Acid-base and metal ion-binding properties of flavin mononucleotide (FMN). Is a ‘dielectric’ effect responsible for the increased complex stability?

Due to contradictions in the literature we have redetermined the acid-base properties of riboflavin (=RiFl; vitamin B₂), i.e. 7,8-dimethyl-10-ribityl-isoalloxazine, and of flavin mononucleotide (FMN²⁻), also known as riboflavin 5′-phosphate, via potentiometric pH titrations (I = 0.1 M, NaNO₃; 25 °C). In contrast to various claims, the isoalloxazine ring cannot be protonated at pH > 1, a result in agreement with an early study (pK_a = −0.2; L. Michaelis, M.P. Schubert and C.V. Smythe, J. Biol. Chem., 116 (1936) 587–607); deprotonation of the ring system occurs in both compounds with pK_a 10. The pK_a value of 0.7 determined for the deprotonation of H₂(FMN) must be attributed to the release of the first proton from the fully protonated phosphate group; its second proton is released with pK_a = 6.18 in agreement with the acidity constants of various other monoprotonated monophosphate esters. The stability constants of the 1:1 complexes formed between Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺ or Cd²⁺ (---M²⁺) and FMN²⁻ were determined by potentiometric pH titrations in aqueous solution (I = 0.1 M, NaNO₃; 25 °C). The log stability constants of all these M(FMN) complexes are about 0.2 log units higher than expected from the basicity of the phosphate group. This slight stability increase cannot be attributed to the formation of a seven-membered chelate involving the ribit-hydroxy group at C-4′ as the stability constants for the M²⁺ 1:1 complexes of glycerol 1-phosphate (G1P²⁻) demonstrate: G1P²⁻ contains the same structural unit which would also allow in this case the formation of the mentioned seven-membered chelate; however, the stability of the M(G1P) complexes is solely determined by the basicity of the phosphate group. Hence, in agreement with earlier conclusions (J. Bidwell, J. Thomas and J. Stuehr, J. Am. Chem. Soc., 108 (1986) 820–825) regarding Ni(FMN) one must conclude that the slight stability increase of the M(FMN) complexes has to be attributed to the isoalloxazine ring. The equality of the stability increase of the complexes for all the mentioned ten metal ions precludes its attribution to an interaction with an N site and makes a specific interaction with an O site also somewhat unlikely. In addition, carbonyl oxygens appear as not very favorable for the formation of macrochelates by a further interaction with already phosphate-coordinated metal ions. Therefore, we propose that the slight but significant stability increase originates from M(FMN) species (with a formation degree of about 30%) in which the hydrophobic flavin residue is close to the metal ion, thereby lowering the ‘effective’ dielectric constant in the microenvironment of the metal ion and thus indirectly promoting the −PO₃²⁻/M²⁺ interaction.     

Studies on the exchange of G-actin-bound calcium with bivalent cations

1. The possibility of the replacement of G-actin-bound calcium by various bivalent cations has been investigated. After the reaction with all cations studied, with the exception of Cu²⁺, action remains active, i.e., contains bound ATP and polymerizes in 0.1 M KCl.

2. The amount of G-actin-bound calcium, as well as the sum of bivalent cation after replacement, not removable by short-time Dowex-50 treatment, accounts to about 1 mole per 50000 g of G-actin.

3. The rate of exchange is of the same order for bivalent cations studied, including calcium.

4. G-actin-bound Ca²⁺ is fully replaced, besides free Ca²⁺, by free Mn²⁺ and Cd²⁺. The replacement with Mg²⁺, Co²⁺, Ni²⁺ and Zn²⁺ is not complete, and there is practically no reaction with Ba²⁺ and Sr²⁺.

5. Assuming the affinity constant of Ca²⁺ as 1, the following affinity constants for other bivalent cations were obtained: Mn²⁺, 0.90; Cd²⁺, 1.07; Mg²⁺, 0.27; Zn²⁺, 0.22; Co²⁺, 0.18; Ni²⁺, 0.08.

6. The results obtained show that there exists a close correlation between the ionic radius of a particular bivalent cation, and its ability to replace bound Ca²⁺.     

