Low Molecular Weight Peak Research Articles

Study of radiation‐induced polymerization of vinyl monomers adsorbed on inorganic substances. X. Influence of H2O on the styrene–silica gel system

AbstractTo elucidate the reaction mechanism of radiation‐induced polymerization of the styrene—silica gel system, the influence of H2O as adsorbed water and inhibitor of cationic polymerization was investigated by two methods. Monomer conversion decreased as H2O increased. In general, percent grafting decreased as H2O increased, but the presence of a small amount of H2O increased the percent grafting. Grafting at 16 Mrad has a maximum value at a water content of about 0.2%. This seems to be due to two effects of H2O: percent grafting increases due to restraint of cationic polymerization by H2O, but the percent grafting decreases due to adsorption water which interrupts the contact of styrene with silica gel. In GPC spectra, the low molecular weight peaks of both graft polymers and homopolymers decreased when H2O was added. The GPC results suggest that the number of positive holes which initiate cationic polymerization is very large.

Study of radiation‐induced polymerization of vinyl monomers adsorbed on inorganic substances. IX. Preirradiation polymerization of styrene–silica gel system

AbstractPreirradiation polymerization of the styrene–silica gel system was studied in detail. Both graft polymers and homopolymers have bimodal GPC spectra. High molecular weight peaks were formed in a radical mechanism and the low molecular weight peaks were formed in a cationic mechanism as same as those in the simultaneous irradiation polymerization. The rate of formation of the low molecular weight peaks was very high compared with that of the high molecular weight peaks. Monomer conversion and percent grafting leveled off at about 1–2 Mrad. Radiation dose dependence of the four peaks were different from each other. Monomer conversion and percent grafting decreased as the preheating temperature of silica gel increased. The amount of the low molecular weight peaks of graft polymers depended on the number of silanol groups, as in the case of the simultaneous irradiation polymerization. A reaction mechanism for the preirradiation polymerization is proposed based on the results obtained.

Photoinduced Ionic Polymerization. VI. Molecular Weight Distribution in Cationic Polymerization of α-Methylstyrene

The photoinduced cationic polymerization of α-methylstyrene was carried out in dichloromethane solution using pyromellitic dianhydride as an electron acceptor, and the molecular weight distributions of the resulting polymer were measured by gel-permeation chromatography. It was found that the molecular weight distribution was bimodal at low monomer concentration and low temperature. The lower molecular weight peak, which increased with decreasing monomer concentration and temperature, was assigned to the free-ion polymerization, and the higher-molecular weight one to the ion-pair polymerization. It was shown that the molecular weight of the polymer produced by the free-ion polymerization decreased with decreasing monomer concentration, but was not affected by temperature, while that of the polymer produced by the ion-pair polymerization decreased with increasing temperature, but was not affected by the monomer concentration. The propagation mechanism was discussed in connection with the conventional catalytic and radiation-induced polymerizatson.

Study of radiation‐induced polymerization of vinyl monomers adsorbed on inorganic substances. VII. Effect of pretreatment temperature of silica gel on styrene–silica gel system

AbstractTo investigate the reaction mechanism of radiation‐induced polymerization of styrene adsorbed on silica gel, the effect of pretreatment temperature of silica gel was studied. Preheating of silica gel was carried out at 200, 500, and 800°C. The number of silanol groups of silica gel surface decreased as preheating temperature increased. The rate of polymerization on the silica gel preheated at 500°C was faster than that at 200°C, but the polymerization rate on the silica gel preheated at 800°C was the lowest. These results suggest that rate of polymerization on the silica gel is affected by the conditions of silica gel surface such as the number of silanol groups and the pore size. At the same monomer conversion, percent grafting decreased as preheating temperature of silica gel increased. The GPC spectra of both graft polymers and homopolymers have two peaks at all preheating temperatures. The monomer conversion of low molecular weight peaks of graft polymers decreased as preheating temperature of silica gel increased. This result suggests that there is a probability that the grafting sites of low molecular weight peaks of graft polymers somehow interact with silanol groups.

Purification and use of the C3d subunit C3.

