Salt precipitation during the freeze-concentration of phosphate buffer solutions

Salt precipitation during the freeze concentration of phosphate solutions was investigated by differential scanning calorimetry (DSC), in view of its practical importance in the cryopreservation or freeze-drying of biological materials. It was found that the fraction of salt precipitated depends on the initial salt concentration; it began to decrease with decreasing concentration at approx. 1 M. Salt precipitation also depends on the cooling rate. In some cases, cooling at approx. 10(3) degree min-1 inhibited salt precipitation which had been observed during slow cooling (0.62 degree min-1), without, however, affecting the shape of the ice melting endotherm. In the case of ternary phosphate buffers, the fraction of salt precipitating depends on the salt composition as well as the initial concentration and cooling rate. Near the composition of the ternary eutectic or the composition where two salts are present at the same concentration, salts were prevented from precipitation.     

Fractionation of spectrin by differential precipitation with calcium

The calcium-stimulated ATPase activity of spectrin can be substantially separated from the magnesium-stimulated activity by partial precipitation of spectrin with calcium. The precipitated Ca-ATPase fraction can be dissolved by dialysis against EDTA, and the resulting solution can be polymerized into characteristic spectrin fibrils. The supernatant contains Mg-ATPase and, either before or after dialysis, contains predominantly “torus” forms. The two fractions are essentially identical on gel electrophoresis, being composed primarily of the high molecular weight peptides with a constant level of minor bands. These results imply that the Ca-ATPase is associated firmly with the high molecular weight peptides.     

Substrate utilization by Legionella cells after cryopreservation in phosphate buffer.

The objective of this study was to evaluate by relatively simple metabolic tests the usefulness of buffers and energy sources commonly used in Legionella growth media. Legionella pneumophila serogroups 1 to 6, Legionella micdadei, and Legionella bozemanii were grown in an enriched charcoal-yeast extract diphasic medium. The cells were washed thrice, suspended in various buffers (pH 6.9) with 1 or 5 mM MgSO4, and used immediately or after controlled-rate cryopreservation. CO2 produced and C incorporated into the cold trichloracetic acid-insoluble fractions from 14C-labeled substrates were determine. Potassium phosphate buffer (0.02 M) was as satisfactory as organic buffers for glutamate metabolism, but the addition of KCl or NaCl reduced activity. Metabolic activity for glutamate was not lost upon cryopreservation, and cryopreserved cells were used to test the utilization of other single or paired substrates. Rates of activity for serine, glutamate, threonine, and pyruvate, in this descending order, were high, and those for alpha-ketoglutarate, succinate, and gamma-aminobutyrate were low. Although glutamine was not used as rapidly as glutamate, when added to glutamate it was preferentially metabolized, possibly because of more rapid transport. When glutamate and serine were combined, glutamate furnished more C for CO2 and less for incorporation, whereas the reverse was true of serine. In conclusion, glutamate as an energy source may in some cases spare other amino acids for synthesis. alpha-Ketoglutarate, a common constituent of Legionella media, may reduce oxygen toxicity but is probably not a chief energy source.     

Stimulation of precipitation of calcium phosphate by matrix vesicles.

The ability of matrix vesicles isolated from the epiphysial growth plate of 6-week-old chicks to facilitate the precipitation of calcium phosphate was studied in vitro. The vesicles lowered the minimum concentration product [ca2+]X[p1] needed to induce crystal formation, thereby showing the vesicles are nucleators of crystallization. After freezing and thawing the vesicles at pH6.0, part but not all of this ability to nucleate disappeared. Freezing and thawing markedly decreased the Ca and Pi content of the vesicles, suggesting that part of the nucleating activity may have been due to mineral already present. After removal of the mineral the residual nucleating activity could be destroyed by extracting the vesicles with lipid solvents or by treatment with enzymes such as phosphoilipase C, neuraminidase or proteinase. Matrix vesicles obtained from chicks treated with 1-hydroxyethane-1, 1-diphosphonate, a compound that inhibits calcification in vivo, showed impaired nucleating activity, both before and after treatment at pH6.0. The vesicle preparation bound some diphosphonate in vitro, probably to the mineral present in the preparation, since no binding could be detected in vesicles preincubated at pH6.0. No difference was found in the nucleating activity of vesicles isolated from rachitic chicks which had or had not received cholacalciferol 48 h before death. These results suggest that matrix vesicles possess intrinsic nucleating activity that may be important in biological calcification.     

Fractionation of Chromatin Depleted of Histone H1

Chromatin depleted of histone HI was prepared after extraction with 0.6 M NaCl. The salt-extracted chromatin was mechanically fragmented, then fractionated on sucrose gradients in two fractions, the fast migrating (heavy) and the slow migrating (light) fractions. The average sizes of the ONA-moieties from heavy and light fractions were found to be similar. However, heavy fractions were enriched in high temperature melting populations, whereas, light fractions exclusively included low temperature melting populations, not seen in the heavy fractions. Comparison of the derivative plots of thermally denatured whole and salt-extracted chromatin as well as heavy and light fractions of salt-extracted chromatin suggests that the DNA regions associated with histone HI in intact chromatin may be co-isolated with the light fraction.     

