Dilute Acid Extraction Research Articles

Purification and properties of some unique low molecular weight basic proteins degraded during germination of Bacillus megaterium spores.

Two major proteins, termed proteins A and B, and one minor species, termed protein C, have been purified to homogeneity from dilute acid extracts of dormant spores of Bacillus megaterium. These three species comprise approximately 80% of the protein in the dilute acid extracts and account for 60 to 75% of the protein degraded during spore germination. All three proteins have low molecular weights (7,000 to 10,000), high isoelectric points (greater than 9.8), alanine as the NH2-terminal amino acid, are more hydrophilic than most proteins, and all lack cysteine, cystine, and tryptophan. In addition all three proteins are extremely sensitive to a wide variety of proteolytic enzymes, much more so than "average" proteins such as serum albumin, lysozyme, and hemoglobin. These proteins also bind to both purified DNA and to a nuclear body from dormant spores. Although this binding gives little or no protection to proteins A and B from proteolysis, it does result in elevation of the melting temperature of the DNA by as much as 20degrees.

Simultaneous Assay for Six Alkaloids in Opium, Using High-Performance Liquid Chromatography

A method is described for the quantitative determination of morphine, codeine, cryptopine, thebaine, papaverine, and narcotine in opium by high-performance liquid chromatography. The alkaloids are isolated from a dilute acid extract by adsorption on an Amberlite XAD-2 resin column and eluted first with methanol and then with chloroform-methanol (3+1). After solvent removal by reduced pressure evaporation, the alkaloids are redissolved in chloroform-methanol (3+1). The sample solution, plus brucine as an internal standard, is injected onto a Corasil II column and eluted with hexane that is gradient programmed with a solution of chloroform-methanol-diethylamine (100+300+1). The absorbances of the separated alkaloids are continuously monitored at 254 nm, using a flow-through ultraviolet double-beam photometer.

Measurements on disulphide bonds in cereal glutelins

AbstractCereal glutelins were divided by dilute acetic acid extraction into‘soluble’and‘insoluble’fractions. The quantities dissolved in the cases of oats and maize were negligibly small. The S.S bonds of these fractions from wheat, rye, barley, oats and maize have been amperometrically titrated after reaction with sulphite in the absence and presence of guanidine hydrochloride to give, respectively, the accessible and total S.S groups. Accessible S.S groups are believed to be mainly inter‐polypeptide chain bonds. The results imply that wheat glutenin resembles the other glutelins in being highly crosslinked. There is some evidence that the density of crosslinking is higher in oats, maize and barley glutelins, provided that the polypeptide chains of the other cereal glutelins are similar in size to those of wheat glutenin.

Isolation and characterization of antigens of Aspergillus fischeri.

Abstract Dilute acid extraction of Aspergillus fischeri mycelia contained two distinct antigens that were purified and separated by ethanol fractionation, concanavalin A precipitation, and chromatography on a Sephadex G-100 column. One antigen which emerges from the column among the early fractions contained 66% hexose determined as galactose and mannose but no demonstrable protein. The second antigen contained mannose as the sole hexose, was inactivated by pronase, and had a molecular weight of approximately 8000. Fungi of the genus Aspergillus are ubiquitous saprophytes capable of causing debilitating disease under certain conditions. Although aspergilli can attack any part of the body, they are most often encountered as respiratory pathogens. Aspergillus fumigatus is the species most frequently responsible for infection. Aspergillus fischeri is an ascosporic species within the aspergilli group, closely related to A. fumigatus This organism grows on Czapek's agar at 25 and 37°C and at 37°C it produces a...

Effects of Adding Charcoal During Dilute Acid Extraction of Manganese from Soils

AbstractAddition of charcoal during extraction of soil with dilute HCl + H2SO4 increased the amounts of manganese and iron extracted but had little or no effect on the copper, zinc, and aluminum extracted concurrently. Experiments indicated that the increase in extractable manganese due to the presence of charcoal was not a consequence of an increase in efficiency of extraction of reduced manganese already present but resulted from a reaction in which forms of manganese of low solubility were reduced and made soluble and in which CO2 and CO were evolved. The amounts of manganese extracted from soils by dilute HCl + H2SO4 in the presence of charcoal and by dilute HCl + H2SO4 containing hydroquinone were highly correlated. The effect due to charcoal increased with the acidity of the extracting solution, the time of extraction, and the amount of charcoal added.

