All-reagent test tablets and method for rapid and selective alpha-amylase iodometric determination

The new type of test tablets (Iodocrom) for alpha-amylase assay contain crosslinked (CL)-amylose or CL-starch (specific substrates for alpha-amylase only) and the reagent (KIO3/KI) generating iodine (in acidic medium, when the reaction is stopped). The method--amyloclastic in nature--is based on selective action of alpha-amylase on the CL-substrate liberating soluble polysaccharide chains large enough and in a conformation suitable to allow the formation of iodine inclusion complexes. Unlike the classical iodometric methods, the reaction is followed by an increase in iodine complex blue color. The method has some common points with the well-known chromogenic (e.g., Phadebas) methods. Both use insoluble substrates which are not susceptible to attack by exoamylases and in both cases the enzymatic reaction is followed by the release of soluble products. The amounts of these released chains and the absorbances of their inclusion complexes with iodine are in a linear dependence with the enzyme concentration (activity).     

Anion effects on the reaction of lecithin-cholesterol acyltransferase with discoidal complexes of phosphatidylcholines . apolipoprotein A-I . cholesterol

Discoidal complexes of phosphatidylcholine (PC) . apolipoprotein A-I . cholesterol were prepared with egg PC, palmitoyloleoylPC, dipalmitoylPC, or dimyristoylPC, and were used as substrates of purified lecithin-cholesterol acyltransferase to investigate the effects of neutral salts on the enzymatic reaction. Sodium fluoride, chloride and bromide concentrations up to 1 M, did not affect the properties of the substrate particles, but caused marked and distinct changes in the activity of the enzyme with the various PC particles. The effects of salts were largely due to the anions, which followed the order of the lyotropic series in their inactivating capacity: F- less than Cl- less than Br- less than NO3- less than I- less than SCN-. Sodium salts (F-, Cl-, and Br-) produced a very large increase in the pH optimum of the enzymatic reaction (7.4 to at least 8.5) essentially obliterating the ionization of a functional group with pK of 8.1. The kinetics of the enzymatic reaction revealed major differences among the PC particles, and different responses of their kinetic parameters with increasing salt concentrations. The conclusions reached in this work are the following: (1) The relative reactivity of PC substrates, in discoidal particles, with lecithin-cholesterol acyltransferase depends strongly on the concentration and type of salts in the medium. (2) Anions (in lyotropic series) rather than cations affect the enzymatic reaction. (3) There are functional groups with pK of 8.1 which are affected markedly in their ionization behavior by anion binding. (4) The active site of lecithin-cholesterol acyltransferase and its interaction with anions are affected by the exact nature of the PC-apolipoprotein interface.     

Anion effects on equilibria and kinetics of the disorder–order transition of κ-carrageenan

The effect of a number of tetramethylammonium salts on the equilibria and kinetics of the disorder to order transition in the polysaccharide κ-carrageenan have been investigated. Data from the temperature dependence of optical rotation show that anion stabilization of the ordered form follows the lyotropic series I^? > Br^? > NO > Cl^? > F^?. Stopped-flow polarimetry was used to study the kinetics of conformational ordering following a rapid increase in salt concentration. The transition to the new equilibrium position was shown to be biphasic for all of the tetramethylammonium salts studied. The rate equation for the fast phase and the temperature dependence of the observed forward rate constant accord with a cooperative dimerization process. Activation parameters for helix nucleation, ΔH^* and ΔS^*, vary with both salt concentration and (at constant ionic strength) the anion type, increasing through the lyotropic series from I^? to F^?. The slow phase shows second-order kinetics, and is interpreted as further stabilization of the ordered form either through limited aggregation or annealing. The rate constant for the slow phase also follows the lyotropic series. Thus we have shown that both the growth and nucleation processes are anion dependent.     