Substrate utilization by Legionella cells after cryopreservation in phosphate buffer

The objective of this study was to evaluate by relatively simple metabolic tests the usefulness of buffers and energy sources commonly used in Legionella growth media. Legionella pneumophila serogroups 1 to 6, Legionella micdadei, and Legionella bozemanii were grown in an enriched charcoal-yeast extract diphasic medium. The cells were washed thrice, suspended in various buffers (pH 6.9) with 1 or 5 mM MgSO4, and used immediately or after controlled-rate cryopreservation. CO2 produced and C incorporated into the cold trichloracetic acid-insoluble fractions from 14C-labeled substrates were determine. Potassium phosphate buffer (0.02 M) was as satisfactory as organic buffers for glutamate metabolism, but the addition of KCl or NaCl reduced activity. Metabolic activity for glutamate was not lost upon cryopreservation, and cryopreserved cells were used to test the utilization of other single or paired substrates. Rates of activity for serine, glutamate, threonine, and pyruvate, in this descending order, were high, and those for alpha-ketoglutarate, succinate, and gamma-aminobutyrate were low. Although glutamine was not used as rapidly as glutamate, when added to glutamate it was preferentially metabolized, possibly because of more rapid transport. When glutamate and serine were combined, glutamate furnished more C for CO2 and less for incorporation, whereas the reverse was true of serine. In conclusion, glutamate as an energy source may in some cases spare other amino acids for synthesis. alpha-Ketoglutarate, a common constituent of Legionella media, may reduce oxygen toxicity but is probably not a chief energy source.     

Formation of directly mutagenic alpha-hydroxy-N-nitrosopiperidine phosphate ester by near-ultraviolet irradiation of N-nitrosopiperidine in phosphate buffer

Previously we found that direct-acting mutagens can be formed from N-nitrosodialkylamines on exposure to near-ultraviolet light in the presence of phosphates. We have now isolated the mutagenic photoproduct formed from N-nitrosopiperidine and inorganic phosphate and identified its structure as the phosphate ester of alpha-hydroxy-N-nitrosopiperidine. This reaction represents a new, non-enzymatic activation of promutagenic N-nitrosodialkylamines.     

Oxidation of low-density lipoprotein by copper and iron in phosphate buffer

We examined the effect of phosphate buffer on the iron- and copper-catalyzed peroxidation of low-density lipoprotein (LDL). The incubation of LDL with CuSO4 in 0.15 M NaCl led to the peroxidation of LDL as evidenced by the detection of thiobarbituric acid-reactive substances (TBARS) and lipid hydroperoxides (LPO). The peroxidation of LDL was also observed with FeSO4 and FeCl3 in 0.15 M NaCl, although there was a lag phase with FeCl3. In 10 mM phosphate buffer, the peroxidation of LDL was observed with CuSO4 to an extent similar to that in 0.15 M NaCl. However, the peroxidation induced by incubation with FeSO4 and FeCl3 was significantly inhibited in phosphate buffer. Iron and copper each formed a complex with lipoprotein during incubation with LDL in 0.15 M NaCl. Although no effect on the formation of copper-LDL complex was observed in phosphate buffer, the formation of iron-LDL complex was reduced in the buffer. These observations suggest there are marked differences in the peroxidation of LDL and in the formation of complexes with LDL between iron and copper in phosphate buffer.     

Antagonism by phosphate buffer of the twitch loss in isolated muscle fibers produced by calcium-free solutions.

In 37 of 41 isolated frog skeletal muscle fiber preparations (one, two, or three fibers) the twitch was eliminated or reduced to less than 10% of control by exposing the fibers to a O-calcium, bicarbonate-buffered solution for 10 min or less. Replacing the bicarbonate by a phosphate buffer either prevented twitch inhibition or increased the O-calcium exposure time required for its production. It is concluded that surface membrane-bound calcium ions (presumably in the t-tubules) are required to couple the action potential to the mechanical response and that phosphate ions inhibit the loss of the membrane-bound calcium ions into an external calcium-free solution.     

Proteoglycan inhibition of calcium phosphate precipitation in liposomal suspensions.

