Buffer Ratio Research Articles

Elimination and addition reactions. Part 33. Formation and behaviour of carbanions derived from sulphones and nitriles bearing β-′onium substituents

Kinetics of elimination reactions of β-arylsulphonylethyl- and β-cyanoethyl-ammonium and -sulphonium salts have been measured in ethanolic triethylamine buffers. The reactions show buffer saturation kinetics; at low buffer base concentrations ionisation to form the intermediate carbanion is rate-determining, but at higher buffer base concentrations the intermediate carbanion is formed in a rapidly established pre-equilibrium step and the observed rate constant does not change with increasing base concentration at constant buffer ratio.

Calcium as a limiting factor in the biology of Biomphalaria pfeifferi (Krauss), (Gastropoda: Planorbidae)

The minimum calcium requirements, relative importance of buffering and optimum ratio of calcium to magnesium, calcium to sodium, and calcium to potassium ions were determined for laboratory populations ofBiomphalaria pfeifferi and related to suggested limiting factors for the natural distribution of this species. Snails were reared in a range of concentrations of both calcium bicarbonate and unbuffered calcium sulphate from 0.5 to 20 mg/l as Ca++ and also in a series of media with a constant concentration of 2 mg/l as Ca++ but with a range of Ca/Mg, Ca/Na and Ca/K ratios of 4.0 to 0.1. Shell growth, survivorship, fecundity, egg fertility, and the net reproductive rate were compared. In calcium bicarbonate cultures a concentration of 2mg/l Ca++ appeared to be the lower limit for the survival of laboratory populations but a concentration of 4 mg/l Ca++ was needed for a population to thrive. The calcium sulphate salt gave much poorer results, emphasizing the importance of the bicarbonate buffer. In the cationic ratio experiments the low Ca/Mg ratios proved to have the most damaging effects on snail populations but the effects of very low Ca/Na and Ca/K ratios could also be measured. A parallel experiment on the hatching rate of snail eggs, using similar experimental solutions, gave comparable results. The significance of these findings to snail ecology is discussed.

Ionized calcium concentrations in squid axons.

Values for ionized [Ca] in squid axons were obtained by measuring the light emission from a 0.1-mul drop of aequorin confined to a plastic dialysis tube of 140-mum diameter located axially. Ionized Ca had a mean value of 20 x 10(-9) M as judged by the subsequent introduction of CaEGTA/EGTA buffer (ratio ca. 0.1) into the axoplasm, and light measurement on a second aequorin drop. Ionized Ca in axoplasma was also measured by introducing arsenazo dye into an axon by injection and measuring the Ca complex of such a dye by multichannel spectrophotometry. Values so obtained were ca. 50 x 10(-9) M as calibrated against CaEGTA/EGTA buffer mixtures. Wth a freshly isolated axon in 10 mM Ca seawater, the aequorin glow invariably increased with time; a seawater [Ca] of 2-3 mM allowed a steady state with respect to [Ca]. Replacement of Na+ in seawater with choline led to a large increase in light emission from aequorin. Li seawater partially reversed this change and the reintroduction of Na+ brought light levels back to their initial value. Stimulation at 60/s for 2-5 min produced an increase in aequorin glow about 0.1% of that represented by the known Ca influx, suggesting operationally the presence of substantial Ca buffering. Treatment of an axon with CN produced a very large increase in aequorin glow and in Ca arsenazo formation only if the external seawater contained Ca.

Hemoglobin function and 2,3-DPG levels of blood stored at 4 C in ACD and CPD: pH effect.

Normal hemoglobin function depends on adequate erythrocyte levels of 2,3‐diphosphoglycerate (2,3‐DPG), a compound which is poorly maintained in acid‐citrate‐dextrose (ACD). Since 2,3‐DPG is better maintained in citrate‐phosphate‐dextrose (CPD) and this preservative has a higher pH (5.5) than ACD (pH = 5.0), these preservatives were prepared at each pH and studied. The CPD preservatives (pH 5.0, 5.5) had similar amounts of phosphate so the differences between them, obtained by altering the buffer ratio, should relate to pH. The ACD solutions (pH 5.0, 5.5) contained no phosphate. Hemoglobin function, expressed as P50 (the Po2 at 50 per cent oxygenation, an inverse but direct measure of oxygen affinity), and 2,3‐DPG were better maintained in ACD and CPD of pH 5.5. The lower pH (5.0) preservatives, whether ACD or CPD, showed rapidly declining hemoglobin function and 2,3‐DPG levels. The values at the higher pH remained close to normal for two weeks and above those of the lower pH preservatives for most of the four‐week storage period.