Electrostatic Interaction between Plastocyanin and P700 in the Electron Transfer Reaction of Photosystem I-Enriched Particles

The nature of the electron transfer reaction between reducedplastocyanin and P700 oxidized by flash illumination was studiedin P700-enriched Triton subchloroplast fraction 1 particles.An addition of monovalent salts to the suspension at neutralpH increased the reaction rate at low concentrations (>20mM). Salts of divalent cations showed a similar effect at muchlower concentrations (>2 mM), This effect was not dependenton the concentration and the valence of anions. The increaseof rate at low salt concentrations was observed at pH's above5, but below pH 5 the rate was decreased by adding salts. Atabout pH 5, the rate was not affected by salts. Apart from thesesalt effects, the optimum pH for the reaction rate was observedbetween 5.5 and 6.5. The reduction rate depended sigmoidally on the added plastocyaninconcentration at pH 6.8 and 4. A Michaelis-Menten type relationshipwas observed at about pH 5. The half-saturation concentrationof plastocyanin became lower as the salt concentration increasedat pH 6.8, while it became higher by adding salt at pH 4. The effects of salts on the rate of electron donation from othermetalloproteins and artificial electron donors to P700 werealso studied. It is concluded, from the analysis with the Gouy-Chapmantheory, that the net charges on the electron donors and themembrane surfaces mainly determine the response of the P700reduction rate to salt addition. The salt addition changes mainlythe local concentration or accessibility of electron donorsto P700. (Received January 12, 1981; Accepted March 6, 1981)     

Effects of divalent cations on bovine testicular hyaluronidase catalyzed transglycosylation of chondroitin sulfates

Glycosaminoglycans were prepared as salts of different divalent cations and tested as donors in bovine testicular hyaluronidase catalyzed transglycosylation reactions. All of the metal cations examined had similar binding efficiency of divalent cations to hyaluronan. However, cations bound with different efficiencies to chondroitin sulfate species and the differences were marked in the case of chondroitin 6-sulfate; the numbers of cations bound per disaccharide unit were estimated to be 0.075 for Mn, 1.231 for Ba, 0.144 for Zn, and 0.395 for Cu. While barium salt of chondroitin sulfates enhanced transglycosylation, the zinc salt of chondroitin sulfates inhibited transglycosylation. Therefore, by selecting the proper divalent cation salt of chondroitin sulfates as a donor in the transglycosylation reaction it is possible to improve the yields of the products.     

Factors relevant in the reaction of pyrroloquinoline quinone with amino acids. Analytical and mechanistic implications

In order to reveal the stability of pyrroloquinoline quinone (PQQ) in complex samples, its reaction on incubation with amino acids was followed spectrophotometrically by monitoring oxygen consumption, and with a biological assay. For several alpha-amino acids, the formation of a yellow coloured compound (lambda max = 420 nm) was accompanied by oxygen uptake and disappearance of biological activity from the reaction mixture. The yellow product appeared to be an oxazole of PQQ, the exact structure depending on the amino acid used. Oxazole formation also occurred under anaerobic conditions with concomitant formation of PQQH2, suggesting that PQQ is able to oxidize the presumed oxazoline to the oxazole. Besides the condensation reaction, there is also a catalytic cycle in which an aldimine adduct of PQQ and the amino acid is converted into the aminophenol form of the cofactor and an aldehyde resulting from oxidative decarboxylation of the amino acid. Addition of NH4+ salts, as well as that of certain divalent cations, greatly stimulated both the cyclic and the linear reaction. With basic amino acids, oxazole formation scarcely occurred. However, as oxygen consumption was observed (provided that certain divalent cations were present), conversion of these compounds took place. A reaction scheme is proposed accounting for the products formed and the effects observed. Since NH4+ ions activate several quinoproteins (PQQ-containing enzymes) and divalent cations (Ca2+, Fe2+, and Cu2+) are additional (co)factors in certain metallo quinoproteins, the effects of metal ions observed here could be related to the mechanistic features of these enzymes. Although all oxazoles were converted to PQQ by acid hydrolysis, PQQ was not detected when hydrolysis was carried out in the presence of tryptophan, a compound which appeared to have a deleterious effect on the cofactor under this condition. The results here described explain why analysis methods for free PQQ in complex samples fail in certain cases, or are not quantitative.     

