Carbonate-bicarbonate Buffer Research Articles

A comparison of the transphosphorylating activities of human liver and intestinal alkaline phosphatases

The relative rates of transphosphorylation by human liver and intestinal alkaline phosphatases from p-nitrophenyl phosphate to Tris and diethanolamine have been measured. The maximum velocities of formation of diethanolamine phosphate by the two enzymes are nearly equal when preparations with equal hydrolytic activities in sodium carbonate—bicarbonate buffer are used. However, Tris phosphate is formed at a lower rate by liver alkaline phosphatase than by the intestinal enzyme. Consequently, the ratio of the activities of the solutions of liver and intestinal alkaline phosphatases varies with the nature and concentration of the buffer system.

Titration of L-cysteine hydrochloride with standard nickel sulphate solution employing 1-(pyridyl-2′-azo)-naphthol-(2) as indicator

The estimation of cysteine hydrochloride can be carried out quantitatively against standard nickel sulphate solution using 1-(pyridyl-2′-azo)-naphthol-(2) in carbonate-bicarbonate buffer medium of pH 10.0. Other natural amino acids have no interference with the titration.

New method for the isolation of membranes from Mycoplasma gallisepticum.

Mycoplasma gallisepticum lysed readily in carbonate bicarbonate buffer at pH 9.2 to 10.5. The hemagglutination titer of the lysates was 2- to 16-fold greater than a cell suspension at the same protein concentration in buffered saline. Membranes prepared from cells lysed by this method at pH 10 were relatively free from cytoplasmic contaminants as shown by electron microscopy of thin sections. The membranes retained their hemagglutination activity, gave reactions in immunodiffusion tests identical to those obtained by osmotic lysis and sonic treatment, and showed a similar pattern of protein bands by polyacrylamide disk electrophoresis. When inoculated into rabbits, the membranes gave rise to antibodies active in growth-, metabolic- and hemagglutination-inhibition tests. On the average, membranes obtained by lysis at pH 10 contained 44% of the original cell protein. The method is simple, giving high yields of membranes, and may be adaptable to other mycoplasmas.

The detection and localization of carbonic anhydrase in the rabbit cornea

A new, sensitive micromethod for the determination of carbonic anhydrase has been applied to the rabbit cornea. In this technique the concentration of carbonic anhydrase is related to the rate of exchange of 18O between labeled bicarbonate and water during the catalyzed dehydration of bicarbonate. Excised rabbit corneas and extracts prepared from the separated layers of rabbit corneas were placed in 18O-enriched carbonate-bicarbonate buffer solution at pH 9·4 and 25°C. The 18O content of the bicarbonate at periodic intervals was determined by mass spectrometry. Carbonic anhydrase was found to be present in the rabbit corneal endothelium at an estimated cellular concentration of 10 −6 m, the same order of magnitude as in most secretory tissues. No carbonic anhydrase was detected in the stroma or epithelium.

Virus in water. II. Evaluation of membrane cartridge filters for recovering low multiplicities of poliovirus from water.

The efficiency of a Millitube MF cartridge filter, a membrane filter, for recovery of poliovirus from 100-gal volumes of both fresh (tap) and estuarine water was determined. In the high multiplicity of virus input-output experiments, recovery of 97% or greater of input virus was achieved in both types of water when the final concentration of divalent cation as Mg(2+) was 1,200 mug/ml and the pH was 4.5. Virus was effectively eluted from the membrane cartridge with 5x nutrient broth in 0.05 M carbonate-bicarbonate buffer at pH 9.0. Four elutions of 250 ml each were used. In the low multiplicity of virus input-output experiments under the same cationic and pH conditions, up to 67% of the input virus was recovered when the virus was further concentrated from the eluates by the aqueous polymer two-phase separation technique. The volume reduction was 126,000-190,000 to 1. The use of the combined techniques, i.e., membrane adsorption followed by aqueous polymer two-phase separation, provided a highly sensitive, simple, and remarkably reliable sequential methodology for the quantitative recovery of poliovirus occurring at multiplicities as low as 1 to 2 plaque-forming units per 5 gal of water.

