A comparison between the coupled spectrophotometric and uncoupled radiometric assays for RuBP carboxylase

The coupled spectrophotometric assay for RuBP carboxylase was compared with the conventional radiometric assay to assess the validity of its use in the measurement of initial and total activities in crude leaf extracts. At high magnesium concentrations both assays gave the same absolute values of initial and total activities, and resolved similarly the changes of total activity and activation state (ratio of initial to total activity) which occurred when the water status and light environment of leaves was altered prior to sampling. Although the magnesium concentration supporting the maximum rate of initial activity in soybean extracts was similar in the two assays, substantial differences of initial activity were observed at sub-optimal concentrations of magnesium. At low magnesium concentrations reaction rates in the spectrophotometric assay exhibited an initial phase of non-linearity which subsequently gave way to a linear rate. In contrast, reaction rates at low magnesium were linear from the time of initiation in the radiometric assay. Inclusion of EDTA in the reaction medium did, however, induce non-linear rates in the radiometric assay. The pre-addition of RUBP to extract immediately prior to dilution into the reaction medium did not eliminate the non-linearity in either assay system. The significance of these observations is discussed briefly in relation to the use of the spectrophotometric assay.     

A general model of reaction kinetics in biological systems

Dynamic mathematical models in biotechnology require, besides the information about the stoichiometry of the biological reaction system, knowledge about the reaction kinetics. Modulation phenomena like limitation, inhibition and activation occur in different forms of competition with the key enzymes responsible for the respective metabolic reaction steps. The identification of a priori unknown reaction kinetics is often a critical task due to the non-linearity and (over-) parameterization of the model equations introduced to account for all the possible modulation phenomena. The contribution of this paper is to propose a general formulation of reaction kinetics, as an extension of the Michaelis-Menten kinetics, which allows limitation/activation and inhibition effects to be described with a reduced number of parameters. The versatility of the new model structure is demonstrated with application examples.     

The stimulatory effect of catalase on the formation in vitro of ethylene by an ethylene-forming enzyme purified from Penicillium digitatum

The dependence of the rate of formation of ethylene on the concentration of an ethylene-forming enzyme was determined with a purified preparation of the ethylene-forming enzyme from Penicillium digitatum IFO 9372. The relationship was n ot linear. When catalase and bovine serum albumin were added to the reaction mixture, the rate of formation of ethylene was directly proportional to the concentration of the enzyme. The non-linearity of the reaction, in the absence of these additives, is probably due to the hydroxyl radical ions (OH) produced by the Fenton reaction which occurs in the reaction mixture when ferrous ions and oxygen are present together under reducing conditions.     

Effect of ligands with selective type of binding on the DNA helix-coil transition

The influence of the extended interacted under adsorption ligands with a selective binding on the DNA helix-coil transition has been theoretically studied. It was found that contact interaction between ligands or/and their extent give rise to a marked non-linearity of the GC-content dependence of the melting temperature. This non-linearity causes a few features of the dependence of the melting range width on ligand concentration [delta T(C0)]. Such as a non-monotony of the delta T (C0) increase in the presence of ligands increasing the difference between the thermostabilities of poly(d(A-T)] and poly[d(G-C)] polymers. The degree of a non-linearity defines the character of changes of the form of the differential melting curves in the presence of ligands.     

Mutagenicity of the reaction products of carbazole in the presence of nitrogen dioxide and nitrocarbazole

The mutagenicity of the photochemical reaction products of carbazole in the presence of nitrogen dioxide (NO2) and nitrocarbazole was investigated using a high-pressure mercury lamp (100 W). Samples extracted from the photochemical reaction products of carbazole with NO2 were more mutagenic than those of acridine and phenazine with NO2 for Salmonella typhimurium strain TA98 in the absence of S9 mix with a trend toward detoxification in the presence of the metabolic system. The mutagenicity of the photochemical reaction products of carbazole with NO2 were higher than those of the reaction products of carbazole with a mixture of NO2 and sulfur dioxide (SO2) and no irradiation. Mononitro- and dinitro-carbazole in the samples extracted from the reaction products were analyzed by mass spectrometry. It was suggested that mononitrocarbazole, which seemed to be weakly mutagenic, and dinitrocarbazole were readily formed by the reaction of carbazole with NO2, and that the other high-potency mutagens were formed by the photochemical reaction of carbazole with NO2 with irradiation by light.     

