Estimation of polymerization efficiency in the formation of polyacrylamide gel,using continuous optical scanning during polymerization

The Ferguson plot and ‘quantitative’ gel electrophoresis (based on the Ferguson plot) depend on a knowledge of accurate gel concentrations. The easiest way to estimate accuracy of gel concentrations, in terms of the degree of completion of the polymerization reaction which gives rise to a gel, is by spectrophotometry. Making use of the apparatus for continuous optical scanning of polyacrylamide gels, the extent and rate of polymerization of cross-linked polyacrylamide were estimated by measuring the absorbance at 275 mm of the reaction mixture subsequent to free radical initiation of polymerization. Under appropriate conditions of monomer concentration, initiator levels and temperature, absorbance decreased monotonically after alag period of 10 min, and after 20–30 min of reaction the absorbance reached a plateau value which provided a measure of polymerization efficiency. Application of a standard curve of absorbance vs. monomer concentration allowed one to quantitate concentrations of residual monomer throughout the course of polymerization. Under a set of arbitrary polymerization conditions (e.g. 6–20% total gel concentration), the reaction went to 63–96% completion. The rate of polymerization was approximately proportional to the square of the monomer concentration (2nd-order reaction kinetics). Absorbance decrease subsequent to the initiation of the polymerization reaction appeared suitable as a measure of efficiency of polymerization since: (1) absorbance spectra of monomers at 0.5%T and residual monomers in a 10%T gel, at a time when polymerization seemed terminated, coincided; (b) values of residual monomer obtained were reasonable (10–30%); (c) bimolecular reaction kinetics were found, in agreement with expectation; and (c) absorbance of incomplete polymerization mixtures, deficient in either initiators or monomers, was constant with time.     

Polyacrylamide gel electrophoresis: reaction of acrylamide at alkaline pH with buffer components and proteins

The chemical reaction of monomeric acrylamide with primary, secondary, and tertiary amines, used as buffer components in polyacrylamide gel electrophoresis systems, was investigated in the basic pH range. Adduct formation proceeded for several minutes up to weeks, depending on the reactivity of the amino groups. A pH shift in the reaction mixture due to an altered pK value of the reaction product was observed. However, a few primary amines (tris(hydroxymethyl)aminomethane, 2-amino-2-methyl-1,3-propanediol) and secondary amines 3-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)-1-propanesulfonic acid, 3-(dimethyl(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid) showed negligible shifts of pH. They are, therefore, useful as components in the polymerization mixture; whereas some tertiary amines showing complete pH stability as well (e.g., triethanolamine) are not suitable, as they acted as accelerators of gel polymerization. Acrylamide can also covalently bind to proteins by reacting with the epsilon-amino group of lysine residues, especially. Bovine serum albumin, having an acidic isoelectric point, and the basic protein cytochrome c were treated with different acrylamide concentrations at alkaline pH yielding modified protein molecules with altered electrophoretic mobilities in different polyacrylamide gel electrophoresis systems. This reaction gave rise to artifacts in alkaline polyacrylamide gels and isoelectric focusing systems when residual acrylamide monomers were still present in the gel matrix after the polymerization process ceased.     

Cytochrome c-induced acceleration of the reaction of muscle actin polymerization

The reaction of rabbit skeletal muscle actin polymerization initiated by the addition of neutral salts is accelerated in the presence of mitochondrial cytochrome c. The observed effect is specific, since the addition of serum albumin does not change the initial velocity of this process. The dependence of the rate of actin polymerization and exogenous cytochrome c concentration correlates within the molar protein ratios from 43:1 to 9:1, respectively. The increase in the polymerization rate occurs immediately after addition of cytochrome c to the reaction mixture; however, the maximal effect is observed only after 5 min coincubation of the proteins. The ability of cytochrome c to stimulate this process is abolished at alkaline values of pH (8.5), which points to the significant role of the molecule positive charge which, in all probability, serves as a primer of the muscle actin polymerization reaction.     

Nucleation and growth phases in the polymerization of coat and scaffolding subunits into icosahedral procapsid shells.