Characterisation and selectivity of divalent metal ions binding by citrus and sugar-beet pectins

A scale of selectivity for the binding of calcium and some heavy metal ions by citrus and sugar-beet pectins was set up by pH-measurements. The same order of selectivity was found for the two pectins, decreasing as follows: Cu²⁺ Pb²⁺ Zn²⁺ > Cd²⁺ Ni²⁺ ≥ Ca²⁺. Binding isotherms for Ca²⁺, Cu²⁺, Ni²⁺, Pb²⁺ and Zn²⁺ ions have shown a greater binding level when the ionic strength decreased and when the pectin concentration increased in the presence of 0.1 M NaNO₃. By comparing binding isotherms, the same order of selectivity was found as by pH-studies. Scatchard plots and Hill index evaluation showed for all ions and all pectins anticooperative interactions in water. In the presence of 0.1 M NaNO₃, citrus pectins displayed cooperative interactions for all metal ions. In contrast, for sugar-beet pectins, cooperative interactions only occured with Cu²⁺ and Pb²⁺. With Ca²⁺, Ni²⁺ and Zn²⁺ sugar-beet pectins displayed Scatchard plots which could not be distinguished from an anticooperative binding. This difference of behaviour could be related to the presence of acetyl groups decreasing the affinity of Me²⁺ for sugar-beet pectins.     

Improvement of the binding capacity of metal cations by sugar-beet pulp. 2. Binding of divalent metal cations by modified sugar-beet pulp

Binding of some divalent cations (Ca²⁺, Cd²⁺, Cu²⁺, Ni²⁺, Pb²⁺ and Zn²⁺) in aqueous solution by saponified and cross-linked sugar-beet pulp was investigated. Saponification doubled the cation-exchange capacity, while cross-linking decreased specific surface area and hydration properties to low and stable values independent of pH and ionic strength conditions. The sorption isotherms indicated a high metal-binding capacity which increased with sorbent concentration, and followed a clear order of selectivity: Cu²⁺˜Pb²⁺ Zn²⁺˜Cd²⁺ > Ni²⁺ > Ca²⁺. The sorption data were better represented by the Langmuir isotherm than by the Freundlich one, suggesting that the monolayer sorption, mainly due to ion-exchange, would not be disturbed by lateral interactions between cations sorbed with similar sorption energies. The same order of selectivity could be drawn from the Langmuir parameters, sorption equilibrium constants (K_L) and maximum binding capacities (Me_Amax). Whatever the cation, K_L decreased with increasing sorbent concentration, while Me_bmax increased. Higher quantities of Cu²⁺ and Pb²⁺ than predicted by the one divalent cation to two carboxyl functions ratio were bound. This was attributed to the partial contribution to the sorption phenomenon of hydroxyl functions close to ionic sites, explaining the higher affinity of such cations for substrates. Cross-linked pulp exhibited higher metalbinding capacity per volume unit than the raw pulp.     

Stability constants of metal complexes of phosphonoacetic acid

The antiviral drug, phosphonoacetic acid (PAA), forms stable complexes with Mg²⁺, Ca²⁺, Cu²⁺ and Zn²⁺. Stability constants of these complexes were determined in aqueous solution (0.15 M in KNO₃, 37°) by potentiometric titration. Mixed ligand complex formation of Cu²⁺ and Zn²⁺ with PAA and glycinate ion, and with PAA and histidinate ion, was studied. In a theoretical model for blood plasma, PAA affects the distribution of Mg²⁺ and, to a lesser extent, Ca²⁺.     

