Saturated Ammonium Sulfate Solution Research Articles

RADIOIMMUNOASSAY OF PLASMA TESTOSTERONE

SUMMARYA radioimmunoassay for the determination of testosterone in human plasma is described. After extraction from plasma, testosterone is separated by paper chromatography. The radioimmunoassay is performed using an antiserum to testosterone‐3‐oxime‐rabbit serum albumin and a saturated solution of ammonium sulphate is used for separation. The reliability criteria of the method in terms of precision, accuracy, sensitivity and specificity have been evaluated.The mean level of testosterone in plasma samples from nineteen normal men (age range 16–73 years) is 527±226 (±SD) ng/100 ml. In ten normal ambulatory females (in the second phase of the cycle) aged 19–35 years the mean plasma testosterone is 27±8±10±6 (±SD) ng/100 ml. In twenty‐four impotent men (aged 18–50) the testosterone level is 431±191 (mean±SD) ng/100 ml. These values are not significantly different from normal subjects.Lastly, the testosterone levels found in a few cases of male hypogonadism are reported.

Preliminary crystallographic data for Escherichia coli β-lactamase

The β-lactamase (penicillinase) from Escherichia coli W3310 has been crystallized from 25% saturated ammonium sulfate solution at pH 6.9. An X-ray examination of the monoclinic crystals shows the space group is C2, with unit cell dimensions a = 47.2 A ̊ , b = 76.9 A ̊ , c = 73.3 A ̊ and β = 98.6°. With Z = 4 and the molecular weight = 22,000 (Melling & Scott, 1972), the Å 3/dalton ratio is 2.98. The crystals are suitable for structure analysis to at least 2.4 Å resolution.

Modified Rapid Screening Method for Aflatoxin in Corn

Abstract A rapid screening method for detecting aflatoxin in cottonseed products was investigated to determine if it was applicable to corn. Other aflatoxin-extracting solvents and protein-precipitating agents were tried with white and yellow corn. When acetone-water (85+15) and a saturated solution of ammonium sulfate were substituted for acetonitrile-water (80+20) and lead acetate in the cottonseed method, fewer interferences and more compact fluorescent zones were observed on the developed minicolumns. The modified method for corn was applicable to samples containing 10 ppb or more aflatoxin.

Isolation and characterization of some of the proteins that shuttle between cytoplasm and nucleus in Amoeba proteus.

AbstractThe Rapidly Migrating Proteins (RMP) which shuttle nonrandomly between nucleus and cytoplasm and equilibrate in approximately equal amounts in each compartment, were isolated from Amoeba proteus by implanting 3H‐protein containing nuclei into unlabeled cells and some time later extracting the labeled material from the cytoplasms of such cells. The labeled material was subsequently fractionated by gel filtration in Sephadex G‐100 columns. The RMP are soluble in dilute salt solutions and appear as a heterogenous group of molecules, one component of which seems to be a single species of protein accounting for ca. one‐third of the RMP fraction. Because of its distinctness this component, called the LR fraction, received the major attention in this study. LR was found to comprise ca. 17% of the aqueous‐soluble proteins of the nucleus and ca. 3–4% of the total cell protein.LR has a very low molecular weight as determined, e.g., by its elution from a Sephadex G‐100 column. Because of its low molecular weight, LR could be purified by taking advantage of the fact that LR is (1) soluble in a saturated Solution of ammonium sulfate and (2) insoluble in butanol, diethyl ether, and 10% trichloroacetic acid.LR migrates toward the anode as a single band when subjected to electrophoresis on “standard disc” and SDS polyacrylamide gels. It does not enter a gel designed to separate basic proteins (at pH 4.0). When subjected to Sephadex G‐25 gel filtration LR migrates through the gel as a single band and elutes from the gel at a position in the middle of the linear separation range that indicates its molecular weight is ca. 2300. The only N‐terminal amino acid found in the LR fraction is proline.Evidence is presented to show that LR is not the product of a non‐specific breakdown of protein produced during its isolation, but the possibility that it results from the cleavage of a single chemical bond of a larger polypeptide, has not been eliminated.When injected into non‐labeled amebae, purified radioactive LR concentrates in the nucleus — just as radioactive RMP concentrates in a recipient cell nucleus when an amino acid‐labeled nucleus is implanted into an unlabeled cell.