Chemical characterization of the proteins and glycoproteins of mouse liver plasma membranes solubilized by sequential extraction with aqueous and organic solvents.

1. A procedure for the stepwise fractionation of the proteins of mouse liver plasma membranes is described. 2. Of the membrane protein 20-25% was soluble in 50mm-sodium carbonate-bicarbonate buffer (pH9.7). This fraction contained a large number of proteins but only 1 major glycoprotein. It was low in sialic acid, amino sugars and phospholipid. 3. Extraction of the alkali-insoluble residue with aq. 33% pyridine solubilized an additional 30-35% of the membrane protein. The pyridine-soluble membrane components were enriched in sialic acid and glucosamine and it was shown that this procedure resulted in the selective extraction of glycoproteins. 4. Gel filtration in sodium dodecyl sulphate resolved the pyridine-soluble proteins into five fractions of decreasing molecular weight and an inverse relationship between molecular weight and sialic acid content was indicated.

Virus in Water. II. Evaluation of Membrane Cartridge Filters for Recovering Low Multiplicities of Poliovirus from Water

The efficiency of a Millitube MF cartridge filter, a membrane filter, for recovery of poliovirus from 100-gal volumes of both fresh (tap) and estuarine water was determined. In the high multiplicity of virus input-output experiments, recovery of 97% or greater of input virus was achieved in both types of water when the final concentration of divalent cation as Mg(2+) was 1,200 mug/ml and the pH was 4.5. Virus was effectively eluted from the membrane cartridge with 5x nutrient broth in 0.05 M carbonate-bicarbonate buffer at pH 9.0. Four elutions of 250 ml each were used. In the low multiplicity of virus input-output experiments under the same cationic and pH conditions, up to 67% of the input virus was recovered when the virus was further concentrated from the eluates by the aqueous polymer two-phase separation technique. The volume reduction was 126,000-190,000 to 1. The use of the combined techniques, i.e., membrane adsorption followed by aqueous polymer two-phase separation, provided a highly sensitive, simple, and remarkably reliable sequential methodology for the quantitative recovery of poliovirus occurring at multiplicities as low as 1 to 2 plaque-forming units per 5 gal of water.

The absorption of CO 2 into chemically reactive solutions at high temperatures

Simultaneous absorption and chemical reaction of carbon dioxide in potassium carbonate-bicarbonate buffer solutions was measured, at 80°C. Rates of absorption were catalyzed by addition of water soluble amines to the solution. Addition of methyl-amino ethanol or morpholine enhances the rate of adsorption by factors of 2 to 2·35 at concentrations of 2·5 per cent. The absorption rates measured were correlated with penetration and surface renewal theory. Reaction rate constants, equilibrium constants and mass transfer coefficients for a postulated reaction mechanism are presented for the cases of high temperature absorption with chemical reaction over reaction times of 3–4 hr. Activation energies are presented for the reaction of CO 2 with hydroxyl ion and with amine molecules in solution. Corroboration of the simultaneous reaction mechanism for amine enhancement is seen and extension of the use of surface-renewal theory to higher temperatures and long periods of absorption is confirmed. Mechanism of buffer catalysis by water soluble amines is discussed.

Coulometric determination of Ce(III) in alkaline solutions with electrogeneated octacyanomolybdate(V)

The coulometric determination of Ce(III) with octacyanomolybdate(V) in concentrated carbonate-bicarbonate buffer solutions was carried out. The current efficiency for electrogeneration of octacyanomolybdate(V) and the optimum conditions for analytical determinations of microquantities of cerium have been investigated.

Conjugates of chitinase with fluorescein isothiocyanate or lissamine rhodamine as specific stains for chitin in situ.