PHOTOSYNTHESIS AND METABOLISM IN MARINE ALGAE: VII. PRODUCTS OF PHOTOSYNTHESIS IN FRONDS OF FUCUS VESICULOSUS AND THEIR USE IN RESPIRATION

Samples of Fucus vesiculcsus fronds were permitted to assimilate 14CO2 for 5 h and were then maintained in alternating periods of light and darkness for 3 days. Samples were collected at intervals, and the radioactivity of various simple and complex compounds was measured. The major product of photosynthesis was mannitol; relatively small amounts of 14C entered other compounds. From its behavior, it appears that mannitol is the major substrate of respiration in these plants; there may be secondary substrates among the complex polysaccharides. The complex polysaccharides are not formed directly from mannitol in light, but from some common precursors, or else from a small isolated pool of mannitol which is separated from the main cellular supplies. In darkness, the complex polysaccharides appear to be derived from stored mannitol. One of the more active metabolites, judged from its behavior, is a component of the residue left after dilute acid and sodium carbonate extraction. This component undergoes turnover, i.e. breakdown and resynthesis from newly-acquired photosynthate in the light, and is formed from stored photosynthate in the darkness.

Determination of the chloride content of small muscle samples

The chloride content of fat-free dry muscle tissue can be determined by dilute nitric acid extraction or alkaline digestion followed by titration with silver ions. If the sample of tissue weighs less than 30 mg a significant exponential relationship is found between the chloride content and sample size. Evidence is presented to suggest that free sulphydryl groups are present in varying amounts in extracts of fat-free dry muscle samples weighing less than 30 mg. These combine with silver ions in a reaction similar to that between silver and chloride ions and this effect accounts for the relationship between chloride content and sample size. This relationship is not observed if alkaline digestion is followed by perborate oxidation, or if chloride is measured by microdiffusion analysis instead of by silver ion titration.

Cholecystokinin and pancreozymin, one single hormone?

AbstractSecretin, cholecystokinin (GCK) and pancreozymin (PZ) are taken up on alginic acid from a dilute acetic acid extract of the heat‐coagulated duodeno‐jejunal mucosa of the hog. After elution they are precipitated with sodium chloride. From the salt cake secretin is taken up in methyl alcohol. From the material insoluble in methanol witli 5 Ivy dog units (IDU) of CCK per mg, the CCK and PZ activities are adsorbed on CMC cellulose and the active material further chromatographed on TEAE cellulose, resulting in a product with 250 IDU of CCK per mg. Further purification was achieved by passage through a Sephadex G 50 column and subsequent chromatography on an Amberlite XE‐64 column. The activity was now 3,000 (6,000) IDU/mg. Since Harper and Raper's original PZ preparation and a commercial sample of Pancreozymin Boots contain 0.3 Ivy dog units of cholecystokinin per mg the cholecystokinin activity has been enriched 10,000‐(20,000‐)fold. Parallel with this rise in strength of the CCK activity, as assayed in guinea pigs, runs an equal rise in the PZ activity, as determined in cats at three different levels of strength, namely at 0.3. 220 and 3,000 (6,000) IDU/mg. This observation strongly supports the assumption that both activities are exerted by one and the same substance. It is also known that, because of the methionine content of the purest polypeptide, both are inactivated through the action of a dilute H2aO2 solution. and both fully reactivated on subsequent reduction with cysteine hydrochloride.

A crystalline avian pancreatic protein

A protein was found to crystallize during dialysis of the 0.4–0.8 ammonium sulfate fraction of a dilute sulfuric acid extract of chicken pancreas. The yield of crystals was approximately 0.6–0.8 mg protein per gram of whole pancreas. The weight average molecular weight is 41,000 as determined by light scattering. Amino acid analysis indicates a molecular weight of about 6900 if the molecule has one methionine. A subunit structure is indicated by the change in sedimentation characteristics in the analytical ultracentrifuge before and after incubation of the protein in cold acidic salt solutions. The protein did not show enzymic, inhibitory, or hormonal activities known to be associated with exocrine and endocrine functions of the pancreas.