A method is described for the preparation of C3d from fresh-frozen CPD plasma. A redissolved EDTA euglobulin precipitate formed from defibrinated plasma is chromatographed on DEAE cellulose. The C3-rich peak is further chromatographed by stepwise elution from hydroxy-apatite. The highly purified C3 and C3b so obtained are then treated with commercial C3b inactivator. G-200 sephadex filtration of this material produces a low molecular weight peak containing C3d greater than 95 per cent purity. The purified C3d, which contains negligible carbohydrate, has a molecular weight of 28,000 (based on SDS polyacrylamide gel electrophoresis). When added at a final concentration of less than 70 mug per ml, it completely inhibits the anti-C3d hemagglutinin reactivity of a potent anti-C3d antiglobulin serum but does not inhibit sera with reactivities against C3c, C4b, and C5b. Two of three rabbits hyperimmunized with purified C3d produced antisera exhibiting only anti-C3d precipitin activity. After absorption of heterologous antibodies, the antisera strongly agglutinated C3d-coated RBC, failed to agglutinate RBC glutinated C3d-coated RBC, failed to agglutinate RBC coated only with C4, and reacted weakly against strongly anti-Rh coated RBC. The latter reactivity was readily absorbed by whole IgG. Preliminary studies with 125I-labeled C3d indicate its potential value in assessing potency of anti-C3d reagents.

Radiation‐induced polymerization of vinyl monomers adsorbed on inorganic substances. VI. Temperature dependence and effects of additives on methyl methacrylate–silica gel system

AbstractTo elucidate the reaction mechanism of radiation‐induced polymerization of methyl methacrylate adsorbed on silica gel, the temperature dependence and effects of acetone and pyridine were investigated. It was found that even at −78°C the polymerization rate was quite fast. The amounts of high molecular weight GPC peaks of both graft polymers and homopolymers increased with increasing irradiation temperature. The activation energy of the adsorbed state polymerization was low compared with that of bulk polymerization. The low molecular weight peaks of homopolymers decreased with acetone addition but were almost unaffected by pyridine. The low molecular weight peaks of homopolymers were thus polymerized by an anionic mechanism. In the methyl methacrylate–silica gel system both radical and anionic polymerization take place at the same time in formation of graft polymers and homopolymers. A reaction mechanism for the methyl methacrylate–silica gel system was proposed based on the results obtained to date.

Study of radiation‐induced polymerization of vinyl monomers adsorbed on inorganic substances. V. Effects of p‐benzoquinone and dose rate on methyl methacrylate–silica gel system

AbstractIn order to elucidate the mechanism of radiation‐induced polymerization of methyl methacrylate adsorbed on silica gel, the effects of p‐benzoquinone addition and dose rate were studied in detail. Most of the polymerization is inhibited by p‐benzoquinone at levels above 10‐2 mole/l. The GPC spectra of both graft polymers and homopolymers show two peaks. The high molecular weight material appears to have been formed by polymerization by a radical mechanism, because these peaks decrease as p‐benzoquinone concentration increases; on the other hand, their low molecular weight polymers seem to be products of an ionic polymerization mechanism because those peaks are almost not affected by p‐benzoquinone. The four GPC peaks differ in dose rate dependences of their polymerization rate. The dose‐rate exponents of polymerization rate were obtained for the four GPC peaks. The behavior of the low molecular weight peaks of graft polymers and homopolymers were quite different, suggesting that the polymers differ considerably in formation mechanism.

In vitro transcription by isolated nuclei of Rhynchosciara americana salivary glands. Characteristics of incorporation and inhibition by alpha-amanitin.

A method for the isolation of polytene nuclei from salivary glands cells of the Diptera Rhynchosciara americana is described. The stage-specific morphological pattern of the chromosome is maintained during the isolation. The isolated nuclei show two distinct RNA polymerase activities, namely I and II, characterized on the basis of ionic requirements and alpha-amanitin sensitivity. Studies of the product under the incubation conditions show that the system allows the synthesis of high-molecular weight RNA, beside a low molecular weight peak which may comprise pre-4S and 5S RNAs. Autoradiographic studies carried out in the presence or absence of the toxin alpha-amanitin showed that micronucleoli contain products of RNA polymerase type I activity (ribosomal RNA) and that the DNA puffs are engaged in alpha-amanitin sensitive RNA synthesis and thus are sites of polymerase type II activity.

Importance of Protease Inhibition in Studies on Purified Factor VIII (Antihaemophilic Factor)

According to some authors factor VIII procoagulant activity may be dissociable from carrier protein (MW approximately 2 X 10(6) by agarose gel filtration, e.g. at high ionic strength. We were able to reproduce this phenomenon. However, addition of protease inhibitor (Trasylol) prevented the appearance of low molecular weight peak of factor VIII procoagulant activity both at high ionic strength and elevated temperature (37 degrees C). We conclude from our results that procoagulant activity and carrier protein (von Willebrand factor, factor VIII antigen) are closely associated functional sites of native factor VIII macromolecule. Consequently, proteolytic degradation should be avoided in functional and structural studies on factor VIII and especially in preparing factor VIII concentrate for therapeutic use.