The fluorescent chitinase technique is based on the specific affinity of the enzyme for its substrate and applicable when an enzyme can be coupled with a fluorescent dye. Fluorescent chitinase specifically stained chitinous structures in several fungi and an insect, but failed to stain other polysaccharides in bacterial and algal cell walls. Freezing-microtome sections of Drosophila and fungal mycelia 6 μ thick were fixed in acetone for 5 min, then stained and mounted in fluorescent chitinase. Staining of smears of unsectioned fungal material required 5 min in absolute acetone, 5 min in 95% ethanol-1 N aqueous acetic acid (1:1), 10 min in 0.2 M phosphate buffer, PH 5.7, 1 sec in enzyme-dye conjugate, and 10 min in carbonate-bicarbonate buffer (0.2 M, pH 10.7, for chitinase-FITC; pH 7.6, for chitinase-LRBC). Preparations are viewed microscopically with ultraviolet light.

The absorption of CO 2 in carbonate—bicarbonate buffer solutions containing hypochlorite catalyst on a sieve plate

The rate of absorption of carbon dioxide into potassium carbonate—bicarbonate buffer solutions, containing sodium hypochlorite and potassium chloride, has been experimentally determined for a 30·5 × 30·5 cm square sieve plate. The values of the first-order reaction rate constant have been measured for the system studied by the laminar jet technique. An anlysis of these results using the Danckwerts pseudo-first order reaction method gives values of specific interfacial area and surface renewal rate comparable with earlier estimates for a similar system[1].

The molecular structure of human ceruloplasmin: Evidence for subunits

1. 1. Human ceruloplasmin (mol. wt. = 143100; s° 20, w = 7.11 S) can be dissociated into a variety of discrete subunits by either of two kinds of treatment which increase its negative charge density. 2. 2. Exposure of ceruloplasmin to carbonate-bicarbonate buffer (pH 10.2) results in dissociation into half-molecule subunits with mol. wt. = 82500 and s° 20, w = 5.48 S, while exposure to borate-NaCl buffer (pH 12.5) results in dissociation into quarter-molecules with mol. wt. 37700 and s° 20, w = 2.63 S. In both cases the dissociation is accompanied by loss of blue color and oxidase activity and by release of prosthetic copper. This loss of “native” properties appears to be irreversible, as there is no recovery of initial color or activity upon neutralization. 3. 3. When ceruloplasmin is reacted with succinic anhydride under neutral conditions so as to modify more than 90% of the free amino groups, it dissociates into half-molecules with mol. wt. = 87900 and s° 20, w = 4.06 S. The dissociation process also results in loss of blue color and oxidase activity and in release of prosthetic copper. 4. 4. Exposure of the succinylated ceruloplasmin half-molecules to borate-NaCl buffer (pH 12.5) results in further dissociation into eighth-molecules having apparently equivalent molecular weights (17200). No new free amino groups are detectable by assay with the ninhydrin reagent, indicating that the eighth-molecule subunitsdo not arise from peptide-bond cleavage. 5. 5. The calculated values of the frictional ratio, f f 0 , for ceruloplasmin half-, quarter-, and eighth-molecules (1.87, 1.64 and 2.06, respectively) are distinctly greate than the corresponding value for native ceruloplasmin (1.48), suggesting a significant increase in asymmetry upon dissociation into subunits. 6. 6. After acrylamide-gel electrophoresis at pH 12.0, the eighth-molecule subunits are resolved into two closely spaced bands, of equal protein-staining intensity. This finding, together with the ultracentrifugal evidence, suggests equal amounts of two polypeptide chains, identical in size but differing slightly in net charge. 7. 7. Double-diffusion experiments in agar gel reveal that either type ceruloplasmin half-molecule gives a reaction of partial identity with rabbit specific anti-ceruloplasmin serum. However, neither the quarter-molecule nor the eighth-molecule gives any reaction with the antiserum. 8. 8. The simplest model for the quaternary structure of human ceruloplasmin therefore is an octamer, α 4 β 4, which may undergo sequential dissociation to produce half-, quarter- or eighth-molecules.