The polymerization of protein subunits into precursor shells empty of DNA is a critical process in the assembly of double-stranded DNA viruses. For the well-characterized icosahedral procapsid of phage P22, coat and scaffolding protein subunits do not assemble separately but, upon mixing, copolymerize into double-shelled procapsids in vitro. The polymerization reaction displays the characteristics of a nucleation limited reaction: a paucity of intermediate assembly states, a critical concentration, and kinetics displaying a lag phase. Partially formed shell intermediates were directly visualized during the growth phase by electron microscopy of the reaction mixture. The morphology of these intermediates suggests that assembly is a highly directed process. The initial rate of this reaction depends on the fifth power of the coat subunit concentration and the second or third power of the scaffolding concentration, suggesting that pentamer of coat protein and dimers or trimers of scaffolding protein, respectively, participate in the rate-limiting step.     

Biocatalytic polyester synthesis: analysis of the evolution of molecular weight and end group functionality

The enzymatic synthesis of polyesters from activated diesters and diols has been investigated. Differences between enzymatic synthesis and traditional chemical condensation processes are discussed. The disappearance of monomers during the initial phase of reaction indicates that enzyme has a higher specificity for transesterification of ester-terminated oligomers. During the intermediate phase, enzymatic polymerization involves a competition between diol and enzyme-bound water for the nucleophilic attack of the acyl enzyme intermediate. Competition between enzymatic transesterification and hydrolysis at different stages of polymerization in nonaqueous media is responsible for termination of polyesters with acid end-groups and also for limiting the polymer molecular weight. The resulting oligoester consists of chains that are either terminated with - OH groups and/or - COOH groups. We have used Matrix Assisted Laser Desorption/Ionization - Time of Flight Mass spectroscopy (MALDI-TOF) along with colorimetric titration techniques to determine the acidity of enzyme-synthesized polyesters. This paper addresses how the enzymatic polymerization proceeds, and compares our results to the growing literature in this field. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 227-239, 1997.     

An NMR investigation of solution aggregation reactions preceding the misassembly of acid-denatured cold shock protein A into fibrils.

At pH 2.0, acid-denatured CspA undergoes a slow self-assembly process, which results in the formation of insoluble fibrils. 1H-15N HSQC, 3D HSQC-NOESY, and 15N T2 NMR experiments have been used to characterize the soluble components of this reaction. The kinetics of self-assembly show a lag phase followed by an exponential increase in polymerization. A single set of 1H-15N HSQC cross-peaks, corresponding to acid-denatured monomers, is observed during the entire course of the reaction. Under lag phase conditions, 15N resonances of residues that constitute the beta-strands of native CspA are selectively broadened with increasing protein concentration. The dependence of 15N T2 values on spin echo period duration demonstrates that line broadening is due to fast NMR exchange between acid-denatured monomers and soluble aggregates. Exchange contributions to T2 relaxation correlate with the squares of the chemical shift differences between native and acid-denatured CspA, and point to a stabilization of native-like structure upon aggregation. Time-dependent changes in 15N T2 relaxation accompanying the exponential phase of polymerization suggest that the first three beta-strands may be predominantly responsible for association interfaces that promote aggregate growth. CspA serves as a useful model system for exploring the conformational determinants of denatured protein misassembly.     

New Aspects of the Spontaneous Polymerization of Actin in the Presence of Salts

The mechanism of salt-induced actin polymerization involves the energetically unfavorable nucleation step, followed by filament elongation by the addition of monomers. The use of a bifunctional cross-linker, N,N′-(1,4-phenylene)dimaleimide, revealed rapid formation of the so-called lower dimers (LD) in which actin monomers are arranged in an antiparallel fashion. The filament elongation phase is characterized by a gradual LD decay and an increase in the yield of “upper dimers” (UD) characteristic of F-actin. Here we have used 90° light scattering, electron microscopy, and N,N′-(1,4-phenylene)dimaleimide cross-linking to reinvestigate relationships between changes in filament morphology, LD decay, and increase in the yield of UD during filament growth in a wide range of conditions influencing the rate of the nucleation reaction. The results show irregularity and instability of filaments at early stages of polymerization under all conditions used, and suggest that an earlier documented coassembling of LD with monomeric actin contributes to the initial disordering of the filaments rather than to the nucleation of polymerization. The effects of the type of G-actin-bound divalent cation (Ca²⁺/Mg²⁺), nucleotide (ATP/ADP), and polymerizing salt on the relation between changes in filament morphology and progress in G-actin-to-F-actin transformation show that ligand-dependent alterations in G-actin conformation determine not only the nucleation rate but also the kinetics of ordering of the filament structure in the elongation phase. The time courses of changes in the yield of UD suggest that filament maturation involves cooperative propagation of “proper” interprotomer contacts. Acceleration of this process by the initially bound MgATP supports the view that the filament-destabilizing conformational changes triggered by ATP hydrolysis and P_i liberation during polymerization are constrained by the intermolecular contacts established between MgATP monomers prior to ATP hydrolysis. An important role of contacts involving the DNase-I-binding loop and the C-terminus of actin is proposed.     