Purification and synthesis of eosinophilotactic tetrapeptides of human lung tissue: identification as eosinophil chemotactic factor of anaphylaxis.

Preferential eosinophil chemotactic activity exhibiting a molecular weight comparable to that released from sensitized human lung fragments challenged with specific antigen and designated eosinophil chemotactic factor of anaphylaxis has been isolated from extracts of human lung fragments by sequential purification on Sephadex G-25, Dowex-1, Sephadex G-10, and paper chromatography. Two eosinophilotactic tetrapeptides of amino acid sequence Val-Gly-Ser-Glu and Ala-Gly-Ser-Glu were recovered from the extracts in 4-12% overall yield of the low molecular weight peak from Sephadex G-25. Purified eosinophil chemotactic factor of anaphylaxis and the synthetic tetrapeptides were maximally active in amounts from 0.1 to 1.0 nmol per chemotactic chamber, and the activity was dependent on both the NH2-terminal and the COOH-terminal residues. Both natural and synthetic peptides were preferentially chemotactic for eosinophils and rendered them unresponsive to a subsequent stimulus.

Study of radiation‐induced polymerization of vinyl monomers adsorbed on inorganic substances. III. Effect of p‐benzoquinone and ammonia on radiation‐induced polymerization of styrene adsorbed on silica gel

AbstractIn order to elucidate the mechanism of radiation‐induced polymerization of styrene adsorbed on silica gel, the effect of p‐benzoquinone and ammonia was investigated. The high molecular weight GPC peaks of both graft polymers and homopolymers decreased with increasing p‐benzoquinone concentration, while the low molecular weight peaks of both graft copolymers and homopolymers decreased with increasing ammonia concentration. The results indicate that the high molecular weight peaks of both graft and homopolymers are formed as a result of a radical mechanism and that the low molecular weight peaks of both types of polymers are formed by a cationic mechanism. In formation of both graft polymers and homopolymers both radical and cationic polymerization take place at the same time.

Insoluble non-collagenous cartilage glycoproteins with aggregating sub-units

Two insoluble non-collagenous glycoprotein fractions (A and G) have been separated from puppy rib cartilage, following extraction of most of the proteoglycan and digestion of the insoluble residue with purified collagenase. After reduction, alkylation and extraction with sodium dodecylsulfate most of each protein is solubilized. Gel electrophoresis of solubilized A or G shows the presence of either one or two bands and gel chromatography shows both high and low molecular weight peaks. The production of a low molecular weight electrophoresis band from the high molecular weight Sephadex fraction indicates that there is aggregation and disaggregation of sub-units in sodium dodecylsulfate. Both A and G are high in aspartate plus glutamate and have a low hydroxyproline content. The insoluble A and G both contain hexose, uronic acid, galactosamine, glucosamine and a small amount of sialic acid, but they differ in their contents of hexose and six amino acids. They both form single bands in CsCl gradients but they differ in density. Electron microscopy shows that both insoluble glycoprotein fractions stain with lead, ruthenium red, or alcian blue plus phosphotungstate and that G contains many fine filaments. Material with the same appearance and staining properties was found to occur on the surface of collagen fibres in the undigested cartilage residue.

Partial purification of osteoclast-activating factor from phytohemagglutinin-stimulated human leukocytes.

Osteoclast-activating factor (OAF) is a soluble mediator found in supernates of human peripheral leukocytes which have been cultured with antigens or phytomitogens. OAF is a potent stimulator of osteoclastic resorption of fetal bone in organ culture. The present studies were designed to characterize OAF chemically. Bone resorbing activity from supernates of leukocytes cultured without added plasma was not lost on dialysis using a membrane with a molecular weight cutoff of 3,500, but was lost when heated to 60 degrees C for 30 min. The activity was lost after treatment with trypsin or pronase but not after treatment with ribonuclease or neuraminidase. Papain, which inactivated parathyroid hormone at a concentration of 25 mug/ml, did not inactivate OAF at 250 mug/ml. OAF did not react with an antibody to bovine parathyroid hormone which cross-reacts with human parathyroid hormone. OAF was also distinguished from active metabolites of vitamin D and from prostaglandin by extraction procedures and immunoassay for prostaglandin E(2). When the medium from activated leukocytes cultured with autologous plasma was fractionated by gel filtration on Sephadex, bone resorbing activity eluated both with plasma proteins and in lower molecular weight fractions. However, when medium from leukocytes cultured without added plasma was chromatographed, all the OAF activity was eluted in a sharp low molecular weight peak located between chymotrypsinogen (25,000 molecular weight) and ribonuclease A (13,700 molecular weight). This peak contained about 4% of the total protein originally present in the supernate. Its activity was destroyed by overnight incubation at 37 degrees C at pH 6 or 8, but not at pH 7.2. After incubation at 4 degrees C, the activity was lost at pH 3 or 10, but not at pH 4-9. The active fraction from Sephadex G-100 was therefore chromatographed at pH 7.2 on DEAE cellulose and carboxymethyl cellulose. The active material was not adsorbed; however, about sevenfold further purification was achieved by removal of contaminants. The material obtained after sequential Sephadex, DEAE and, carboxymethyl cellulose chromatography stimulated resorption of fetal rat bone in culture at concentrations of 0.75-3 mug protein/ml, indicating that this preparation of OAF was nearly as potent as bovine parathyroid hormone in this system.