Exchange of structurally bound phosphate in muscular activity

1. When rectus abdominis and sartorius muscles of the frog are incubated with (32)P-orthophosphate and subsequently minced, extracted with carbonate-bicarbonate buffer and water, and converted to acetone powders, considerable radioactivity remains associated with the solid material.2. Muscles labelled with (32)P and subjected to contracture at room temperature with KCl or ACh yield acetone powders which, in comparison with those of uncontracted control muscles, show a significantly decreased radioactivity. There is no evidence for a corresponding decrease in the total phosphate content.3. Relaxation of a contracted muscle restores the (32)P content of its acetone powder at least to the level for a resting, control muscle.4. When two recti are incubated with 5 x 10(-4)M-DNP after labelling with (32)P, subsequent depolarization of one muscle with KCl usually results in an increase in the activity of the bound phosphate fraction, with little or no contracture in addition to that already caused by the DNP.5. It is concluded that the bound phosphate fraction undergoes exchange during activation, shortening and relaxation of muscle, and may represent a source of energy for muscular activity.

酵素によるペクチン酸の分解(第4報)

(1) ペクチン酸は温アルカリ溶液では安定であるが,ペクチンは不安定でtranseliminationによってすみやかに分解する.しかし低温になると反応は遅れ,0°ではtranseliminationがほとんど起きないうちに,けん化が完了する.この際transelimination以外の原因による粘度の低下が認められた. (2) CPGによるペクチン酸の分解限度は二重結合が多いほど低い.しかし二重結合を含んでいない, PEでけん化したのちアルカリ処理を行なってつくったペクチン酸の場合でも,分解ははなはだ不完全であった. CPGは不飽和ペクチン酸には作用しない.これらの結果はCPGはペクチン酸の非還元性末端から作用し,分子の中の中性糖類ばかりでなく,基質調製中に生成する非還元性末端の4, 5-不飽和ガラクチュロン酸によっても作用が阻止されることを示している. (3) PEによるペクチンのけん化は不完全であると言われている.しかし温州みかんの果皮からつくったPEをペクチンに作用させ,けん化の度合をクロモトロップ酸法の改良法で調べると, PEでもアルカリ同様ペクチンの完全なけん化が起きることがわかった.

CLOTTING OF CITRATED PLASMA BY A PROTEOLYTIC ENZYME ASSOCIATED WITH AN ANTIBACTERIAL AGENT FROM SERUM.

Two to 4 mg/ml of an antibacterial agent occurring in the serum of humans and animals caused the clotting of citrated rabbit, human, calf, ox, sheep, goat, guinea pig, rat, mouse, horse, chicken, pigeon and swine plasma. Heparinized plasmas of the same species were found resistant to the clotting action of the antibacterial agent. Citrated plasma previously heated at 56 C for 30 min, treated with 640 units/ml tyrosinase for 60 min, or absorbed with 0.2M Ca3(PO4)2 and 0.2M Mg(OH)2 was also found resistant to the clotting action of the antibacterial agent. The clotting action of the antibacterial agent was not affected by heating at 66 C for 60 min, nor by multiple passage through Seitz filters. The coagulation of citrated plasma proceeded most rapidly with 0.2M Tris(hydroxymethyl)-amino-methane buffer, pH 7 to 9, or distilled water as the antibacterial agent solvent; 0.2M phosphate buffer, pH 6 to 8, reduced the clotting action of the antibacterial agent, while 0.2M citrate-phosphate buffer, pH 4.0, or 0.2M carbonate-bicarbonate buffer, pH 10.0, inhibited entirely the clotting activity of this agent.

