Standard apparent reduction potentials of biochemical half reactions and thermodynamic data on the species involved

Standard apparent reduction potentials E' degrees of half reactions of enzyme-catalyzed reactions are useful because they provide a global view of the apparent equilibrium constants of redox reactions. A table of E' degrees at a specified pH shows at a glance whether a given half reaction will drive another half reaction or be driven by it. This table can be used to calculate apparent equilibrium constants. Standard Gibbs energies of formation of species in a half reaction can be used to calculate E' degrees values at pHs in the range 5-9 and ionic strengths in the range of 0-0.35 M. My previously published values of E' degrees values for 42 half reactions has been extended by 22 new E' degrees values in this paper. When DeltafG degrees and DeltafH degrees are both known for all the species in an enzyme-catalyzed reaction at 298.15 K, it is possible to calculate all the standard transformed thermodynamic properties of the reaction over a range of pHs, ionic strengths, and temperatures.     

A new detector for fully automatic peptide synthesis

A new method of monitoring the rate of reactions in solid phase peptide synthesis is described. A conductivity detector in the reaction cell enables the deprotection, washing, and subsequent coupling stages to be examined in detail. The half lives of the reactions can be calculated and hence the optimum reaction times predicted. The aggregation of peptide chains and subsequent collapse of the resin is observed. Difficult sequences are sensed and appropriate action taken completely automatically.     

False dopa reaction in studies of mammalian tyrosinase: some characteristics and precautions

In cytochemical studies of B16 melanoma cells with the dopa reaction for tyrosinase, a false positive result that can easily be confused with authentic reactions has been observed. A preliminary characterization of this false reaction in comparison to authentic reactions with respect to stereospecificity, morphological characteristics, and subcellular localization has been conducted. Modifications of the classic dopa method have been devised that minimize occurrence of the false reaction.     

False Dopa Reaction in Studies of Mammalian Tyrosinase: Some Characteristics and Precautions

In cytochemical studies of B16 melanoma cells with the dopa reaction for tyrosinase, a false positive result that can easily be confused with authentic reactions has been observed. A preliminary characterization of this false reaction in comparison to authentic reactions with respect to stereospecificity, morphological characteristics, and subcellular localization has been conducted. Modifications of the classic dopa method have been devised that minimize occurrence of the false reaction.     

Effect of pressure on electron transfer reactions in inorganic and bioinorganic chemistry

Kinetic and thermodynamic studies involving the application of different high-pressure techniques, are very useful in gaining mechanistic information on the basis of volume changes that occur during inorganic and bioinorganic electron transfer reactions. The most fundamental type of electron transfer reaction concerns self-exchange reactions, for which the overall reaction volume is zero, and activation volumes can be measured and discussed. In the case of non-symmetrical electron transfer reactions, intra- and intermolecular processes can be studied and volume profiles can be constructed. Precursor complex formation can in some cases be recognized kinetically in such systems. Typical values of activation and reaction volumes are reviewed for various reversible and irreversible electron transfer reactions. Mechanistic conclusions reached on the basis of these parameters are presented. Volume profiles for electron transfer reactions enable a simplistic presentation of the reaction mechanism on the basis of intrinsic and solvational volume changes along the reaction coordinate.     

Positional isotope exchange

The detection of intermediates in enzyme-catalyzed reactions can be accomplished by several techniques. For those intermediates which do not have easily observed electronic spectra, use can be made of isotope exchange phenomena if the chemistry of the reaction is appropriate. Recently, the technique of positional isotope exchange (intramolecular isotopic scrambling) has been used to study several reactions which have been thought to involve high-energy intermediates in their mechanisms. A review of some of these reactions and the limitations of the method are presented in this article.     

Enzyme-catalyzed and binding reaction kinetics determined by titration calorimetry

BackgroundIsothermal calorimetry allows monitoring of reaction rates via direct measurement of the rate of heat produced by the reaction. Calorimetry is one of very few techniques that can be used to measure rates without taking a derivative of the primary data. Because heat is a universal indicator of chemical reactions, calorimetry can be used to measure kinetics in opaque solutions, suspensions, and multiple phase systems and does not require chemical labeling. The only significant limitation of calorimetry for kinetic measurements is that the time constant of the reaction must be greater than the time constant of the calorimeter which can range from a few seconds to a few minutes. Calorimetry has the unique ability to provide both kinetic and thermodynamic data.Scope of reviewThis article describes the calorimetric methodology for determining reaction kinetics and reviews examples from recent literature that demonstrate applications of titration calorimetry to determine kinetics of enzyme-catalyzed and ligand binding reactions.Major conclusionsA complete model for the temperature dependence of enzyme activity is presented. A previous method commonly used for blank corrections in determinations of equilibrium constants and enthalpy changes for binding reactions is shown to be subject to significant systematic error.General significanceMethods for determination of the kinetics of enzyme-catalyzed reactions and for simultaneous determination of thermodynamics and kinetics of ligand binding reactions are reviewed. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Time hierarchy, equilibrium and non-equilibrium in metabolic systems.