Influence of co-cations on biosorption of lead and zinc – a comparative evaluation in binary and multimetal systems

The influence of co-cations (cadmium, copper, cobalt and nickel) on lead and zinc biosorption by Streptoverticillium cinnamoneum and Penicillium chrysogenum in binary and multimetal systems was evaluated. The metal sorption capacity of S. cinnamoneum was higher than P. chrysogenum for all the metals tested. Both the biomasses exhibited preferential uptake of lead in a multimetal situation. Even though mutual inhibition was seen for all binary systems containing zinc, systems containing lead exhibited unequal inhibition. The extent of metal sorption was dependent on metal chemistry, affinity for binding sites and the type of metal binding. In multimetal systems, S. cinnamoneum and P. chrysogenum exhibited preferential sorption orders: Pb²⁺ > Zn²⁺=Cu²⁺ > Cd²⁺ > Ni²⁺ > Co²⁺ and Pb²⁺ > Cu²⁺ > Zn²⁺ > Cd²⁺ > Ni²⁺ > Co²⁺. The order of metal biosorption in a multimetal system could be predicted well on the basis of Langmuir parameters evaluated in binary metal systems.     

A new macrocyclic tetraamine, 2,4-dinitrophenylcyclen (= 1-(2,4-dinitrophenyl)-1,4,7,10-tetraazacyclododecane):synthesis, cation reporter properties, and recognition of 1-methylthymine by its zinc(II) complex

A new functional macrocyclic ligand, 2,4-dinitrophenylcyclen (= 1-(2,4-dinitrophenyl)-1,4,7,10-tetraazacyclododecane), has been synthesized and isolated as its trihydrochloric acid salt (L·3HCl). The protonation constants (log K_n) for three secondary nitrogens of L were determined by potentiometric pH titration to be 10.10, 7.33 and <2 with I = 0.10 (NaNO₃) at 25°C. The 2,4-dinitrophenylaniline chromophore was proven to be a good reporter signaling proton- and metal-binding events in the macrocyclic cavity. The UV absorption band (λ_max 370 nm, 8200) of the 2,4-dinitrophenylaniline moiety at pH ≥ 9 becomes quenched as pH is lowered (to pH 3.1, where the major species is L·2H⁺), due to the strong protonation effect extended to the aniline moiety within the macrocyclic cavity. This is in sharp contrast to the pH-independent UV absorption (λ_max 390 nm, 14 000) of a reference compound, N,N-diethyl-2,4-dinitroaniline. The UV absorption band of L is shifted to lower wavelengths with Zn²⁺ (λ_max 320 nm), Cd²⁺ (λ_max 316 nm) and Pb²⁺ (λ_max 317 nm), while it almost disappears with Cu²⁺ and Ni²⁺. The 1:1 Zn²⁺ and Cu²⁺ complexes with L were isolated and characterized. The Zn²⁺ complex recognizes 1-methylthymine anion (MT⁻) in aqueous solution at physiological pH to yield a stable ternary complex ZnL-MT⁻. The X-ray crystal structure of ZnL-MT⁻ showed that Zn²⁺ is four-coordinate with three secondary nitrogens of L and the deprotonated imide anion that is cofacial to the 2,4-dinitrophenyl ring.     

Activities and relative affinities of divalent metals in unmodified and phosphorothioate-substituted hammerhead ribozymes

The roles of metals in the phosphodiester bond cleavage reaction performed by the hammerhead ribozyme are under investigation. In this study, the apparent affinities and the abilities of several different metals to support ribozyme activity are reported. The relative affinities of divalent cations for the hammerhead ribozyme are determined by measuring their ability to release bound Mn²⁺. The EPR-detected Mn²⁺ competition studies give an order of apparent affinity of Mn²⁺ Co²⁺ Zn²⁺>Cd²⁺Mg²⁺. This ordering generally follows the trend of maximum rates of cleavage determined at pH 7.0, 0.1 M NaCl, and saturating metal concentrations, of Mn²⁺>Co²⁺>Cd²⁺>Mg²⁺. The maximum rate is observed for Mn²⁺ under these conditions and may be related to the high affinity, low pK_a and low ΔH_hyd of this ion. Substitution of phosphorothioates 5′ to each of the nine adenosines in the enzyme strand yields a change in the Mn²⁺ binding properties of the hammerhead complex. In the phosphorothioate-substituted hammerhead complex, eight to nine Mn²⁺ bind in two types of classes: ‘type 1’ (n=1±0.3, K_d=1.1±1 μM) and weaker ‘type 2’ (n=7.7±0.3, K_d=125±27 μM). The multiple phosphorothioate substitutions result in the loss of two to three of the higher affinity sites observed in the unmodified ribozyme. Metal competition studies with the phosphorothioate-substituted ribozyme indicate that the relative affinities of the metals are Cd²⁺>Zn²⁺>Co²⁺, Mg²⁺ with the number of Mn²⁺ displaced and apparent affinity of the thiophilic Cd²⁺ most affected by the phosphorothioate substitutions.     