Radical-capturing reaction of 5,7,3',4'-tetramethylquercetin with the AIBN radical initiator

In order to clarify the mechanism for the radical-capturing reaction which is initiated at the C3-hydroxyl group of flavonols, 5,7,3',4'-tetramethylquercetin (TMQ) was reacted with the 2,2'-azobis-isobutyronitrile (AIBN) radical initiator in benzene. Six products, one depside and its two hydrolytic products, one nitrile adduct, and two others, were isolated from the reaction mixture, and their structures were determined by instrumental analyses. The quantitative change to the four main products against the reaction time was measured by an HPLC method. The radical-capturing reaction pathway for TMQ with AIBN is proposed from these products and their quantitative changes. The pathway dividing into two clearly reveals that one subpath formed the depside and its hydrolytic products, while the other formed the nitrile adduct. The reactivity of each two sub-path was nearly the same, different from the case of TMQ and the 2,2'-azobis-2,4-dimethylvaleronitrile (AMVN) radical initiator.     

Measuring the Intangibles: A Metrics for the Economic Complexity of Countries and Products

We investigate a recent methodology we have proposed to extract valuable information on the competitiveness of countries and complexity of products from trade data. Standard economic theories predict a high level of specialization of countries in specific industrial sectors. However, a direct analysis of the official databases of exported products by all countries shows that the actual situation is very different. Countries commonly considered as developed ones are extremely diversified, exporting a large variety of products from very simple to very complex. At the same time countries generally considered as less developed export only the products also exported by the majority of countries. This situation calls for the introduction of a non-monetary and non-income-based measure for country economy complexity which uncovers the hidden potential for development and growth. The statistical approach we present here consists of coupled non-linear maps relating the competitiveness/fitness of countries to the complexity of their products. The fixed point of this transformation defines a metrics for the fitness of countries and the complexity of products. We argue that the key point to properly extract the economic information is the non-linearity of the map which is necessary to bound the complexity of products by the fitness of the less competitive countries exporting them. We present a detailed comparison of the results of this approach directly with those of the Method of Reflections by Hidalgo and Hausmann, showing the better performance of our method and a more solid economic, scientific and consistent foundation.     

The study of volt-ampere characteristics of ionic channels formed by gramicidin A

A method of measurement of the non-linearity coefficient of volt-ampere characteristics of the type i(U) approximately = U(1 + beta U2) has been developed for ionic channels formed by gramicidin A, using the third harmonic of the membrane current. The shape of the volt-ampere characteristics (VA) of ionic channels formed by gramicidin A did not depend on the antibiotic concentration in the membrane. The coefficient beta of non-linearity of VA of membranes modified by gramicidin A depended on electrolyte concentration "c" and it increased proportionally with the lg c from -17 V-2 at 0.03 mol/l KC1 to 8 V-2 at 3.4 mol/l KCl, and it was zero at co = 0.3 - 1 mol/l KCl. Egg lecithin and glycerol monooleate (GMO) membranes differ in their co values. The substitution of K+ for Li+ of the membrane solvent (n-heptane for n-hexadecane) did not influence the value of beta; the same applied for GMO membranes without any solvent. In a number of membranes, spontaneous change of the non-linearity coefficient with time observed after the membrane formation, as well as jumps of the non-linearity coefficient at a practically unchanged membrane conductivity. An analysis of some theoretical models of the ion transport through the channel has shown that, at voltages above 200 mV, these models provide rather small values of beta, or extremely high VA non-linearity.     

Mutagenicity of the reaction products of dibenzo-p-dioxin with nitrogen oxides.

Dibenzo-p-dioxin (DD) was made to react with various concentrations of nitrogen oxides in the dark. The mutagenicities of the reaction products were tested using Salmonella typhimurium strains TA98, TA100, TA98NR and TA98/1,8-DNP6 in the presence or absence of a mammalian metabolic activation system (S9 mix). DD-NOx (molar ratios 1:3, 1:6 and 1:18) reaction products exhibited mutagenic potency in strains TA98 and TA98/1,8-DNP6 without S9 mix. In a gas chromatography/mass spectrometry study, 2-nitrodibenzo-p-dioxin (NDD) was identified with authentic sample in the mutagenic reaction products. DD-NOx (1:18) reaction products were reduced by sodium hydrogen sulfide and the reduction mixture was analyzed by HPLC. 2,7-Dinitrodibenzo-p-dioxin (DNDD) and 2,8-DNDD were identified as corresponding diamino-DDs in the reduction mixture. 2-NDD, 2,7-DNDD and 2,8-DNDD were also mutagenic in strains TA98 and TA98/1,8-DNP6 without S9 mix and the mutagenicity of DD-NOx reaction products was largely accounted for by the nitro-DDs.     