A method for the determination of plasma testosterone by competitive protein-binding after chromatography on Sephadex LH-20

A method is described for the determination of testosterone in plasma by a competitive protein-binding technique. The procedure includes ether extraction and chromatography on Sephadex LH-20. Sex steroid-binding plasma protein (SBP), present in pregnancy plasma, was used as the binding protein and bound testosterone was precipitated with saturated ammonium sulphate solution. The mean plasma testosterone found in normal females was 45.5 ng/100 ml (range 27.6–68.7) and in normal males: 427.8 ng/100 ml (range 280–597.5). Values for a number of endocrinological disorders are also reported. The suitability of the method for clinical use is discussed.

高湿下における干しのり成分の変化-I

It is well known that the color and flavor of “Nori” (dried laver) rapidly deteriorate under humid conditions. In collective surveys of the contents of the various “Nori” components in relation to the storage humidity, the authors have made a comparison of the contents of pigments, organic acids, free amino acids, sugars, and lipids of “Nori” which had been stored under dry and humid conditions. 1) Six lots of 90 g each of “Nori” prepared from Porphyra yezoensis UEDA were stored separately at 20°C for 10, 25, and 45 days under dry and humid conditions, controlled by concentrated sulfuric acid and saturated solution of ammonium sulfate, respectively. 2) Chlorophyll is relatively stable under dry, but not under humid conditions. In the latter case the calculated contents of chlorophyll differed somewhat depending on the method of estimation. 3) Carotenoid is more stable than chlorophyll. The rate of decomposition is greater under humid than under dry conditions, and after 25 days storage a difference of about 15% was observed between the two storage conditions. 4) Phycobilin seems to be most stable among the examined pigments, and humid atmosphere has little effect on its stability.

Purification, Crystallization and Some Properties of Intracellular Pullulanase from Aerobacter aerogenes

Intracellular pullulanase was entirely extracted with sodium dodecylsulfate from the cells and was purified by means of ammonium sulfate fractionation and DEAE-cellulose and Sephadex chromatography. Crystalline pullulanase was precipitated with saturated ammonium sulfate solution. Intracellular pullulanase was purified over 150 fold in 17% yield to a final specific activity of 7000 per mg protein from the enzyme solution obtained by SDS-extraction. On ultracentrifugation analysis, the enzyme showed a symmetrical peak. The sedimentation coefficient, s20, w was 6.29 S. Polyacrylamide disc electrophoresis gave a main band and a sub-band, and both showed activity. Molecular weight of intracellular pullulanase was estimated to be (8±1) × 10,000 from gel filtration with Sephadex G-200 and to be (9±1) × 10,000 from sedimentation equilibrium. These values were higher than that (6~7 × 10,000) of extracellular pullulanase. Both enzymes differed slightly in thermal- and pH-stabilities.

Oxygen-dependent cleavage of the vinyl-ether linkage of plasmologens. 1. Cleavage by rat-brain supernatant.

The vinyl‐ether linkage of plasmalogen was cleaved by rat brain homogenates; practically all activity was localized in the soluble fraction of the homogenate. The cleavage of the vinyl‐ether linkage was tightly coupled to the reduction of molecular oxygen; about 3.5–4 mol oxygen were reduced for each mol vinyl‐ether cleaved. Both the reduction of oxygen and the cleavage of vinyl‐ether ceased if the reaction was run in an atmosphere of nitrogen or if antioxidants such as N,N‐diphenyl‐p‐phenylenediamine, butylated hydroxytoluene or Quercetin were used, in the presence of oxygen. Either reaction was also inhibited by the addition of the following chelators: EDTA, citrate, dipyridyl, desferal and o‐phenanthroline. A detergent such as sodium deoxycholate was required for optimal activity. The reaction (i.e., vinyl‐ether cleavage or oxygen uptake) was directly proportional to protein concentration and time and had an optimal pH at about 7.3. When reaction rates were plotted against substrate concentration, hyperbolic curves of the Michaelis‐Menten type were obtained. The active component of rat brain supernatant was dialyzable, was not inactivated when heated for 10 min at 100 °C and did not precipitate in a saturated ammonium sulfate solution. This suggests that cleavage of the vinyl‐ether linkage of plasmalogen is catalyzed by a thermostable, low‐molecular‐weight, non‐protein component of rat brain supernatant.