A metabolic system consists of cooperating biochemical reactions. The motion is described by differential equations in the metabolites. The right-hand sides of these equations are linear combinations of the velocities of the individual reactions. These velocities depend in a non-linear manner on the metabolite concentrations (according to the law of mass action). A characteristic "metabolic" time may be defined for the motion of the whole system. It scales the essential metabolic events whose evolution time is comparable to this metabolite time unit. The constituent reactions of the metabolic system have an individual characteristic time which need not coincide with the general metabolic time. The individual time characterises the approach to the individual equilibrium of the isolated undisturbed reaction. According to the ratio of these two time scales, a single reaction may be fast, or slow, or essential, as compared with the metabolic events. Characteristic time of a single reaction and its steady-state deviation from equilibrium are closely related. It can be shown that the relative deviation from equilibrium of a reaction within the metabolic network is of the same numerical order as the ratio between individual time to metabolic time. The interaction of many reactions with different characteristic times introduces a time hierarchy into the system. This can be made transparent by appropriate scaling and by linear transformation of the system. The subsystem of fast cooperating reactions (dehydrogenases, phosphotransferases) attains a state which is near to the individual equilibrium and reestablishes this state after perturbation. The equilibration is fast; an ultrarapid phase of cofactor equilibrium can be distinguished from the fast phase of substrate equilibrium (exchange of metabolic material between different pathways). During the slower metabolic phase these near-equilibria manifest themselves as stoichiometric linkage between unrelated metabolites. The latter cease to be independent variables and combine to metabolic pools. It can be strictly shown that the essential variables at the metabolic time scale are carrier pools and the degree of occupancy of these carriers by metabolic groups. Chemically different types of carrier pools may be functionally linked together by fast reactions. A consequence of such an arrangement of reactions are distance effects: Changes at one end of a metabolic map may be directly conveyed to other pathways via stoichiometric linkage brought about by fast equilibration of cofactor reactions.     

A stopped-flow mixer device for a batch microcalorimeter application to NAD-NADase reaction

A new molded polypropylene, diamond-like carbon (DLC)-coated mixing cell has been developed for use in the batch microcalorimeter. Reagent volume can be varied from 25 microliters to 100 microliters. A 10 microcalorie reaction heat can be measured to 5%. Repeat reactions can be done as often as every 10 min for a fast reaction. Reactions can be started within 1 h or less after loading. A pre-equilibrator and a temperature-controlled syringe drive unit permit solutions to be stored at 4 degrees C while being run at any temperature from -20 degrees C to 40 degrees C. The kinetics and enthalpy of reaction of NAD-NADase have been measured. delta H is about 21 kcal/mol endothermic.     

Electrophoretic light scattering as a probe of reaction kinetics

A new method is proposed for determining chemical rate contants in dimerization reactions of globular proteins. Light scattering from a solution of charged macromolecules in and applied electric field gives a series of bands whose widths can be used to deduce the reaction rate contants. This method should be applicable to other types of peactions. First order reactions are also considered.     

Basic Principles of Reactivity in Free Radical Chemistry

This review is concerned with an overall survey of reactivity in free radical chemistry. A concise classification is given of elementary reaction steps which can be combined in different ways to account for overall chemical transformations: radical forming reactions, radical transformations, and radical destroying reactions. From this is derived the concept of the chain reaction which leads on to an up-to-date theory for understanding reactivity in free radical processes. Finally, a few aspects of autoxidation are discussed.     

Basic Principles of Reactivity in Free Radical Chemistry

This review is concerned with an overall survey of reactivity in free radical chemistry. A concise classification is given of elementary reaction steps which can be combined in different ways to account for overall chemical transformations: radical forming reactions, radical transformations, and radical destroying reactions. From this is derived the concept of the chain reaction which leads on to an up-to-date theory for understanding reactivity in free radical processes. Finally, a few aspects of autoxidation are discussed.     

INFLUENZA VACCINE,ASIAN STRAIN—Reactions Following Its Use in Adults

A study of reactions following influenza vaccine inoculation of 327 employees of Peralta Hospital, 55 men and 272 women, showed a very low value for significant or severe reactions. The reaction rate as observed with the present monovalent vaccine containing 200 CCA units of Asian strain, Type A influenza virus, was considerably lower than that reported with previous polyvalent vaccines containing up to 1,400 or 1,500 CCA units of total virus content.The absenteeism rate was 1.1 per cent for women, nil for men.The incidence of reactions was much greater in women than in men. Local reactions such as pain, swelling, or redness at the site of injection occurred in 29.1 per cent of men and 35.7 per cent of women. The incidence of systemic reactions—fever, aching, chilliness, headache, nausea and vomiting—was 3.6 per cent in men and 8.8 per cent in women. About 9 per cent of men and 30 per cent of women had both local and systemic reaction. Some 58 per cent of men and 25 per cent of women had no reaction.The greater majority of reactions appeared within five hours after inoculation with influenza vaccine.In adults the prevention of anaphylactic reactions due to the small amount of egg protein in influenza vaccine, can be accomplished by screening for history of hypersensitivity to egg, chicken or chicken feather. In questionable cases, intradermal testing can be done.The reaction rate observed in this study for the present influenza vaccine was so low that it ought not deter immunization.     