Molecular dynamic simulations study of the effect of salt valency on structure and thermodynamic solvation behaviour of anionic polyacrylate PAA in aqueous solutions

The effect of salt concentration and valency on intermolecular structure and solvation thermodynamic properties of aqueous solution containing polyacrylicacid (PAA) chains and multi-valent salts calcium chloride (CaCl₂) and aluminium chloride (AlCl₃) as a function of charge density was investigated using atomistic molecular dynamic simulations with explicit solvent. Salt-free solution favours the self-association of uncharged (acidic form) PAA chains facilitated by inter-chain hydrogen bonds. The ionised (charged) PAA chains are not associated in salt-free aqueous solutions and undergo self-association in the salt solutions due to bridging effect induced by condensed salt ions in agreement with scattering investigations available in literature. The collapse behaviour of PAA in presence of CaCl₂ and re-expansion behaviour of PAA chains in case of AlCl₃ salt solutions are observed. The rigidity of PAA chains decrease with increase in salt concentration, in agreement with experimental results available in literature. The trivalent salt favours relatively the greater extent of shrinking of PAA chains as well as inter-chain interactions as compared to divalent salts as evident from radius-of-gyration, H-bond and pair-wise solvation enthalpy data. The conformation and hydration behaviour of the acid form of PAA chains are not significantly altered by added salt ions. The hydration behaviour of ionised PAA chains is significantly reduced by added salts due to screening effect of the condensed salt ions. The pair correlation functions of solutions species such as Ca²⁺, Al³⁺, Na⁺ and Cl^? with respect to PAA oxygen show the greater affinity of PAA units with the higher valency Al³⁺ ions over Ca²⁺ and Na⁺ in solution. With increase in concentration of AlCl₃ and CaCl₂ salts, a decrease in effective charge density of ionised PAA chains is observed from the existence of unfavourable PAA–water, PAA–Ca²⁺ and PAA–Al³⁺ interactions.     

Polynucleotide adsorption to negatively charged surfaces in divalent salt solutions

Polynucleotide adsorption to negatively charged surfaces via divalent ions is extensively used in the study of biological systems. We analyze here the adsorption mechanism via a self-consistent mean-field model that includes the pH effect on the surface-charge density and the interactions between divalent ions and surface groups. The adsorption is driven by the cooperative effect of divalent metal ion condensation along polynucleotides and their reaction with the surface groups. Although the apparent reaction constants are enhanced by the presence of polynucleotides, the difference between reaction constants of different divalent ions at the ideal condition explains why not all divalent cations mediate DNA adsorption onto anionic surfaces. Calculated divalent salt concentration and pH value variations on polynucleotide adsorption are consistent with atomic force microscope results. Here we use long-period x-ray standing waves to study the adsorption of mercurated-polyuridylic acid in a ZnCl2 aqueous solution onto a negatively charged hydroxyl-terminated silica surface. These in situ x-ray measurements, which simultaneously reveal the Hg and Zn distribution profiles along the surface normal direction, are in good agreement with our model. The model also provides the effects of polyelectrolyte line-charge density and monovalent salt on adsorption.     

Neutral salt effects on the velocity and activation volume of the lactate dehydrogenase reaction: Evidence for enzyme hydration changes during catalysis