Formation of dipeptides--a side reaction in racemization of phenylalanine

It was shown that acetylated dipeptides, Ac-D-Phe-D-Phe-OH, Ac-L-Phe-L-Phe-OH, Ac-D-Phe-L-Phe-OH, and Ac-L-Phe-D-Phe-OH, are formed during D-phenylalanine racemization. The overall content of these dipeptides in the reaction mixture ranged from 40 to 60% depending on the reaction conditions. We concluded that, like alpha-aminoisobutyric acid, phenylalanine is prone to polymerization under racemization conditions.     

Polymerase chain reaction engineering

A mathematical model for polymerase chain reaction (PCR) is developed, taking into account the three steps in this process: melting of DNA; primer annealing; and DNA synthesis (polymerization). Activity and deactivation of the polymerase enzyme as a function of temperature is incorporated in the kinetic model to get a better understanding of the amplification of DNA. Computer simulation of the model is carried out to determine the effects of various parameters, such as the cycle number, initial DNA concentration (copynumber), initial enzyme concentration, extension time, temperature ramp, and enzyme deactivation on the DNA generation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 359-366, 1997.     

Actin polymerization. The mechanism of action of cytochalasin D

Fluorescence changes using actin covalently labeled with N-(1-pyrenyl)iodoacetamide have been used to determine the effect of cytochalasin D on actin polymerization. A mechanism for the effect of cytochalasin D on actin polymerization is presented, which explains the experimental observation of a cytochalasin D-induced increase in the initial rate of polymerization and a decrease in the final extent of the reaction. Central to this mechanism is the Mg2+-dependent formation of cytochalasin D-induced dimers. The dimers serve as nuclei to enhance the polymerization rate. Binding of Mg2+ to a low affinity site on the dimer induces a conformational change which can be observed as a rapid fluorescence increase. A subsequent time-dependent fluorescence decrease observed prior to polymerization appears to represent ATP hydrolysis resulting in dissociation of the dimer and release of actin monomers containing ADP. We postulate that a slow rate of exchange of ATP for bound ADP relative to hydrolysis results in the accumulation of monomers containing ADP. As these monomers have a high critical concentration, the final extent of polymerization is reduced dramatically. The Mg2+ dependence of the final extent of polymerization in the presence of cytochalasin D is also explained in the context of this mechanism.     

Chemical alterations taken place during deep-fat frying based on certain reaction products: A review

Deep-fat frying at 180°C or above is one of the most common food processing methods used for preparing of human kind foods worldwide. However, a serial of complex reactions such as oxidation, hydrolysis, isomerization, and polymerization take place during the deep-fat frying course and influence quality attributes of the final product such as flavor, texture, shelf life and nutrient composition. The influence of these reactions results from a number of their products including volatile compounds, hydrolysis products, oxidized triacylglycerol monomers, cyclic compounds, trans configuration compounds, polymers, sterol derivatives, nitrogen- and sulphur-containing heterocyclic compounds, acrylamide, etc. which are present in both frying oil and the fried food. In addition, these reactions are interacted and influenced by various impact factors such as frying oil type, frying conditions (time, temperature, fryer, etc.) and fried material type. Based on the published literatures, three main organic chemical reaction mechanisms namely hemolytic, heterolytic and concerted reaction were identified and supposed to elucidate the complex chemical alterations during deep-fat frying. However, well understanding the mechanisms of these reactions and their products under different conditions helps to control the deep-fat frying processing; therefore, producing healthy fried foods. By means of comprehensively consulting the papers which previously studied on the chemical changes occurred during deep-fat frying process, the major reaction products and corresponding chemical alterations were reviewed in this work.     

Kinetics of the polymerization reaction of tobacco mosaic virus protein: transient-saturation type polymerization reaction.