Trypsin immobilization by direct adsorption on metal ion chelated macroporous chitosan-silica gel beads

Silica gel bead coated with macroporous chitosan layer (CTS-SiO₂) was prepared, and the metal immobilized affinity chromatographic (IMAC) adsorbents could be obtained by chelating Cu²⁺, Zn²⁺, Ni²⁺ ions, respectively on CTS-SiO₂, and trypsin could be adsorbed on the IMAC adsorbent through metal–protein interaction forces. Batch adsorption experiments show that adsorption capacity for trypsin on these IMAC adsorbent variated with change of pH. The maximal adsorption reached when the solution was in near neutral pH in all three IMAC adsorbents. Adsorption isothermal curve indicated that maximal adsorption capacity could be found in the Cu²⁺-CTS-SiO₂ with the value of 4980 ± 125 IU g⁻¹ of the adsorbent, while the maximal adsorption capacity for trypsin on Zn²⁺ and Ni²⁺ loaded adsorbent was 3762 ± 68 IU g⁻¹ and 2636 ± 53 IU g⁻¹, respectively. Trypsin immobilized on the IMAC beads could not be desorbed by water, buffer and salt solution if the pH was kept in the range of 5–10, and could be easily desorbed from the IMAC beads by acidic solution and metal chelating species such as EDTA and imidazole. The effect of chelated metal ions species on CTS-SiO₂ beads on the activity and stability of immobilized trypsin was also evaluated and discussed. Trypsin adsorbed on Zn-IMAC beads retained highest amount of activity, about 78% of total activity could be retained. Although the Cu-IMAC showed highest affinity for trypsin, only 25.4% of the calculated activity was found on the beads, while the activity recovery found on Ni-IMAC beads was about 37.1%. A remarkable difference on stability of trypsin immobilized on three kinds of metal ion chelated beads during storage period was also found. Activity of trypsin on Cu-IMAC decreased to 24% of its initial activity after 1-week storage at 4 °C, while about 80% activity was retained on both Ni-IMAC and Zn-IMAC beads. Trypsin immobilized on Zn-CTS-SiO₂ could effectively digest BSA revealed by HPLC peptide mapping.     

Catalysis of catechol oxidation by metal-dithiocarbamate complexes in pesticides

Dithiocarbamate (DTC)-based pesticides have been implicated in Parkinson’s disease (PD) through epidemiological links to increased risk of PD, clinical reports of parkinsonism following occupational exposure to the DTC-based pesticide maneb, and experimental studies showing dopaminergic neurodegeneration with combined exposure of rats to maneb and paraquat. We hypothesize that the manganese-ethylene-bis-dithiocarbamate (MnEBDC) complex in maneb may produce oxidative stress by catalyzing catechol oxidation. We tested this hypothesis by performing a structure-function analysis of metal-EBDC and metal-diethyldithiocarbamate (DEDC) complexes of Mn²⁺, Zn²⁺, and Cu²⁺ to catalyze oxidation of N-acetyldopamine (NA-DA) and 3,4-dihydroxyphenylacetic acid (DP) in the presence and absence of N-acetylcysteine (NAC), a model of glutathione. Both Mn-DTCs retained the capacity of the parent ion to catalyze one-electron oxidation of NA-DA, but lost the ability to catalyze DP oxidation. Strikingly, while Zn²⁺ did not catalyze catechol oxidation, both Zn-DTCs catalyzed one-electron oxidation of NA-DA but not DP. While Cu²⁺ catalyzed oxidation of both catechols, Cu-DTCs were inert. Similar results were obtained with MnEBDC and dopamine or norepinephrine; however, zinc-ethylene-bis-dithiocarbamate was less efficient at catalyzing oxidation of these catechols. Our results point to the potential for manganese- and zinc-containing EBDC pesticides to promote oxidative stress in catecholaminergic regions of the brain.     