TECHNICAL CONSIDERATIONS IN THE PREPARATION OF FLUORESCENT-ANTIBODY CONJUGATES.

A comparison was made of (NH(4))(2)SO(4), HCl, ethodin, and ethanol for fractionation of rabbit antiserum prior to conjugation with fluorescein isothiocyanate. Fractionation with the salt was found to be the method of choice from the standpoints of simplicity and recovery of antibody effective in conjugates prepared from the fractions. Effects of pH, temperature, dye-protein ratio, and molarity and type of buffer upon conjugation were studied. These technical factors were adjusted to produce conjugates for Corynebacterium diphtheriae which possessed higher specific titers than did reagents obtained by previously employed techniques.

Technical Considerations in the Preparation of Fluorescent-Antibody Conjugates

A comparison was made of (NH(4))(2)SO(4), HCl, ethodin, and ethanol for fractionation of rabbit antiserum prior to conjugation with fluorescein isothiocyanate. Fractionation with the salt was found to be the method of choice from the standpoints of simplicity and recovery of antibody effective in conjugates prepared from the fractions. Effects of pH, temperature, dye-protein ratio, and molarity and type of buffer upon conjugation were studied. These technical factors were adjusted to produce conjugates for Corynebacterium diphtheriae which possessed higher specific titers than did reagents obtained by previously employed techniques.

Micromethod for the methyl red test.

SUMMARY: A micromethod for the methyl red (mr) test was worked out for suspensions of bacterial cells. The most important factors in the test are (1) the density of the cell suspension; (2) the ratio of glucose to buffer concentrations. Adaptation of the cells to glucose is essential for the success of the test, and cells from glucose-free media are not satisfactory. At the optimal glucose: buffer ratio Bacterium coli and Klebsiella aerogenes suspensions have different effects on the final pH value attained in the system. Because the mr microtest is dependent on a fine balance between glucose and buffer concentrations, and is so greatly affected by even twofold differences in cell concentration, it is not really suitable for routine work in the characterization of bacteria.

An Improved Histochemical Technic for Acid Phosphatase

Causes of failure in the histochemical technic for acid phosphatase were studied. It was found that the ratio of buffer to substrate in the incubating mixture is an important factor in the constancy of results. In the case of glycerophosphate as a substrate, the optimal ratio is 5 to 6; with other substrates it may be different. The mechanism of the effect of this ratio on the results is explained.

Inactivation of Crystalline Pepsin

CRYSTALLINE pepsin, in solutions of constant ionic strength on the alkaline side of its stability maximum, inactivates unimolecularly at a rate which is inversely proportional to the fifth power of the hydrogen ion concentration. This unusual relationship has been demonstrated over a velocity interval of 1 to 5,000 in nearly a hundred kinetic experiments at two temperatures with four different buffers. The rate varies with the buffer, but the fifth-power relation does not. As the buffer ratios change by different amounts in the same pH range, general basic catalysis is therefore excluded. It is likewise improbable that simple hydroxyl ion catalysis determines the rate.

Effect of certain electrolytes on the buffering power of bacterium coli.

In the present studies we have developed a technique for the measurement of the power of the bacterial cell to combine with hydrogen and hydroxyl ions. The bacteria show a general tendency to simulate amphoteric colloids and we have therefore measured their amphoteric property by the use of titration curves. A bacterial suspension in water or in a particular salt solution is titrated electrometrically with hydrochloric acid and with sodium hydroxide and similar titration curves are obtained for the menstruum alone. From these two sets of curves it is possible to calculate a series of “buffer ratios” by taking the quotients of the amounts of acid or alkali added to shift the PH one unit in the bacterial suspension and in the menstruum alone. Such buffer ratios have been calculated for each PH unit zone between PH 2 and PH 12. The titration cells which have been developed for these experiments are illustrated in the accompany figure. The buffer ratios for Bact. coli in distilled water, 0.725, M NaCl, 0.145 M CaCl2 and in 0.580 M NaCl + 0.145 M CaCl2 solutions are presented in Table I. 1. In distilled water suspensions of Bacterium coli show a marked tendency to resist a change in hydrogen-ion concentration in the zones of PH which are of physiological interest, i. e., PH 4-10. In more acid and more alkaline solution this buffering power is weaker and the buffer ratios approximate unity at PH 3-4 and at PH 10-11.