The kinetics of the endothermic polymerization reaction of tobacco mosaic virus protein in the mild acid region was studied by means of temperature-jump (rising time of 6 sec)-turbidimetry, electron microscopy, and computer simulation. The time course profile of the turbidity increase changed from a normal one to an anomalous one as the size of the temperature-jump was made greater. The anomalous type polymerization profile, which we named the "transient-saturation" type, could be characterized by a rapid increase of turbidity and its transient saturation, and a slow increase to the final level. At a higher concentration of the protein, this transient-saturation effect was more marked, whereas the slow turbidity in the second phase occurred with a higher rate. This transient-saturation type polymerization profile was observed also in a pH-induced polymerization reaction. It was not observed in the case of the N-bromosuccinimide modified tobacco mosaic virus protein under a similar environmental change. By an electron microscopic study and computer simulation, it was revealed that in the first phase, a large number of short polymers were formed, and the concentration of the polymerizing units was rapidly reduced to the equilibrium value, and the polymerization reaction stopped transiently. In the second phase, polymer-polymer associations took place slowly and longer polymers were formed. The revlevance of the present study to the polymerization reaction of actin, myosin, and to a transient-overshoot type polymerization are discussed.     

Reaction conditions for laccase catalyzed polymerization of catechol

Poly(catechol) was synthesized in batch runs with laccase from Trametes versicolor (ATCC 200801). The polymerization reaction was conducted in a closed, temperature controlled system containing acetone and sodium acetate buffer for pH control. The effects of the solvent mixture, monomer (catechol), enzyme, medium pH and temperature on the polymerization rate were investigated with respect to initial reaction conditions and depletion rate of dissolved oxygen in the medium. Maximum initial reaction rate was attained with 10% (v/v) acetone-sodium acetate buffer at pH 5.0, 25 degrees C, 0.02 U/ml enzyme and 250 mg/l initial catechol and 10 mg/l dissolved oxygen. A general saturation enzyme kinetics response was observed for catechol substrate. Temperature rise supported the rate increase up to 45 degrees C, after which the rate tended to be stable due to a drop in dissolved oxygen concentration as well as enzyme instability.     

Hot start of the polymerase chain reaction using DNA helicase]

A novel method for the hot start of PCR using DNA helicases is developed. The addition of a DNA helicase prevents the random annealing of primers and synthesis of nonspecific products during the preparation of the reaction mixture and initial heating. The hot start of PCR occurs automatically after inactivation of the DNA helicase upon heating of the reaction mixture.     

Pseudomonas cytochrome c peroxidase. Initial delay of the peroxidatic reaction. Electron transfer properties.

A delay of some seconds is observed in the reaction of Pseudomonas cytochrome c peroxidase if the reaction is initiated by adding the enzyme to the reaction mixture containing reduced electron donor and hydrogen peroxide. This lag phase is avoided if the enzyme is incubated with the reduced electron donor and the reaction is started by adding hydrogen peroxide. The nature of the initial delay has been studied and it is shown that the peroxidase is reduced before a steady-state rate in the peroxidatic reaction is reached. The ability of the peroxidase to accept electrons from various electron donors emphasizes its cytochrome-like properties.     

Preparation,purification and properties of a crosslinked trimer of G-actin

Phenylenebismaleimide has been used to form crosslinks between actin monomers [Knight, P. and Offer, G. (1978) Biochem. J. $\text{175}$, 1023–1032]. We have purified a trimer of actin monomers as well as a dimer and a mixture of higher molecular weight oligomers. The trimer is much more effective than the dimer in enhancing the rate of polymerization while higher oligomers do not appear to be any more effective than the trimer. A lag in the polymerization process, as measured fluorescence enhancement of trace pyrene-actin, still occurs in the presence of trimers serving as the nuclei, suggesting that the mechanism for polymerization is more complex than nucleation followed by elongation.     

Involvement of superoxide anion in the reaction mechanism of haemoglobin oxidation by nitrite.