Gas absorption with chemical reaction on a sieve tray

The rate of adsorption of carbon dioxide into four different sodium carbonate—bicarbonate buffer solutions and a sodium hydroxide solution has been experimentally determined for a six-inch diameter sieve tray. An analysis of these results using the penetration theory yields unexpectedly high values of interfacial area and contact time. Similar results had previously been obtained for packed columns [7]. It is proposed that this discrepancy is owing to the fact that the entire froth is not well mixed. This leads to some of the interfacial liquid becoming saturated with gas and thus ineffective for absorption. An analysis has shown that the fraction of ineffective area may be as much as 40 per cent of the total.

PHOTOSYNTHESIS IN CELL DEVELOPMENT.

Abstract Non-synchronized suspensions of the green, high-temperature alga Chlorella 7-11-05 were subjected to fractional centrifugation, and two size groups of cells were separated. The small-cell fraction was presumed to consist largely of younger cells and the large-cell fraction predominantly of older cells. Manometric measurements in phosphate buffer at pH 4.5, in bicarbonate buffer at neutral pH, and in carbonate-bicarbonate buffer at pH 9.3 indicated that younger cells invariably possessed higher photosynthetic activity than older cells, provided the separation of cells into size fractions was reasonably good and the large cells were prevented from dividing during the process of separation. The superior activity of younger cells was ascertained at various light intensities and at different temperatures. Previous observations on the decline in photosynthetic activity with the age of synchronized cells were thus substantiated in the absence of a synchronizing agent, and the decline in photosynthetic capacity must be assumed to be characteristic of normal cell development. The decline in metabolic activity in the course of cell development is discussed in connection with metabolic turnover and is viewed as a demonstration of aging of cells.

Determination of beryllium by means of hexamminecobalt(III) carbonatoberyllate—II : High-precision indirect determination of beryllium by titration of cobalt with edta

A highly precise method of determining beryllium, based on the precipitation of hexamminecobalt(III) hexacarbonato-oxo tetraberyllate, is described. The complex is decomposed by fuming with sulphuric acid and the cobalt(II) produced determined by weight titration with standard 0.1 M EDTA and back-titration with 0.01 M cobalt(II). The titration is carried out at pH 8.5–9.0 in a potassium carbonate-bicarbonate buffer and in the presence of potassium thiocyanate, tetraphenylarsonium chloride and chloroform. The end-point is indicated by the appearance in the chloroform layer of the blue colour of the ion-association pair, tetraphenylarsonium tetrathiocyanatocobaltate(II). A precision of 0.02% has been obtained, which is much better than that possible with any other method for determining beryllium. There appears to be a negative bias not exceeding 0.1% which cannot yet be explained.

Hydrogen sulphide chemical absorption

The chemistry of the processes of H 2S absorption in aqueous hydroxide solutions and in aqueous carbonate-bicarbonate buffer solutions is studied, and theoretical equations for the rate of absorption are derived. Experimental results are in good agreement with theory.

Dialysis Phosphatase Method for Milk and All Dairy Products

Summary The principles, directions, and comparative results of a new phosphatase method for milk and milk products are presented. Dialysis through cellulose membranes, leading to a rapid separation of phenol accumulating in the cellulose bag from phosphatase activity, is the basis of the method. The separation occurs without attendant problems of protein interference or turbidity when color is developed with 2,6-dichloroquinone-chloroimide (CQC) in the receiving aqueous solution. Specifically, 5ml milk or 1g cheese are added to 10ml concentrated carbonate-bicarbonate buffer substrate residing in a small, single-service, knotted cellulose bag. The bag is incubated for 1 hr at 37C in 10ml of diluted copper-sulfate solution, after which it is removed and discarded. Two drops CQC are added to the clear liquid remaining and color permitted to develop for 5min. Phenol concentration is determined at this point, to complete the analysis. The Dialysis Phosphatase Test requires few reagents and its operation is basically simple. Comparative results between four laboratories, on standard fluid milk and Cheddar cheese samples, show good reproduction and indicate detection of less than 0.1% raw milk addition or a reduction of 1-2F in high-temperature—short-time pasteurization is readily possible.