The effects of different neutral salts on the maximal velocity (V) and activation volume ($\text{Δ}\text{V3}$) of the M₄-lactate dehydrogenase reaction were studied to determine the mechanistic basis of the inhibitory effects of these salts. For salting-in salts (which increase protein group solubility), increasing salt concentrations led to reductions in V and increases in $\text{Δ}\text{V3}$, with the order of salt effectiveness following the Hofmeister (lyotropic) series: KSCN > KI > KBr. A 50% reduction in V was associated with an approximately 17 cm³ mol^?1 increase in $\text{Δ}\text{V3}$ for different concentrations of the same salt and for equal concentrations of different salting-in salts. Salting-out salts were also inhibitory, but no uniform correlation between changes in V and $\text{Δ}\text{V3}$ was observed. The strongly salting-out salt KF decreased $\text{Δ}\text{V3}$ at all concentrations. The weaker salting-out salt K₂SO₄ increased $\text{Δ}\text{V3}$ at concentrations below 0.1 m and decreased $\text{Δ}\text{V3}$ at higher concentrations. KCl increased $\text{Δ}\text{V3}$ as the salt concentration was raised to approximately 0.2 m; further increases in KCl concentration were without effect on $\text{Δ}\text{V3}$. The rate and volume effects of these neutral salts, especially the highly regular covariation in V and $\text{Δ}\text{V3}$ found for salting-in salts, seem difficult to explain in terms of salt-induced changes in the geometry of the active site. We propose instead that these salt effects can all be explained in terms of the energy and volume changes which accompany transfers of protein groups (amino acid side chains and peptide backbone linkages) between the hydrophobic interior of the enzyme and the enzyme-water interface during catalytic conformational changes.     

The effect of ions on the superstructures of reconstituted collagen

The effect of a variety of electrolytes on the stabilization and destabilization of the rod-like superstructures in reconstituted collagen membranes have been studied by small-angle light scattering (SALE). Structural orders at two levels are effected: association–disassociation at the micron level and the helix-coil transitions at the mollecular level. In addition to lyotropic salts, several transition metal ions were also studied. They act to disassociate collagen aggregates by the formation of stable complexes.     

Continuum electrostatics of the C-peptide: anatomy of the problem.

A computational study of the role of all ionizable groups of the C-peptide in its helix-coil transition is performed within the framework of continuum electrostatics. The method employed in our computations involves a numeric solution of the Poisson equation with the Boundary Element Method. Our calculations correctly predict the experimentally observed trends in the helix-coil equilibrium of the C-peptide, and suggest that the mechanisms involved are more complex than usually presumed in the literature. Our results suggest that electrostatic interactions in the unfolded conformation are often more important than in the helix, total electrostatic contribution to the helix-coil transition due to the side chains of the C-peptide destabilizes the helix, changes in the helix stability produced by the changes in the ionization state of the side chains are dominated by side chain effects, the effect of the helix dipole on the energetics of the helix-coil transition of the C-peptide is either minor or similar to other contributions in magnitude; while the formation of a salt bridge is electrostatically favorable, formation of the hydrogen bond between a charged and a polar side chains is not. Factors limiting the accuracy of the computations are discussed.     

Salt dependence and ion specificity of the coil–helix transition of furcellaran

The conformational transition and the cation-binding properties of aqueous furcellaran (a gel-forming, low-sulfated polysaccharide of the carrageenan family) in various salts and salt mixtures was studied by optical rotation and by ¹³³Cs-nmr. The results were compared with theoretical predictions based on the Poisson–Boltzmann cell model (PBCM). The conformational transition of furcellaran occurs in a single step, which implies a nonblocklike distribution of sulfate groups along the polymer chain. The chloride salts of sodium, lithium, and tetramethylammonium are equally potent in inducing helix formation of furcellaran, indicating that these ions act by nonspecific electrostatic interactions. In contrast, the potassium and cesium ions specifically promote helix formation and aggregation (gelation) of furcellaran. The divalent calcium and magnesium ions are nonspecific, but more potent than the nonspecific monovalent ions in inducing helices. Anions differ in their capacity to stabilize the furcellaran helix in the sequence Cl^? < NO < Br^? < SCN^? < I^?. The iodide and thiocyanate anions impede aggregation and gel formation. ¹³³Cs-nmr chemical shifts indicate specific binding of cesium ions to the furcellaran helix. Thus, with respect to its ion specificity and ion-binding properties, furcellaran, with 0.6 sulfate group per repeating disaccharide, resembles κ-carrageenan (1 sulfate/disaccharide) but differs from ι-carrageenan (2 sulfates/disaccharide). The conformational transition temperatures of furcellaran are, however, generally higher than those of κ-carrageenan under comparable conditions, and in mixtures of the two polysaccharides, separate transitions still occur, indicating that no mixed helices are formed. The observed ion sensitivity and cation-binding properties of furcellaran agree with predictions, by the PBCM, for a K-carrageenan with a reduced charge density.     