Action of Ammonium Sulfate Solution on Dry Milk Powders

Milk powders dispersed in concentrated solution of ammonium sulfate maintain their structural configurations in spite of loss of lactose and calcium. In 95% saturated ammonium sulfate solutions, protein is not removed but lactose and calcium are rapidly and completely removed. In 50% saturated ammonium sulfate, some whey proteins were also removed without affecting the particles physical appearance. Some fragmentation occurred in all foam spray-dried powders probably from coalescence of interior gas bubbles in these less dense particles. Agglomerates of instantized skimmilk powder were broken down to their fundamental particles by the ammonium sulfate solutions, appearing thereafter much the same as noninstantized skimmilk particles. These phenomena indicate that, in the terminal stages of drying, proteins may interact to form a continuous phase in spray-dried milk and that lactose may be the bridging material in the formation of powder particle aggregates during instantizing.

Purification, Crystallization and Some Properties o-Extracellular Pullulanase from Aerobacter aerogenes

Extracellular pullulanase was purified and crystallized from the culture fluid of Aerobacter aerogenes. Pullulanase was purified by means of ammonium sulfate fraction, DEAE-cellulose column chromatography and Sephadex column chromatography. Crystalline pullulanase was formed when saturated ammonium sulfate solution was added to the purified enzyme solution. The crystalline enzyme appeared as colorless fine rods. On ultracentrifugation analysis, the enzyme showed a single sharp and symmetrical Schlieren peak. The sedimentation coefficient, s20, w was 4.39S. Polyacrylamide gel electrophoresis at pH 8.4 gave a main band with two sub-bands and the molecular weight of the main enzyme was estimated to be 66, 000 from polyacrylamide gel electrophoresis and to be 58, 000 from sedimentation equilibrium. The optimum pH and temperature for the enzyme action were pH 6.5 and 50°C, respectively.

Preparation of two antigens of human liver isoenzymes of alkaline phosphatase

A convenient standard technique has been developed to prepare human liver isoenzyme (alkaline phosphatase) antigens based on an initial butanol treatment of a tris-homogenate of liver, followed by acetone precipitation, fractionation with ammoniumzsulfate and Sephadex G-200 gel filitration. The bulk of the enzyme was distributed in a bimodal manner, one moiety separating out in 0–30% saturated ammonium sulfate solution and the other, in 50–60% saturated ammonium sulfate. The latter, after Sephadex gel filtration is designated variant A and the former, variant B. Both variants have been characterized on starch gel electrophoresis and with respect to sensitivity to Mg 2+, l-phenylalanine and l-homoarginine. Variant A (mol. wt. 280000) corresponds to the fast liver band (6 cm) seen in sera rich in liver isoenzyme and variant B mainly with the isozyme band at or near the origin. These two variants represent two characterized liver antigens capable of producing the desired specific antisera.

Separation of free and bound steroid in the competitive protein binding assay for testosterone: Accuracy and reproducibility

Sephadex, dextran-charcoal and saturated ammonium sulfate solution were compared as agents for separating free and bound steroids in the plasma testosterone assay over the range of 0 to 200 ng per 100 ml. The accuracy and sensitivity of the three methods were essentially identical, but the reproducibility as well as the simplicity of the ammonium sulfate procedure were superior and commend it as the method of choice.