Extraction of Total Available Carbohydrates from Grass and Legume Tissue

Carbohydrates are the primary source of reserve energy stored in the vegetative organs of biennial and perennial forage plants. Reserves of available carbohydrates are essential to survival and to the production of plant tissues during periods when carbohydrate utilization exceeds photosynthetic activity. Total available carbohydrate3 levels and trends in storage organs are useful in indicating the periods of storage and usage and are useful in evaluating the potential of plants for regrowth and production following defoliation. A number of methods have been used to extract total available carbohydrates from forage plant tissue. However, the method of extrac-tion used will influence greatly the values obtained because forages vary in the type of carbohydrate stored. The common perennial and biennial forage legumes, such as alfalfa, red clover, white clover, and sweetclover, are characterized by the accumulation of sucrose and starch (3, 9, 1 ). Extraction of total available carbohydrates from these species has been accomplished largely by the takadiastase enzyme methad developed by Weinmann(16, 17) -or with acid solutions (8, 10). The common perennial forage grasses belong to 2 groups according to the type of reserve carbohydrate stored. Grasses native to tropical and subtropical latitudes, such as bermudagrass, bahiagrass, and dallisgrass, are characterized by the accumulation of sucrose and starch. Extraction of total available carbohydrates has been done as described above for legumes (17, 18). Grasses native to temperate latitudes, such as timothy and orchardgrass, accumulate sucrose and fructosan ( 1, 4, 7, 12, 13). Extraction of total available carbohydrates has been accomplished by acid hydrolysis (8) or with water (1, 14,15) since fructosan is soluble in water. Starch, however, is largely water-insoluble. This study was initiated to compare enzyme, dilute acid, and water extraction methods for the estimation of total available carbohydrates in forage plant tissue. Timothy stem bases and alfalfa roots were used as the test tissues. Timothy stores the largest proportion of its reserve carbohydrates as fructosan in the stem bases (4, 7). Alfalfa stores most of its reserve carbohydrates as starch in the roots (3). Materials and Methods

PHOTODIMERIZATION OF UROCANIC ACID IN VITRO AND IN VIVO.

Abstract 1. 1. Irradition of urocanic acid or urocanate frozen in aqueous solution with 280–320-mμ light produced a photodimer which was isolated by gradient elution from an anion-exchange resin. 2. 2. A similar substance was shown to be present in dilute acid extracts of guinea-pig epidermis after irradiation with 280–320-mμ light. 3. 3. The photodimer formed with 280–320-mμ light when irradiated in solution with 254-mμ light was converted to urocanic acid and smaller amounts of several other products. 4. 4. Irradiation of crystalline urocanic acid or its solutions does not produce an accumulation of the photodimer.

Non‐protein‐nitrogen in wheat flour

AbstractNon‐protein‐nitrogen (NPN) fractions obtained from a water extract of wheat flour by dialysis, heat or trichloroacetic acid (TCA) precipitation were compared. Dialysis was found to give a smaller and more reproducible fraction (about 25 mg. of NPN per 100 g. of flour), containing a higher proportion of amino‐nitrogen than the other two methods. A polysaccharide‐protein complex comprising about 30% of the non‐heat‐coagulable nitrogen, which is non‐dialysable but may be precipitated with TCA after incubation with poly‐saccharases, was studied in relation to the observed differences in NPN fractions.Free glutamic acid, aspartic acid, lysine, glycine, serine, alanine, proline, γ‐amino‐butyric acid, threonine, tyrosine, valine, iscleucine, leucine, glutamine, asparagine and tryptophan were found in the diffusible NPN fraction, and hydrolysis of peptides shown to be present, resulted in the release of no other amino‐acids.The formation of NPN on storage of water or dilute acid extracts of flour is attributed to the combined action of indigenous flour enzymes and microbial contaminants, as it was found to be partially inhibited by boiling and by a selection of antimicrobial agents.

Participation of neurohypophysial hormone in oviposition in the hen.

Posterior lobes of pituitary were removed from White Leghorn hens before and after oviposition (expulsion of egg), and their dilute acetic acid extracts were assayed for their activities on isolated rat uterus and on frog water balance. Both these activities significantly decreased at the time of oviposition. The contents of oxytocin and arginine vasotocin in the posterior lobe extracts were estimated by calculation, taking account of the potency ratios of the 2 hormones for the rat uterus and for the frog water balance. It became clear that the content of arginine vasotocin greatly decreased at the time of oviposition, while the content of oxytocin showed a little decrease. The oviposition in the hen may be caused by the prompt discharge of arginine vasotocin from the posterior lobe of pituitary.

STUDIES ON THE CONSTITUTING PEPTIDES OF THE HEALTHY LUNG TISSUES.

1) The constituting peptides of the protein obtained by dilute sulfuric acid extraction from defatted healthy lung tissues and caseosus lesions, have been studied.2) On the basis of counter current distribution study of the performic acid oxidized DNP-proteins of the original samples could have separated into several single peptides.3) Qualitative analysis of the N-terminal residues together with that of constituting amino acids of the individual single peptides, have shown ultimately, that the constituting peptides of the protein of the healthy lungs were : aspartyl, alanyl, and leucyl peptides in contrast to the aspartyl, beta-alanyl, and histidyl peptides from the caseosus focci respectively.