Ovotransferrin possesses SOD-like superoxide anion scavenging activity that is promoted by copper and manganese binding

The embryo of oviparous species is confronted by a highly oxidative stress generating as it grows and must rely on effective antioxidant system for protection. Proteins of avian egg albumin have been suggested to play the major redox-modulatory role during embryo development. Recently, we found that ovotransferrin (OTf) undergoes distinct thiol-linked self-cleavage in a redox-dependent process. In this study, we explore that OTf is SOD mimic protein with a potent superoxide anion (O₂⁻) scavenging activity. The O₂⁻ scavenging activity was investigated using the natural xanthine/xanthine oxidase (X/XOD) coupling system. OTf exhibited O₂⁻ scavenging activity in a dose-dependent manner and showed remarkably higher scavenging activity than the known antioxidants, ascorbate or serum albumin. The isolated half-molecules of OTf exhibited higher scavenging activity than the intact molecule, whereas the N-lobe showed much greater activity. OTf dramatically quenched the O₂⁻ flux but had no effect on the urate production in the X/XOD system, indicating its unique specificity to scavenge O₂⁻ but not oxidase inhibition. Strikingly, metal-bound OTf exhibited greater O₂⁻ dismutation capacity than the apo-protein, ranging from moderate (Zn²⁺-OTf and Fe²⁺-OTf) to high (Mn²⁺-OTf and Cu²⁺-OTf) activity with the Cu²⁺-OTf being the most potent scavenger. In a highly sensitive fluorogenic assay, the metal-bound OTf exhibited significant increase in the rate of H₂O₂ production in the X/XOD reaction than the apo-OTf, providing evidence that Zn²⁺-, Mn²⁺- and Cu²⁺-OTf possess SOD mimic activity. This finding is the first to describe that OTf is an O₂⁻ scavenging molecule, providing insight into its novel SOD-like biological function, and heralding a fascinating opportunity for its potential candidacy as antioxidant drug.     

食品添加剂和金属离子对高粱泡红色素稳定性的影响

探讨了5种常用食品添加剂和7种金属离子对高粱泡红色素稳定性的影响。结果表明:葡萄糖、蔗糖和苯甲酸钠对高粱泡红色素稳定性无不良影响,其中葡萄糖、蔗糖有不同程度的增(护)色效果;柠檬酸能显著提高色素的稳定性;而VitC能促进色素的氧化降解,有明显的破坏作用。金属离子中,Na⁺、Mg²⁺、Al³⁺、Zn²⁺等对高粱泡红色素稳定性无影响,且有一定增色作用;较高浓度(≥0.0025mol/L)Mn²⁺有一定不良影响,而Cu²⁺、Fe³⁺则有明显的破坏作用。     

Inactivation of chloroplast photosynthetic electron-transport activity by Ni

Ni²⁺ inhibits electron-transport activity of isolated barley chloroplasts and this inhibition of electron transport by Ni²⁺ is distinctly different from other heavy metal ion (e.g., Pb²⁺, Cd²⁺, Zn²⁺)-induced inhibition of chloroplast function. Ni²⁺ inactivates Photosystem II (PS II) activity at a lower concentration than that required for the same extent of inhibition of Photosystem I (PS I)-mediated electron flow. Ni²⁺ induces changes in chlorophyll a (Chl a) emission characteristics and brings about a lowering of the Chl a fluorescence yield, and this lowering of Chl a fluorescence intensity is not relieved by the exogenously supplied electron donor NH₂OH which donates electrons very close to the PS II reaction centres. Immobilization of the chloroplast membrane structure with glutaraldehyde fails to arrest the Ni²⁺-induced loss of PS II activity. Also, Ni²⁺-treated chloroplasts do not regain the ability to photoreduce 2,6-dichlorophenolindophenol even after washing of chloroplasts with buffer. These results indicate that unlike Zn²⁺ or Pb²⁺, Ni²⁺ induces alterations in the chloroplast photosynthetic apparatus resulting in an irreversible loss of electron-transport activity.     