Effect of Metal Cations on the Viscosity of a Pectin-Like Capsular Polysaccharide from the Cyanobacterium Microcystis flos-aquae C3-40

The properties of purified capsular polysaccharide from the cyanobacterium Microcystis flos-aquae C3-40 were examined by capillary viscometry. Capsule suspensions exhibited similar viscosities between pH 6 and 10 but were more viscous at pH <=4 than at pH 6 to 11. At pH 7, a biphasic effect of metal ion concentration on capsule viscosity was observed: (i) capsule viscosity increased with increasing metal ion concentration until a maximal viscosity occurred at a specific concentration that was a reproducible characteristic of each metal ion, and (ii) the viscosity decreased with further addition of that ion. Because the latter part of the biphasic curve was complicated by additional factors (especially the precipitation or gelation of capsule by divalent metal ions), the effects of various metal chlorides were compared for the former phase in which capsule viscosity increased in the presence of metal ions. Equivalent increases in capsule viscosity were observed with micromolar concentrations of divalent metal ions but only with 10 to 20 times greater concentrations of Na(sup+). The relative abilities of various metal salts to increase capsule viscosity were as follows: CdCl(inf2), Pb(NO(inf3))(inf2), FeCl(inf2) > MnCl(inf2) > CuCl(inf2), CaCl(inf2) >> NaCl. This pattern of metal efficacy resembles known cation influences on the structural integrity of capsule in naturally occurring and cultured M. flos-aquae colonies. The data are the first direct demonstration of an interaction between metal ions and purified M. flos-aquae capsule, which has previously been proposed to play a role in the environmental cycling of certain multivalent metals, especially manganese. The M. flos-aquae capsule and the plant polysaccharide pectin have similar sugar compositions but differ in their relative responses to various metals, suggesting that capsular polysaccharide could be a preferable alternative to pectin for certain biotechnological applications.     

On the affinity of rabbit-muscle glycogen phosphorylase for highly branched macromolecular subtrates

The rapid actions of mammalian muscle phosphorylases on glycogen and amylopectin may not result from their high affinity for the polysaccharide unit chains but from the high concentration of chain ends at the polysaccharide surface. When set free by the debranching action of pullulanase the linear unit chains of amylopectin are acted on at a low rate by the mammalian enzymes in contrast to the rapid rate of reaction catalyzed by potato phosphorylase. These findings suggest that the conformation of the active site of the mammalian phosphorylases compensates for the weak binding of individual chain ends by allowing the enzyme to act, without hindrance, on the densely packed polysaccharide chain ends at a near-maximum velocity.     

THE LACK OF SPECIFICITY TOWARDS SALTS IN THE ACTIVATION OF CHOLINE ACETYLTRANSFERASE FROM HUMAN PLACENTA

Abstract— The effects of monovalent and divalent anions on the choline acetyltransferase reaction have been determined at high (5.0 mM) and low (0.58 mM) choline. At 0.58 mM-choline, both monovalent and divalent anions activate the enzyme ±9 fold; however, at 5.0mM-choline, monovalent anions activate the enzyme ±25 fold, while divalent anions activate ±9 fold. Both monovalent and divalent anions show uncompetitive activation with respect to choline. When either dimethylaminoethanol, N -(2-hydroxyethyl)- N -methyl piperidinium iodide, or N -(2-hydroxyethyl)- N -propyl pyrrolidinium iodide was substituted for choline, activation by monovalent or divalent anions was only 2.5-4 fold. With AcCoA as substrate the ChA reaction can be increased ±20 fold by increased salts; however, with acetyl dephosphoCoA as substrate, the reaction is insensitive to the salt concentration. Similar salt effects on the ChA reaction, as measured in the direction of acetylcholine synthesis, have been demonstrated in the reverse reaction. In addition, inhibition of the forward reaction by acetylcholine has been measured as a function of sodium chloride concentration. Although the K₁ for acetylcholine increases with increasing salt, this change in K ₁, parallels the increase in the K _m for choline. These results support the hypothesis that both monovalent and divalent anions activate choline acetyltransferase by the same singular mechanism; which is to increase the rate of dissociation of coenzyme A from the enzyme.     