Concentration of parathyroid hormone activity by chromatography on carboxymethylcellulose

A method for the further purification of a fraction salted-out from a dilute hot hydrochloric acid extract of untreated ground bovine parathyroid glands is described. Chromatography on carboxymethylcellulose using a NaCl gradient rising to I M at pH 4.68 results in an active Ca-mobilizing fraction with a 6-fold increase in specific activity in moderate yield. This can be further increased 2- to 6-fold by recycling and eluting with a gradient rising to 0.5 M salt. The method is applicable to starting material containing either high or low amounts of activity.

Inositol Hexaphosphate: I. Quantitative Determination in Extracts of Soils and Manures

AbstractA procedure was developed for the quantitative determination of inositol hexaphosphates in extracts of soils and manures. The inositol hexaphosphates were separated from other phosphorus compounds by anion‐exchange chromatography in a simplified adaptation of the technique developed originally by Smith and Clark. Dilute hydrochloric acid extracts of manures can be added directly to the exchange columns, but extracts of soils obtained by the usual acid‐alkali extraction procedure must be given a preliminary treatment to remove organic matter that otherwise would flocculate under the acid conditions in the exchange columns.

Effect of Spermine on Tissue Oxidations.

Little is known of the biological rôle of spermine (N, N′-dipropylamino-diaminobutane) despite its widespread occurrence in mammalian tissues (human semen, bovine pancreas, spleen, thyroid, lung, ovary, brain, ocular tissue, muscle, liver, and intestine). We have isolated spermine from dilute acid extracts of swine duodenal mucosa. The fact that these extracts produce a hyperglycemia in rabbits led us to investigate the effects of the pure base on the blood sugar level. The intramuscular injection of 15–25 mg per kilo of spermine into fasted, male rabbits weighing about 2 kilos, induces a definite and prolonged hyperglycemia. In order to illuminate further the effects of spermine on the intact organism we have undertaken a general study of the action of the base on the respiration of isolated mammalian tissues. The addition of spermine to guinea-pig brain brei suspended in a phosphate-saline medium has little immediate effect on the respiration as measured by the direct Warburg technic. However, in an extensive series of experiments, a slight increase of oxygen consumption in the presence of spermine was noted, especially during the third and fourth hour following the addition of the amine. If glucose is present as a substrate for either brain brei or brain slices (cortex), the addition of spermine causes a marked inhibition of the oxygen consumption (for typical experiments see Table I. Exps. 11, 13, 81). This inhibition (calculated as % inhibition of the extra oxygen consumption due to glucose) shows a tendency to decrease with time, especially when brain slices are used. The averaged data from a series of 8 experiments on brain brei gave values of 75, 74, 73, and 65% inhibition of oxygen consumption by 0.0060 M spermine in each of the 4 thirty-minute periods following addition of the amine.

Biological and Chemical Studies of Extracts of the Anterior Lobe Pituitary

ABSTRACT A buff-coloured precipitate is obtained by the addition of N/20 iodine solution to dilute acetic acid extracts of fresh anterior lobe substance. The volume of the precipitate depends upon the volume of the iodine solution added. Similar acetic acid extracts of tissues and extracts of the anterior lobes prepared with other extracting media do not give the same precipitate with iodine. The precipitate indicates the presence and, within limits, gives an approximate measure of the metamorphic factor in fresh gland extracts of the anterior lobe. Dilute acetic acid extracts of the precipitates after removal of the iodine by shaking with alcohol are biologically active and characterised by their phosphate content. Similar extracts of the posterior lobe give an iodine precipitate but the phosphate content is much lower than that of the anterior lobe precipitate. The iodine in these precipitates is adsorbed or loosely combined. The adsorbed iodine is chemically more reactive than dissolved iodine or iodine crystals giving rapid evolution of nitrogen from sodium azide compared with the failure of the others. The biological activity of anterior lobe extracts varies with the time of extraction and the concentration of the extracts. There is a maximum for the time of extraction at any particular concentration and acid strength after which a loss of potency occurs. There is a continuous extraction—rapid at first—of the melanophore stimulant. The amount increases with the period of extraction and concentration, but is independent of the strength of the acid used in extraction. The metamorphic factor is slowly destroyed by pepsin but rapidly by trypsin. The biological activity is diminished by boiling, or by boiling with dilute hydrochloric acid, sodium hydroxide, or sodium carbonate.