Manganese and copper promote the binding of dopamine to “serotonin binding proteins” in bovine frontal cortex

The authors previously reported that Fe²⁺ is capable of increasing the binding of dopamine and of serotonin to “serotonin binding proteins” which are present in soluble extracts from calf brain. In this study, it is shown that Mn²⁺ and Cu²⁺ are also capable of increasing the binding, but for dopamine only. As for Fe²⁺, Mn²⁺ and Cu²⁺ are likely to promote the binding by virtue of their ability to enhance the oxidation of dopamine into dopamine-O-quinone, a derivative which is known to undergo covalent association with sulfhydryl groups of proteins. Data such as the irreversible nature of the majority of the binding, the inhibitory action of reducing agents (sodium ascorbate) and of reagents which contain, or modify sulfhydryl groups (reduced glutathione) are compatible with such a mechanism. The three metal ions are also capable of inactivating part of the binding sites on SBP directly; this effect is more pronounced for Cu²⁺ than for Fe²⁺ and it is only weak for Mn²⁺. The Fe²⁺-mediated binding of dopamine is inhibited by the superoxide dismutase enzyme, and it was therefore suggested that Fe²⁺ enhances the oxidation of dopamine by virtue of its ability to produce superoxide radicals out of dissolved molecular oxygen. Such a mechanism does not appear to take place in the case of Mn²⁺ and Cu²⁺. Instead, it is likely that Cu²⁺ and dopamine form a complex which is highly susceptible towards oxidation by dissolved molecular oxygen. Mn²⁺, on the other hand, can easily be oxidized into Mn³⁺, which is capable to oxidize dopamine by itself. Chronic manganese intoxication (from exposure to manganese) and Wilson's disease (related to inadequate elimination of copper) go along with neurological symptoms which are very similar to those encountered in Parkinson's disease. Our data indicate that manganese and copper ions accelerate the oxidation of catecholamines to produce toxic quinones. These quinones could, at least in part, account for the degeneration of dopamininergic neurons in such pathologies.     

Binding of ions to chitosan—selectivity studies

Selectivity coefficients for binding of negative and positive ions to chitosans of different chemical composition have been determined by equilibrium dialysis. Chitosans with different fraction of acetylated units (F_A of 0.01 and 0.49) generally behaved similarly in their selectivity towards both negative and positive ions. No selectivity was found in the binding of chloride and nitrate ions, while chitosan showed a strong selectivity towards molybdate polyoxyanions, with selectivity coefficients around 100. Chitosan showed a strong selectivity towards copper (Cu²⁺) compared to the metal ions zinc (Zn²⁺), cadmium (Cd²⁺) and nickel (Ni²⁺), with selectivity coefficients from 10 to 1000, while little or no selectivity could be detected with the other metal ions. Ionic strength and pH did not influence the selectivity coefficients of the chitosans towards the metal ions.     

Thermostability of β-xylosidase from Aspergillus sydowii MG49

Heating of Aspergillus β-xylosidase at 85°C ± 1°C and pH 5.5–6.0 (optimum for activity), causes irreversible, covalent thermoinactivation of the enzyme, involving oxidation of the thiol groups that are required for catalysis. Exogenous addition of cysteine, DTT, GSH and mercaptoethanol stabilizes the enzyme by extending its half-life. A similar effect is also exhibited by bivalent cations like Mg²⁺, Mn²⁺, Co²⁺, Ca²⁺and Zn²⁺ while, on the other hand Cu²⁺ accelerates thermoinactivation. Chemical modification of crude β-xylosidase with cross-linking agents like glutaraldehyde or covalent immobilization to a nonspecific protein like gelatin and BSA also enhances enzyme thermostability. These results suggest that addition of thiols and bivalent metal ions to a crude β-xylosidase preparation or immobilization/chemical modification enhances its thermal stability, thus preventing loss of catalytic activity at elevated temperatures.     