Characterization of gastric mucosal membranes. VI. The presence of channel-forming substances

Abstract Highly purified gastric membranes were extracted by ionic or non-ionic detergents. Further fractionation was carried out on acrylamide gel electrophoresis. Some defined bands, incorporated into artificial bilayer membranes, produced discrete conductance changes characteristics of channel activity. A low molecular weight peak, purified through two cycles of electrophoresis was characterized as an anion-selective, positively voltage dependent channel, and a model to account for the action of the transport ATPase incoporating this channel concept is suggested.

Polymerization and Crystallization of Polytetrahydrofuran and Poly(1,4-epoxycyclohexane)

Abstract The polymerization of tetrahydrofuran and 1,4-epoxycyclo-hexane initiated by triethyloxonium hexachloroantimonate has been studied at temperatures below 0°C. It has been found that during polymerization of tetrahydrofuran in bulk at temperatures of -25°C and below the polymer crystallizes from a solution of its own monomer. The crystallization accelerates the polymerization somewhat. A chain transfer reaction takes place at higher conversion according to molecular weight and molecular weight dis-tribution measurements. The crystallization of poly(1, 4-epoxycyclohexane) during polymerization at -15°C in methylene chloride starts im-mediately after polymerization has been initiated. At very low conversion the distribution of molecular weight is unimodal and narrow; at higher conversion the distribution of molecular weight is bimodal, the high molecular weight peak becomes more prominent, and the distribution broader with increasing conversion. It is suggested that the low molecular weight peak corresponds to the polymer fraction

The presence and reactions of high and lower-molecular-weight procoagulant factor VIII in the plasma of patients with von Willebrand's disease after treatment: Significance for a structural hypothesis for factor VIII

The levels of procoagulant factor VIII activity in the plasma of four patients with von Willebrand's disease (v Wd) obtained 24 hours after treatment with cryoprecipitate or fresh-frozen plasma were higher than those of factor-VIII-related antigen. Analysis of the plasma by gel chromatography on sepharose 60 showed two peaks of factor-VIII procoagulant activity. A lower-molecular weight peak was not seen in normal plasma or that of treated haemophiliacs. In one patient hitherto diagnosed as v Wd an intermediate pattern of results was observed but this patient was otherwise not typical and may have been suffering from a variant of haemophilia or v Wd. In the patients tested the lower-molecular weight procoagulant factor VIII did not precipitate with rabbit antiserum to factor-VIII-related antigen and a plasma sample which contained excess lower-molecular weight procoagulant activity did not neutralise the precipitating capacity of rabbit antiserum. It did however neutralise the procoagulant inhibitory activity of rabbit antiserum and human inhibitor. The findings are consistent with the hypothesis that “factor VIII” consists of a high-molecular weight unit (HMW) and a lower-molecular weight unit (LMW). It is suggested that HMW, autosomally controlled, gives precipitin reactions with the rabbit antisera and stimulates the formation or release of LMW. It is further suggested that LMW, controlled by the X-chromosome, is complexed to or carried by HMW. The evidence indicates that LMW has procoagulant activity alone or when carried by HMW and the appearance of free LMW in treated v Wd supports the proposed hypothesis.

The uptake of testosterone and zinc in vitro by the human benign hypertrophic prostate.