Selektive Anreicherung von Staphylokokken-Panton-Valentine-Leukozidin mit Aluminiumoxid-Cholesterin / Selective Enrichment of Staphylococcus “Panton-Valentine”-Leukocidine with Aluminium Oxide Cholesterol

A selective concentration of “Panton-Valentine”-leukozidine (PVL) from the culture-supernatant Staphylococcus aureus was achieved through adsorption of most of the a-hemolysin, coagulase, eggyolk-opacity factor and fibrinolysin to aluminiumoxide cholesterol. PVL was further concentrated through dialysis against a saturated ammonium sulfate solution and subsequent pressure-filtration. Through pressure-filtration relatively low-molecular staphylococcal substances, such as a part of a nuclease, could be separated from PVL.

Studies on Determination of Cadmium by Atomic Absorption Spectroscopy Using a APDC-MIBK Extraction System

A method for the atomic absorption spectroscopic determination of cadmium extracted with the APDC (ammonium pyrrolidinedithiocarbamate)-MIBA (methyl isobutyl ketone) is described. We examined various condition of the extraction of cadmium and of the atomic absorption analysis.Cadmium was efficiently extracted from the aqueous solution adjusted to the pH 3.5-10.3 range and to the salt concentration over 2.5M with saturated ammonium sulfate solution. Under these conditions, cadmium in 25ml of the aqueous solution was completely extracted with 10ml of MIBK by a single extraction. When the aqueous solution increased to 50ml, the extractability was still 97 per cent.The presence of a large amount of zinc, lead and mangane decreased the absorbance of cadmium owing to the poor solubililties of APDC-chelates in MIBK.

Change of orientation by thermal contraction of polyacrylonitrile fiber

AbstractPolyacrylonitrile polymer powder was dissolved in 70% nitric acid and spun into isotropic filament through a glass nozzle of 0.5 mm. diameter in a coagulating bath of 30% nitric acid. Stretching was carried out in two stages: the first stretching was done in water at 20°C. followed by drying, and the second stretching was done in a boiling saturated solution of ammonium sulfate. The total stretching ratio was 23. These filaments were shrunk freely in water at 70–180°C. The change in orientation factors was traced by x‐ray, infrared dichroism, visible dichroism, and sonic modulus methods. The relation between the reciprocal absolute temperature of thermal contraction and the logarithm of fiber length is a straight line which has two inflection points at 93 and 175°C. The orientation factors by x‐ray and infrared dichroism remain unchanged up to 175°C. On the contrary, the orientation factors by visible dichroism and sonic modulus drop suddenly at about 90°C. This indicates the occurrence of relaxation of the amorphous chain at the glass transition temperature and shows the polymer is not perfect single‐phase material. Orientation of crystalline and amorphous phases is stable from 100 to 170°C. in spite of considerable thermal contraction. The stability of orientation can be explained by the growth of a folded structure in the polymer.

Occurrence of Tanned Erythrocyte Agglutinating Factor in Various Mammalian Sera

WE have already described a haemagglutination reaction between erythrocytes treated with cold tannic acid and guinea-pig serum (diluted to about 1 : 100). We have also observed a marked increase in the tanned erythrocyte (TE) agglutinating activity in the serum of guinea-pigs at progressive stages of tuberculosis1–5. In a preliminary attempt to identify the TE agglutinating factor (TEAF), serum proteins of guinea-pig and several other mammals were separated by fractional precipitation with saturated ammonium sulphate solution. TEAF was found to be widespread in occurrence and to vary markedly in its distribution pattern in serum proteins between various species. There was also evidence for the presence of inhibitors.

Extraabdominal serum protein synthesis in the rhesus monkey

Summary Serum proteins of the Rhesus monkey were separated by preparative acrylamide gel-electrophoresis. After hepatectomy, the animals still incorporated significant amounts of radioactivity into a heterogeneous group of alpha and beta globulins. This was not altered by enterectomy or nephrectomy. After fractionation of the serum by precipitation with 50% saturated ammonium sulfate solution, radioactive alpha and beta globulins were found in both the precipitate and the supernate.