Surface photochemistry of the herbicide napropamide. The role of the media and environmental factors in directing the fates of intermediates.

The photochemical behaviour of the herbicide napropamide is studied on cellulose and silica surfaces, using steady-state and laser-flash diffuse reflectance techniques. The results are used to probe how the reaction sites of the host matrices influence the photo-reactive pathways. Napropamide undergoes reaction when irradiated with UV (lamps) or visible (sunlight) radiation on both solid supports. The nature of the intermediates and final products depend strongly on the presence or absence of molecular oxygen. The triplet state of napropamide adsorbed on cellulose is detected by both time-resolved luminescence and transient absorption spectroscopies. The triplet sate was not observed on silica, but transients which include the participation of molecular oxygen are detected during flash photolysis studies. The keto intermediates of the photo-Claisen rearrangement products are observed on both solids. Substituted 1-naphthols from photo-Claisen reactions and 1-naphthol are among the main reaction products. 1,4-Naphthoquinone is a major photoproduct in the presence of molecular oxygen and is expected to be prevalent when napropamide undergoes photodegradation in the environment (i.e., after being applied to plants and fields).     

Electrical excitability of artificial enzyme membranes: III. Hysteresis and oscillations observed with immobilized acetylcholinesterase membranes

Experimental evidence for memory and oscillations in artificial acetylcholinesterase membranes is presented. When acetylcholine is injected on one side of an artificial proteinic membrane bearing acetylcholinesterase, a potential difference is recorded as a function of time. The steady-state potential due to the enzyme activity for increasing and decreasing substrate concentrations exhibits a hysteresis loop. The non-linearity of the enzyme reaction coupled with the diffusion constraints cause also some instabilities, such as oscillations of the membrane potential.     

Quantitative electrospray ionization mass spectrometry of zinc finger oxidation: the reaction of XPA zinc finger with H(2)O(2)

Oxidation plays an important role in the functioning of zinc fingers (ZFs). Electrospray ionization mass spectrometry (ESI-MS) is a very useful technique to study products of ZF oxidation, but its application has been limited largely to qualitative analysis of reaction products. On the other hand, ESI-MS has been applied successfully on several occasions to determine binding constants in metalloproteins. We used a synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf), which corresponds to the Cys4 ZF sequence of human nucleotide excision repair protein XPA, to find out whether ESI-MS might be used quantitatively to study ZF reaction kinetics. For this purpose, we studied oxidation of the Zn(II) complex of XPAzf (ZnXPAzf) by H(2)O(2) using three techniques in parallel: high-performance liquid chromatography (HPLC) of covalent reaction products, 4-(2-pyridylazo)-resorcinol monosodium salt (PAR)-based spectrophotometric zinc release assay, and ESI-MS. Single and double intrapeptide disulfides were detected by ESI-MS to be the sole reaction products. All three techniques yielded independently the same reaction rate, thereby demonstrating that ESI-MS may indeed be used in quantitative kinetic studies of ZF reactions. The comparison of experimental information demonstrated that the formation of the Cys5-Cys8 single disulfide was responsible for zinc release.     

Kinetic mechanism of the hydroxypyruvate-lactate dehydrogenase-NADH system.

Chicken liver lactate dehydrogenase L-lactate : NAD+ oxidoreductase, EC1.1.1.27) reversibly catalyses the conversion of hydroxypyruvate to L-glycerate. The variation of the initial reaction rate with the substrate or coenzyme (NADH) concentration together with the inhibition caused by the reaction products and excess substrates, reveal that the kinetic mechanism of the reaction, with hydroxypyruvate as substrate, is of the rapid-equilibrium, ordered-ternary-complex type; NADH is the first substrate in the reaction sequence. Rate equations have been developed for the hydroxypyruvate.E.NADH system without inhibitors, with excess substrates, and with reaction products. Comparison of the rate equations obtained with those calculated theoretically from an ordered-ternary-complex mechanism reveals the existence of E.NAD.NADH,E.NAD-hydroxypyruvate and E.hydroxypyruvate complexes.     