SUMMARY The uptake of 65Zn and [1,2-3H]testosterone by minced tissue of human benign hypertrophic prostates and the subcellular distribution of radioactivity were examined. The nature of steroid and 65Zn binding by the cytosol (105000 g supernatant) fraction was investigated by gel filtration, ion-exchange chromatography and electrophoresis. It was found that steroid binding after incubation at 4°C was specific. One or two regions of steroid binding were observed after gel filtration of the cytosol using Sephadex G-200, depending upon incubation conditions. Binding of 65Zn was found in the low molecular weight peak after G-200 gel filtration. Equimolar CdCl2 and 65ZnCl2 were incubated with [1,2-3H]testosterone and minced tissue and the cytosol was subjected to gel filtration. Compared with control values, without CdCl2, reduction of 65Zn binding by about 50% occurred, while binding of 3H-labelled steroid was unaffected. Electrophoresis and ion-exchange chromatography showed that 65Zn and 3H-labelled steroid were bound to different proteins. A sample of the zinc-binding protein was prepared by ion-exchange chromatography and the homogeneity was checked by electrophoresis.

Molecular weight distributions in radiation‐induced polymerization. VI. γ‐ray‐induced polymerization of “dry” styrene in the solid state

AbstractThe γ‐ray‐initiated polymerization of styrene in the solid state has been studied over the temperature range −35°C to −55°C for samples exhaustively purified and dried to remove residual water (“dry” samples). Comparison with kinetic results previously reported for dry samples in the liquid state indicates a sharp decrease in the rate of polymerization resulting from the liquid to solid state transition. The molecular weight distributions for in‐source polymerization at −35°C and −40°C are bimodal in nature, and the appearance of a third peak is noticeable at −47°C and −55°C. In the case of postpolymerization at −35°C the molecular weight distribution is bimodal as in the case of in‐source samples. In the former case, however, the high molecular weight peak is predominant whereas the low molecular weight peak predominates in the latter. These results have been tentatively attributed to the postulated coexistence of two distinct propagating species which are radical and cationic in nature.

Studies on indoleacetic acid oxidation by liquid medium from crown gall tissue cells: The role of malic acid and related compounds

1. 1. Indoleacetic acid oxidation by liquid medium from crown gall tissue culture cells has been studied. The reaction has a pH optimum of 4.5 and requires Mn 2+ and a monohydric phenol. A short lag phase is routinely observed. The appearance of peroxidase and indoleacetic acid oxidising activity in the medium of a tissue culture was followed over a 3 week time course. One function of this enzyme may be to prevent the accumulation of excess inhibitory concentrations of indoleacetic acid. 2. 2. Gel filtration of the medium through Sephadex G-25 produced 3 peaks. The high molecular weight fraction contained peroxidase but had almost completely lost the ability to oxidise indoleacetic acid unless recombined with the first low molecular weight peak. Malic and other compounds of related structure also reactivated in the same way as the natural activator. Mn 2+ and a monohydric phenol were required in each case. Neither effect was due to inhibition of endogenous catalase. The second low molecular weight peak inhibited indoleacetic acid oxidation by extending the lag phase. 3. 3. Both substrate activation and inhibition were observed in the presence of low levels of activator but were overcome with increasing amounts of activator. There was a non-linear dependence of reaction rate on enzyme concentration which was not apparent in the presence of 3.6 M urea. 4. 4. H 2O 2 is not an obligatory intermediate although it greatly decreased the lag phase and enhanced the rate of oxidation. Mn 2+ was unnecessary for these effects. Competitive studies indicated that both malic acid and H 2O 2 may act on the same process in the reaction mechanism. 5. 5. A model is put forward, based on the hypothesis that the reaction is autocatalytic with a cyclic mechanism involving unstable intermediates, for the participation of malic acid and Mn 2+ as well as monohydric phenols and H 2O 2 in the enzymic oxidation of indoleacetic acid by oxygen. 6. 6. In addition to malic acid, a wide range of non-phenolic compounds was tested for activating ability. The structural characteristics of activators are discussed.

Possibility of the silylation technique in gas liquid chromatography of fulvic and humic acids

The silylation technique was applied to produce volatile and thermally stable tri-methyl (TMS) derivatives suitable for gas liquid chromatography of fulvic and humic acid extracted from a Greenville loam. The chromatograms appeared to be useful for fingerprinting the different organic matter preparations. They had two distinct regions, a region between 0–15 min retention time consisting of a group of peaks due to low molecular weight organic components and another region between 29–45 min retention time consisting of a group of peaks due to higher molecular weight components. The hot water extract was identified with infrared as a polysaccharide, possessing a gas-chromatogram dominated by the low molecular weight components. The HCl extract was similar, but had in addition somewhat more higher molecular weight components. Fulvic acid had both low and high molecular weight components represented in the gas-chromatograms but had a polysaccharide infrared spectrogram. Humic acid chromatograms were dominated by high molecular components, although some low molecular weight peaks were noted. One particular HCl- and/or H 2O polysachharide component seemed to prevail in fulvic as well as in humic acid. There is some evidence that different pretreatments might cause a change in the nature and number of components extracted.