were compared in their mode of action with corresponding immobilized preparations based on microporous ceramics. The binding via amino groups and via carboxyl groups lead, by itself, to changes in the mode of action of the enzyme, consisting of a decrease in randomness of glycosidic linkage splitting. The changes were greater in microporous support conjugates due to additional size-exclusion effects. The action pattern of endopolygalacturonase bound by means of epoxy groups was modulated exclusively by the porosity of the support, whereas the binding alone did not play any role.     

Chemical modification of a beta-glucosidase from Schizophyllum commune: evidence for essential carboxyl groups

The beta-glucosidase from Schizophyllum commune was purified to homogeneity by a modified procedure that employed Con A-Sepharose. The participation of carboxyl groups in the mechanism of action of the enzyme was delineated through kinetic and chemical modification studies. The rates of beta-glucosidase-catalyzed hydrolysis of p-nitrophenyl-beta-D-glucoside were determined at 27 degrees C and 70 mM ionic strength over the pH range 3.0-8.0. The pH profile gave apparent pK values of 3.3 and 6.9 for the enzyme-substrate complex and 3.3 and 6.6 for the free enzyme. The enzyme is inactivated by Woodward's K reagent and various water-soluble carbodiimides; chemical reagents selective for carboxyl groups. Of these reagents, 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide iodide in the absence of added nucleophile was the most effective and a kinetic analysis of the modification indicated that one molecule of carbodiimide is required to bind to the beta-glucosidase for inactivation. Employing a tritiated derivative of the carbodiimide, 44 carboxyl groups in the enzyme were found to be labelled while the competitive inhibitor deoxynojirimycin protected three residues from modification. Treatment of the enzyme with tetranitromethane resulted in the modification of five tyrosine residues with approx. 28% diminution of enzymic activity. Titration of denatured enzyme with dithiobis(2-nitro-benzoic acid) indicated the absence of free thiol groups. Reaction of the enzyme with diethyl pyrocarbonate resulted in the modification of four histidine residues with the retention of 78% of the original enzymatic activity. The divalent transition metals Cu2+ and Hg2+ were found to be potent inhibitors of the enzyme, binding in an apparent irreversible manner.     

Interaction between chitosan and uranyl ions: Part 1. Role of physicochemical parameters

This work is devoted to the comprehension of the sorption mechanism of uranyl ions on chitosan particle dispersions. The uranyl concentration measurements were obtained by inductively coupled plasma atomic emission spectrometry (ICP-AES) and we considered the role of various physicochemical parameters (pH; nature and concentration of added salts; degree of acetylation, DA). The use of appropriate calculation software allowed us to determine the chemical nature of uranyl species in solution in relation to these different parameters. The optimal pH of fixation has been found to be within 6.5–7.5 and can be related to the necessity of having both deprotonated amino groups and no carbonate ions, which are a strong complexant of uranyl ions, thus inhibiting their interaction with chitosan. The decrease of metal uptake with an increase of DA and the lack of influence of ionic strength, confirm the results obtained with pH and allowed us to suppose the formation of a complex with chitosan amino groups rather than interactions of an electrostatic nature.