Regulating the Catalytic Dynamics Through a Crystal Structure Modulation of Bimetallic Catalyst

The surface of solid catalysts is one of the most important factors where the interface with reaction products governs the reaction kinetics. Herein, the crystal phase of palladium–copper nanoparticles (PdCu NPs) is controlled to modulate their surface atomic arrangement, which will govern the growth dynamics of discharge products on their surfaces and thus the catalytic performances in non‐aqueous lithium–oxygen (Li‐O₂) batteries. First‐principles calculations and experimental validations reveal that homogeneous nucleation and distribution of discharge products are observed on the surface of body‐centered cubic PdCu NPs, promoting the oxygen reduction/evolution reaction (ORR/OER) activities in Li‐O₂ batteries. However, the agglomerates formed on the surface of its face‐centered cubic homologue deteriorates ORR/OER activities, which worsen the battery performances. For the first time, this work theoretically and experimentally demonstrates how the crystal phase modulation regulates the nucleation behaviors and growth dynamics of discharge products for ORR/OER.     

Reaction of proteinases with alpha 2-macroglobulin from the American horseshoe crab, Limulus.

The products generated by the reaction of Limulus alpha 2-macroglobulin with trypsin were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Unreacted Limulus alpha 2-macroglobulin had a subunit molecular mass of 185 kDa. Trypsin-reacted samples contained two prominent peptides smaller (85 and 100 kDa) and three peptides larger (200, 250, and 300-350 kDa) than the unreacted subunit. Reaction of methylamine-treated Limulus alpha 2-macroglobulin with trypsin resulted in the same two prominent reaction products smaller than 185 kDa, but all of the reaction products larger than 185 kDa were absent. The covalent binding of biotinylated trypsin with Limulus alpha 2-macroglobulin was detected by probing Western blots with horseradish peroxidase-avidin. Surprisingly, the only reaction products that contained trypsin were bands at 100 and 120 kDa. The staining of these bands with horseradish peroxidase-avidin was weak: most of the biotinylated trypsin that remained associated with alpha 2-macroglobulin during gel filtration chromatography was located at the dye front following reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The reaction products larger than 185 kDa did not contain trypsin. Methylamine-reacted Limulus alpha 2-macroglobulin failed to bind any biotinylated trypsin. In contrast to the reaction of trypsin with Limulus alpha 2-macroglobulin, all high molecular mass bands generated by the reaction of human alpha 2-macroglobulin with biotinylated trypsin stained intensely with horseradish peroxidase-avidin. Thus, Limulus alpha 2-macroglobulin forms thiol ester-dependent, high molecular mass products involving isopeptide bonding between trypsin-generated fragments, without the incorporation of trypsin into the complexes. Most of the alpha 2-macroglobulin-associated trypsin is non-covalently trapped rather than covalently cross-linked.     

Studies on the mechanism of alkaline peroxide delignification of agricultural residues

Alkaline solutions of hydrogen peroxide partially delignify wheat straw and other lignocellulosic materials, leaving a cellulosic residue that is highly susceptible to enzymatic digestion by cellulase. The delignification reaction is strongly dependent upon the pH of the reaction mixture, with an optimum at pH 11.5-11.6, pKa for the dissociation H(2)O(2) right harpoon over left harpoon H(+) + HOO(-). The data are consistent with a mechanism in which H(2)O(2) decomposition products such as .OH and O(2) (-)., rather than H(2)O(2) or HOO(-), are the primary lignin oxidizing species. During the course of the delignification reaction, O(2) is evolved from the reaction mixture indicating active H(2)O(2) decomposition. At a given concentration of H(2)O(2), the rate of O(2) evolution is proportional to the amount of lignocellulosic substrate present in the reaction mixture. However, the total amount of O(2) evolved is inversely proportional to the amount of substrate present, indicating that some of the peroxide oxygen becomes incorporated into lignin degradation products. The amount of peroxide oxygen incorporated can range as high as 2 O(2) per lignin C(9) unit, depending upon the initial concentration of lignocellulosic substrate.     