Stabilization of alpha-helix structure by polar side-chain interactions: complex salt bridges,cation-pi interactions,and C-H em leader O H-bonds

It is generally understood that helical proteins are stabilized by a combination of hydrophobic and packing interactions, together with H-bonds and electrostatic interactions. Here we show that polar side-chain interactions on the surface can play an important role in helix formation and stability. We review studies on model helical peptides that reveal the effect of weak interactions between side chains on helix stability, focusing on some nonclassical side-chain-side-chain interactions: complex salt bridges, cation-pi, and C-H em leader O H-bonding interactions. Each of these can be shown to contribute to helix stability, and thus must be included in a comprehensive catalogue of helix stabilizing effects. The issue of the structure of the unfolded states of helical peptides is also discussed, in the light of recent experiments showing that these contain substantial amounts of polyproline II conformation.     

Self-assembly of amylose-grafted carboxymethyl cellulose

In this study, we performed the self-assembly of the amylose-grafted carboxymethyl cellulose sodium salt (NaCMC) for the formation of nanofiber films under aqueous conditions. The introduction of amylose graft chains was conducted by the chemoenzymatic approach including phosphorylase-catalyzed enzymatic polymerization. The product had the rigid NaCMC main chain, which further assembled leading to nanofibers by the formation of double helix between the long amylose graft chains in the intermolecular NaCMC chains of the products. The lengths of the fibers were depended on degrees of polymerization of amylose chains. The nanofiber films were constructed by drying the alkaline solutions of the amylose-grafted NaCMC. The lengths of the nanofibers strongly affected their arrangements in the films. The nanofibers were merged further by washing out alkali to produce the robust nanofiber films.     

HDV ribozyme activity in monovalent cations

Activity of the two ribozymes from hepatitis delta virus in monovalent salts was examined and compared to activity in Mg2+. Both ribozymes self-cleaved in high concentrations of monovalent cations, and an active site cytosine was required for cleavage activity under those conditions. Cleavage rates were 30-50-fold higher for reactions in LiCl than for reactions in NaCl or NH4Cl, and a thio effect indicated that chemistry was rate-determining for cleavage of the HDV genomic ribozyme in LiCl. Still, in LiCl, there was a more than 100-fold increase in the rate when MgCl2 was included in the reaction. However, the pH-rate profiles for the reactions in LiCl with and without MgCl2 were both bell-shaped with the pH optima in the neutral range. These findings support the idea that monovalent cations can partially substitute for divalent metal ions in the HDV ribozymes, although a divalent metal ion is more effective in supporting catalysis. The absence of a dramatic change in the general shape of pH-rate profiles in LiCl, relative to the profile for reactions including Mg2+, is in contrast to earlier data for the reactions in NaCl and limits our interpretation of the specific role played by the divalent metal ion in the catalytic mechanism.     

Suppressive actions of inorganic ions on the bioactivity of aminoglycoside antibiotics.

Many independent studies have shown that inorganic salts can suppress the inhibitory activities of aminoglycoside antibiotics against various bacterial species. From studies on the nature and mechanism of this suppressive ion effect, several conclusions can be drawn. Both monovalent and divalent cation salts can inhibit aminoglycoside action. At least two different mechanisms are apparently involved. The first is divalent cation dependent and has been described with $\text{Pseudomonas aeruginosa}$ and $\text{Mycobacterium smegmatis}$. With $\text{M. smegmatis}$, the effect aappears to be dependent more on a double positive charge than on particular metal ion species. The second mechanism is related to ionic strength and is elicited by both monovalent and divalent cation salts against many different organisms. However, based on studies with $\text{M. smegmatis}$, it appears that each of the two mechanisms involves interferance with the initial instantaneous electrostatic binding of drug at the cell surface.