THERMAL SHRINKAGE OF POLYACRYLONITRILE FILAMENT AND CHANGE IN THE ORIENTATION DEGREE OF THE AMORPHOUS REGION

A sample of polyacrylonitrile of molecular weight 7.8×104 was dissolved in 70% nitric acid and spun into filament through a glass nozzle of 0.5mmφ, using 30% nitri_??_ acid as a coagulating bath. Three samples of filament were prepared by two-stage stretching: the first stretching was made in water at 60°C, followed by drying, and the second stretching was made in the boiling saturated solution of ammonium sulphate. The total degrees of stretching were 12.3, 18.5 and 23.1 folds. These samples were shrunk freely by heating in water at T=60_??_170°C; the degree of shrinkage S amounted to about 20% at maximum. The change in the orientation degree was traced dichromatically (fD) and by X-ray method (fx). The following results were obtained:(1) Plotting logS against 1/T, a linear relation is found, on which 4 transition points appear at about 94_??_96, 115, 130_??_140 and 160°C respectively.(2) fD-T is also linear with transition points on it; the transition temperatures correspond well with these on the logS-1/T line with a few exceptions.(3) fD drops from 0.80 to 0.15, while fx from 0.89 to 0.86 after the maximum shrinkage.Consequently it may be concluded that the thermal shrinkage occurs with disorientation which was predominant in the amorphous region, and it proceeds discontinuosly at the transition points.

On the Heterogeneity of Chicken Hemoglobin

The nuclear fraction of hemoglobin was obtained from chicken red blood cells by hemolytic procedure and the heterogeneity of this hemoglobin was studied by means of paper electrophoresis.Chicken hemoglobin was separated into two components; main and small components. There was no any significant difference between the migrating patterns of the nuclear and the cytoplasmic fractions of hemoglobin. But in densitometric analysis, there was the increase of main component in nuclear fraction as compared with cytoplasmic fraction. These results suggested that the ghost hemoglobin remained in nuclear fraction after hemolysis is composed of two components, the concentrations of which are different in proportion from those of dissolved cytoplasmic fraction.The supernatant of hemoglobin solution which was dialysed against the saturated ammonium sulphate solution contained main component only, whereas the pellet exhibited two components. It might be supposed that the solubility of chicken hemoglobin, especially the small component, is lowered by sulphate ion.The main and small components showed the absorption spectra characteristic of oxy-hemoglobin and marked trytophan notches.

Studies on a strain of raspberry ringspot virus occurring in England

SUMMARYA strain of raspberry ringspot virus (RRV‐E) infecting blackberry in Essex was transmitted by inoculation of sap to twenty‐six herbaceous species, and caused symptoms in Nicandra physaloides, Datura metel and Gomphrena globosa that differentiated it from the Scottish strain of the virus (RRV‐S). In sap, with an infection end‐point of 10‐5, it was inactivated in 10 min. at 74° C, and in 5 weeks at 23° C. It was precipitated without inactivation by a 30% acetone or 30%‐saturated ammonium sulphate solution, but was inactivated when acidified to less than pH 4. Partially purified preparations of RRV‐E contained approximately equal amounts of three components with sedimentation rates of 50, 90 and 127S; electron micrographs of preparations mounted in neutral sodium phosphotungstate showed three kinds of particles with diameters about 30 mμ but with different internal structure. A preparation of RRV‐S contained a main component with a sedimentation rate of 129s, and a minor one with 50S. Petunia hybrida, cucumber and French bean seemed equally susceptible to infection with RRV‐E when grown in soil containing the nematode vector Longidorus macrosoma, but inoculation of sap infected the roots of P. hybrida much more readily than roots of the other two species. Cucumber seedlings became infected when exposed for 1 day to infective L. macrosoma, and virus‐free L. macrosoma acquired the virus from plants in 4 days. Infective L. macrosoma transmitted readily at 20° C, occasionally at 25° C, and not at 30° C; feeding seems inhibited at 30° C. RRV‐E was transmitted to seedlings grown in soil containing L. macrosoma that had been kept free from plants for 34 days. Of sixty‐eight extracts of infective L. macrosoma inoculated to Chenopodium quinoa plants, only one caused infection.