Subzero temperature quenching and electrophoretic methods for the isolation of protein reaction intermediates

A quenching technique for the study of rapid protein reactions is described which consists of injecting a small volume of aqueous solution of reactants into a large volume (× 10) of hydro-organic solvent cooled at subzero temperature and mechanically shaken. The protein reaction intermediates, stabilized at subzero temperature and brought into a hydro-organic solution, can then be separated by subzero temperature electrophoretic methods, such as isoelectric focusing, in the same solvent. The alkaline hydrolysis of 2,4-dinitrophenylacetate was studied by the use of this quenching technique in order to compare the quenching and the rate constants of the reaction with those obtained by normal rapid quenching methods. It was found that first-order reactions having rate constants up to about 5 s^?1 can be satisfactorily studied by this technique. The technique is not suitable for the study of faster reactions because of the high value of the quenching time (40–100 ms). The hybridization reaction of carboxyhemoglobins A and C in aqueous solution at 22°C was studied as an example of the application of this quenching technique and of the isoelectric focusing method at subzero temperature to the isolation of unstable intermediates in a protein reaction.     

A stochastic analysis of first-order reaction networks

A stochastic model for a general system of first-order reactions in which each reaction may be either a conversion reaction or a catalytic reaction is derived. The governing master equation is formulated in a manner that explicitly separates the effects of network topology from other aspects, and the evolution equations for the first two moments are derived. We find the surprising, and apparently unknown, result that the time evolution of the second moments can be represented explicitly in terms of the eigenvalues and projections of the matrix that governs the evolution of the means. The model is used to analyze the effects of network topology and the reaction type on the moments of the probability distribution. In particular, it is shown that for an open system of first-order conversion reactions, the distribution of all the system components is a Poisson distribution at steady state. Two different measures of the noise have been used previously, and it is shown that different qualitative and quantitative conclusions can result, depending on which measure is used. The effect of catalytic reactions on the variance of the system components is also analyzed, and the master equation for a coupled system of first-order reactions and diffusion is derived. All authors contributed equally to this work.     

A Kinetic Analysis of Coupled (or Auxiliary) Enzyme Reactions

As a case study, we consider a coupled (or auxiliary) enzyme assay of two reactions obeying the Michaelis–Menten mechanism. The coupled reaction consists of a single-substrate, single-enzyme non-observable reaction followed by another single-substrate, single-enzyme observable reaction (indicator reaction). In this assay, the product of the non-observable reaction is the substrate of the indicator reaction. A mathematical analysis of the reaction kinetics is performed, and it is found that after an initial fast transient, the coupled reaction is described by a pair of interacting Michaelis–Menten equations. Moreover, we show that when the indicator reaction is fast, the quasi-steady-state dynamics are governed by three fast variables and one slow variable. Timescales that approximate the respective lengths of the indicator and non-observable reactions, as well as conditions for the validity of the Michaelis–Menten equations, are derived. The theory can be extended to deal with more complex sequences of enzyme-catalyzed reactions.     

Kinetics of coupled enzyme reactions

A theory has been developed for the kinetics of coupled enzyme reactions. This theory does not assume that the first reaction is irreversible. The validity of this theory is confirmed by a model system consisting of enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) with 2,4-decadienoyl coenzyme A (CoA) as a substrate. This theory, in contrast to the conventional theory, proves to be indispensible for dealing with coupled enzyme systems where the equilibrium constant of the first reaction is small and/or the concentration of the coupling enzyme is higher than that of the intermediate. Equations derived on the basis of this theory can be used to calculate steady-state velocities of coupled enzyme reactions and to predict the time course of coupled enzyme reactions during the pre steady state.     

A method for sequencing restriction fragments with dideoxynucleoside triphosphates.

A rapid enzymatic approach is described for the sequence analysis of a 5' terminally labelled restriction fragment. It involves limited nicking of the strands of the molecule throughout the sequence by pancreatic DNAase I. The 3' hydroxyl groups exposed by each nick are then used to prime chain extension by DNA polymerase I in four separate reactions. Each reaction uses one of the four chain terminating dideoxynucleoside triphosphates (ddNT-PSs), together with the four deoxynucleoside triphosphates (dNTPs). In a single reaction all the 3' ends are terminated in positions of the same base, which is different for each of the four reactions. When the products of these reactions are resolved by gel electrophoresis according to size, a sequence can be deduced from the pattern of radioactive bands. Sequences can be determined onwards from 10-20 residues from the 5' labelled end. The length of sequence which can be determined is only limited by the resolution of the gel.     

Spectrophotometric determination of tryptophan and tyrosine in peptides and proteins based on new color reactions

A new spectrophotometric method for quantitative determination of tryptophan and tyrosine in peptides and proteins is described. It is based on two specific color reactions, the reaction of tryptophan with formaldehyde and the reaction of tyrosine and tryptophan with hydroxylamine and ceric cations. By combination of these two reactions both tyrosine and tryptophan can be determined simultaneously. Tyrosine and/or tryptophan bound in peptides and/or proteins react independently of the rest of the peptide or protein molecule. The method is simple, accurate, and sensitive. Hydrolysis is not necessary.