Binding of copper and zinc ions to polypeptides containing glutamic acid and tyrosine residues

The binding of Cu⁺⁺ and Zn⁺⁺ ions to three polypeptides containing Glu and Tyr residues, (Glu Tyr Glu)_n, (Glu-Glu-Tyr-Glu)_n and (Glu-Tyr-Tyr-Glu)_n has been investigated by absorption spectroscopy, fluorescence and circular dichroism. Difference absorption spectra show that Zn^+- slightly perturbs the absorption spectrum of the tyrosyl residue whereas Cu⁺⁺ binding is accompanied by the appearance of a strong absorption band around 245 nm. The fluorescence of the tyrosyl residue is enhanced by Zn⁺⁺ ions while it is quenched by Cu⁺⁺ ions. Cation binding induces a conformational change of the polypeptides from a random coil to an -helix, Mg⁺⁺ ions do not elicit any of these phenomena.     

The use of chemically modified and unmodified cassava waste for the removal of Cd, Cu and Zn ions from aqueous solution

The use of different chemically modified cassava waste biomass for the enhancement of the adsorption of three metal ions Cd, Cu and Zn from aqueous solution is reported in this paper. Treating with different concentrations of thioglycollic acid modified the cassava waste biomass.

The sorption rates of the three metals were 0.2303 min⁻¹ (Cd²⁺), 0.0051 min⁻¹ (Cu²⁺), 0.0040 min⁻¹ (Zn²⁺) and 0.109 min⁻¹ (Cd²⁺), 0.0069 min⁻¹ (Cu²⁺), 0.0367 min⁻¹ (Zn²⁺) for 0.5 and 1.00 M chemically modified levels, respectively. The adsorption rates were quite rapid and within 20–30 min of mixing, about 60–80% of these ions were removed from the solutions by the biomass and that chemically modifying the binding groups in the biomass enhanced its adsorption capacity towards the three metals. The results further showed that increased concentration of modifying reagent led to increased incorporation, or availability of more binding groups, in the biomass matrix, resulting in improved adsorptivity of the cassava waste biomass. The binding capacity study showed that the cassava waste, which is a serious environmental nuisance, due to foul odour released during decomposition, has the ability to adsorb trace metals from solutions.     

Purification and characterization of an endocellulase from the thermophilic fungus Chaetomium thermophilum CT2

Chaetomium thermophilum CT2 produced endocellulases at 50 °C, when grown on 2% microcrystalline cellulose, 1% soluble starch, and 0.4% yeast extract medium. A major endocellulase component was purified to homogeneity by fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE-Sepharose, Phenyl-Sepharose hydrophobic interaction chromatography and gel filtration on Sephacryl S-100. The molecular weight of the enzyme was estimated to be 67.8 kDa and the enzyme was found to be a glycoprotein containing 18.9% carbohydrate. The K_m of the purified enzyme for carboxymethyl cellulose, sodium salt (CMC), was 4.6 mg ml⁻¹. The enzyme displayed highest activity towards CMC and significantly lower activities towards phosphoric acid swollen cellulose and filter paper. The activity was enhanced in the presence of Na⁺, K⁺ and Ca²⁺ but inhibited by Hg²⁺, Zn²⁺, Ag⁺, Mn²⁺, Ba²⁺, Fe²⁺, Cu²⁺, Mg²⁺ and NH₄⁺. Optimum activity was at 60 °C and pH 4.0. The enzyme was stable over 60 min incubation at 60 °C and half-life at 70, 80 and 90 °C was approximately 45, 24 and 7 min, respectively.     

Specific effect of manganese on the allantoinase of Vigna Radiata

Vigna radiata seedlings germinated in the presence of Mn²⁺ show an unusual increase in allantoinase activity which is proportional to Mn²⁺ concentration up to 5 mM. Though Mn²⁺ is not an activator for V. radiata allantoinase, it specifically protects allantoinase against thermal as well as papain-catalysed inactivation. Evidence is presented to show that the primary effect of Mn²⁺ is a protective one, both in vitro and in vivo, and that this is reflected in the observed enhancement of allantoinase activity in Mn²⁺ grown seedlings. That this unusual effect of Mn²⁺ is a specific one is indicated by the lack of a similar effect with Mg²⁺. Cu²⁺ is shown to destabilize V. radiata allantoinase in vitro as well as in vivo.