The sigmoidal time course of haemoglobin oxidation by nitrite, involving an initial slow reaction accompanied by a subsequent rapid reaction, was extensively explored. The initial slow reaction was much prolonged by the addition of superoxide dismutase to the reaction mixture. On the other hand, in the presence of superoxide anion generated by xanthine oxidase systems, the slow phase disappeared and the reaction changed to first-order kinetics. The oxidation of intermediate haemoglobins [defined as haemoglobin tetramer in which different chains (alpha- or beta-) are in the ferric state and in the ferrous state] such as (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 also proceeded in a sigmoidal manner. Similar effects of superoxide anion on these reactions were observed. Since the intermediate haemoglobins such as (alpha 2+ beta 3+)2 and (alpha 3+ beta 2+)2 were found to be produced by the oxidation of haemoglobin by nitrite, the changes in oxyhaemoglobin, intermediate haemoglobins and methaemoglobin during the reaction were followed by isoelectric-focusing electrophoresis. The amounts of (alpha 2+ beta 3+)2 were larger than those of (alpha 3+ beta 2+)2 at the initial stages of the reaction, suggesting that there is a functional difference between alpha- and beta-chains in the oxyhaemoglobin tetramer. On the basis of these results, a reaction model of the haemoglobin oxidation by nitrite was tentatively proposed. The changes in oxyhaemoglobin, intermediate haemoglobins and methaemoglobin were well fitted to the simulation curves generated from the reaction model. Details of the derivation of the equations used for kinetic analysis have been deposited as Supplement SUP 50112 (5 pages) with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K. from whom copies may be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Synthesis of poly(L-lactide) and polyglycolide by ring-opening polymerization

This protocol describes the synthesis of poly(L-lactide) by ring-opening polymerization of L-lactide using tin(II) 2-ethylhexanoate catalyst as well as the synthesis of polyglycolide by ring-opening polymerization of glycolide. Ring-opening polymerization of cyclic diesters synthesized from alpha-hydroxycarboxylic acids gives high-molecular-weight polyester in high yield. Tin(II) 2-ethylhexanoate catalyst is the most common catalyst for ring-opening polymerization of diesters owing to its high reactivity and low toxicity. Purity of monomers and the amount of water and alcohol in the reaction system are significant factors for increasing molecular weight and conversion of polyesters. The molecular weight of the polyesters is also dependent on reaction temperature and reaction time. This protocol can be completed in 3 d for the synthesis of poly(L-lactide) and 2 d for the synthesis of polyglycolide.     

Inhibitory Effects of Melanin Monomers,Dihydroxyindole-2-Carboxylic Acid (DHICA) and Dihydroxyindole (DHI) on Mammalian Tyrosinase,With a Special Reference to the Role of DHICA/DHI Ratio in Melanogenesis

DOPAchrome tautomerase (DCT) is known to control the ratio of DHICA/DHI formed within the melanocyte, but physiologic significance of this activity is not yet fully elucidated. In this study the two melanin monomers are shown to inhibit with different efficacy the initial, tyrosinase-controlled, melanogenic reaction, namely conversion of L-tyrosine to DOPAchrome (2-carboxy-2,3-dihydroindole-5,6-quinone). This is demonstrated in the test tube assay system whereby formation of DOPAchrome is catalyzed by i) isolated premelanosomes (PMS), ii) tyrosinase-rich PMS glycoproteins, or iii) tyrosinase purified from fibroblasts transfected with human tyrosinase gene. Both DHI and DHICA suppress the conversion of L-tyrosine to DOPAchrome when added to reaction mixture but the inhibitory effect is far more strongly pronounced by DHI. DHI inhibits both activities of tyrosinase—tyrosine-hydroxylation and DOPA-oxidation—more strongly than DHICA. The different extent of inhibition is shown to reflect i) the ability of the two monomers to compete with tyrosinase substrates for the enzyme's active center and ii) the rate of interaction between melanin monomers and DOPAquinone. Consequently, we demonstrate that the tyrosinase-catalyzed DOPAchrome formation can be modulated by the ratio of DHICA/DHI among melanin monomers with the increased proportion of DHICA resulting in more efficient DOPAchrome formation. These results raise the possibility that DOPAchrome tautomerase plays a role in positive control of the tyrosinase-catalyzed early phase of melanogenesis.     

Enantioseparation by ligand-exchange using particle-loaded monoliths: capillary-LC versus capillary electrochromatography

Particle-loaded monoliths containing a polymethacrylamide backbone were prepared by suspending a silica-based chiral phase in the mixture of the monomers followed by in-situ polymerization in the capillary. As chiral selector l-4-hydroxyproline chemically bonded to 3 microm silica particles was used following the separation principle of ligand-exchange. Electrolytes containing Cu(II) ions were used. Amino acid enantiomers were separated by capillary-LC and CEC, whereby the latter showed the better resolution properties. For the chiral separation of alpha-hydroxy acids the EOF was reversed by copolymerizing diallyldimethylammonium chloride instead of vinylsulfonic acid as charge providing agent. Short columns of 6 cm were found to be sufficient in the case of CEC for baseline separations of amino acids with alpha values up to 5.