Ultrastructural electron probe X-ray microanalytical reaction product identification of three different enzymes in the same mouse resident peritoneal macrophage

Simultaneous cytochemical enzyme localization procedures for peroxidase (PO) plus acid phosphatase (AcP-ase) and/or aryl sulphatase (AS) have been investigated at the ultrastructural (EM) level. Electron probe X-ray microanalysis (EPMA) will identify and differentiate the reaction products. Dual reaction product localization of PO plus AcP-ase or alternatively PO plus AS have been obtained in the same mouse resident peritoneal macrophage. This has been acquired by first performing a PO-reaction followed by AcP-ase or followed by AS. In both cases PO-related reaction products (PODAB/Os or PODAB/Pt) were localized in nuclear envelope (NE) and rough endoplasmic reticulum (RER). Cells were identified by this reaction product as resident macrophages. Reaction products from the AcP-ase related cerium (AcP-aseCe), localized in lysosomes have been identified and differentiated from the PO-related osmium containing products. Similarly AS related barium (ASBa), localized in lysosomal structures and (R)ER was identified and differentiated. Triple reaction product localization of PO followed by AcP-ase plus AS could also be obtained. In this case, PO-related platinum containing reaction products (PODAB/Pt or PODAB/Os) in NE and RER has been identified and differentiated from the AcP-ase related lysosomal cerium (AcP-aseCe) and the AS related barium localized in lysosomal and (R)ER structures. Reversing the sequences in both dual cytochemical procedures: AcP-aseCe or ASBa followed by PODAB/Os (or PODAB/Pt) resulted in AcP-aseCe or ASBa activity related reaction products only. Reversing the sequence in the triple reaction procedures (ASBa followed by AcP-aseCe) resulted in the absence of the barium containing reaction products. By application of OsO4 postfixation with aminotriazole (ATR) additives the detrimental effects upon the various precipitates have been confirmed. In LM studies, using rat intestine and non-metal identification reactions for two of the enzymes (pararosaniline for AcP-ase, DAB for peroxidase), the influences of the metal ions used in EM were tested on the appearance of the coloured reaction products. Cerium ions used in EM for detection of AcP-aseCe activity have been shown to influence the PODAB visibility in LM and EM experiments. From the AS reaction media components neither barium ions nor p-nitro catachol sulphate influenced the LM visibility of the PO reaction.     

Ultrastructural localization of carbonic anhydrase activity in the rat choroid plexus epithelial cell

Summary Localization of carbonic anhydrase activity was studied electron microscopically on cells of the rat choroid plexus epithelium. For the ultracytochemical detection of these activities, Yokota's technique (1969), which is the modification of Hansson's method (1967) was employed. Numerous electron dense reaction products were observed in the microvilli of the choroidal epithelial cell. The reaction deposits were also remarkably present in the infoldings of the basal plasmalemma but to a lesser extent than in the microvilli. The localization sites were mainly on the plasma membrane, but some reaction products were also observed in the cytoplasm near the plasma membrane. Hardly any reaction product was found in the intracellular organelles except for the mitochondria in which reaction products were occasionally observed on the cristae. These activities were completely inhibited by acetazolamide. As the carbonic anhydrase activity was histochemically seen in the microvilli and the basal infoldings, it is likely that carbonic anhydrase is related to an active transport process in the secretion of cerebrospinal fluid as is Na⁺, K⁺-ATPase (Masuzawa et al. 1980).     

Modification of serine residues during sequential degradation of proteins and peptides by the phenylisothiocyanate method

A novel method of serine determination during Edman degradation of proteins and peptides which is based on the reaction of serine degradation products with alkyl thiols is described. The physicochemical characteristics and the structures of the reaction products, i.e., of 5-[alkyl-thio)methyl)-3-phenyl-2-thioxo-4-imidazolinones, were determined. The procedure described permits the products of serine degradation to be obtained in a 70-80% yield.     

Investigation of the reactions of iron(III) halobisdithiocarbamates with halogens: a novel series of fluxional homobinuclear iron(III) complexes with two different coordination spheres around the magnetic centers

In addition to the variety of products formed during the reaction of iron(III) halobisdithiocarbamates with halogens, some novel fluxional homobinuclear iron(III) complexes with two different coordination spheres around the magnetic centers have also been synthesized and studied. The formation of these products depends on the nature of both the molecular halogen and the reagent complex, as well as on the reaction conditions. The new compounds have been characterized chemically and by means of spectroscopic methods and magnetic susceptibility measurements. The volatility characteristics and thermogravimetric analysis data for the complexes were also studied. Finally, a general mechanism accounting for the variety of products formed in the reactions of iron(III) halobisdithiocarbamates with molecular halogens is proposed.