The major proteoglycan in cartilage (aggrecan) is a complex macromolecule with numerous chondroitin sulphate, keratan sulphate, and oligosaccharide substituents. It has been proposed that this macromolecule has an important role in regulating mineralization in this tissue, a process which is initiated by the deposition of apatite in matrix vesicles. We have used a liposome-centred endogenous precipitation method as a model for matrix vesicle mineralization to study the effect of the rat chondrosarcoma aggrecan and its chondroitin sulphate and core protein components on apatite formation from solution. Precipitation was initiated by encapsulating buffered (pH 7.4) 50 mmol/l KH2PO4 solutions in the aqueous centres of 7:2:1 phosphatidylcholine:dicetylphosphate:cholesterol liposomes, adding 2.25-2.65 mmol/l Ca2+ and 1.5 mmol/l total inorganic phosphate (PO4) to the suspending medium (pH 7.4, 22 degrees C), then making the intervening lipid membranes permeable to the Ca2+ ions with the calcium ionophore X-537A. Aggrecan (0.5%) in the suspending medium had no effect on intraliposomal precipitation, but severely reduced (approximately 70% reduction at 24 h) its subsequent spread into the medium. The chondroitin sulphate and core protein were similarly inhibitory. The degree to which aggrecan and its constituent parts inhibited precipitation correlated with their capacity to bind Ca2+ ions. These findings suggest that functional groups in aggrecan blocked apatite growth by linking via Ca2+ bridges to growth sites on the crystal surfaces. Similar Ca-mediated interactions may well have a critical regulatory role in cartilage mineralization.     

The solubility of sickle and non-sickle hemoglobins in concentrated phosphate buffer.

A new turbidimetric method for the direct measurement of the solubility of oxy- and deoxyhemoglobins (Hb) in concentrated phosphate buffer has been established. The principle of the method is the formation of a homogeneous emulsion when hemoglobin is introduced in concentrated phosphate buffer. The solubility of the oxy and deoxy forms of Hb A, Hb S, Hb C, Hb F, and Hb CHarlem (beta 6Glu leads to Val, beta 73Asp leads to Asn) has been studied. The solubility of deoxy-Hb S was the lowest and the solubility curve was broader than those of the other hemoglobins indicating that the aggregates of deoxy Hb S require more water to be dissolved. The solubility of oxy- and deoxyhemoglobins depends on temperature and pH. The solubility of hemoglobins is increased as the temperature is lowered and the pH is raised. The pH dependency of the solubility of deoxy-Hb S in high phosphate buffer was opposite to that of the minimum gelling concentration of deoxy-Hb S. The order of the solubility of Hb CHarlem, Hb FS, Hb AS, Hb CS, and Hb S in concentrated phosphate buffer corresponds to the order of minimum gelling concentration of these hemoglobins or hemoglobin mixtures. Solubility studies of a 1:1 mixture of deoxy-Hb A and deoxy-Hb S show that deoxy-Hb A aggregates in 2.42 M phosphate buffer in which pure deoxy-Hb A is totally soluble. This result indicates that deoxy-Hb S interacts with deoxy-Hb A and decreases its solubility.     

Ozone inactivation of Cryptosporidium parvum in demand-free phosphate buffer determined by in vitro excystation and animal infectivity.

Inactivation of Cryptosporidium parvum oocysts by ozone was performed in ozone demand-free 0.05 M phosphate buffer (pH 6.9) in bench-scale batch reactors at 7 and 22 degrees C. Ozone was added to each trial from a concentrated stock solution for contact times ranging from 5 to 15 min. The viability of the control and treated oocysts was determined by using in vitro excystation and infection in neonatal CD-1 mice. It was found that excystation consistently underestimated inactivation when compared with animal infectivity (P < or = 0.05). As inactivations increased, the difference between excystation and infectivity also increased. The inactivation kinetics of C. parvum by ozone deviated from the simple first-order Chick-Watson model and was better described by a nonlinear Hom model. The use of the Hom model for predicting inactivation resulted in a family of unique concentration and time values for each inactivation level rather than the simple CT product of the Chick-Watson model.     

Hypochlorite inactivation kinetics of Listeria monocytogenes in phosphate buffer

The inactivation kinetics of Listeria monocytogenes in a phosphate buffer (PB) was determined at different hypochlorite concentrations, pH values and temperatures. D-values, using a linear regression, of L. monocytogenes in PB (pH 6.5) were 23.54, 17.40, 14.24 and 12.00s at 5, 10, 50 and 100 mg l(-1) hypochlorite, respectively, at 30 degrees C. The k-values ranged from 0.098 to 0.192s(-1) and 0.007 to 0.018s(-1) for hypochlorite concentrations (from 5 to 100 mg l(-1)) in PB (pH 6.5) and PB containing 0.1% peptone (pH 6.5), respectively, at 30 degrees C. D-values of L. monocytogenes exposed to hypochlorite were decreased with decreasing pH of PB (pH from 8.5 to 4.5). Hypochlorite showed higher antimicrobial activity at higher temperature. Not only the effect of hypochlorite concentration on the inactivation of L. monocytogenes but also other parameters like temperature, pH and suspending solutions effect the inactivation rates.     

Molecular properties of R-phycocyanin subunits from Polysiphonia urceolata in potassium phosphate buffer.

The two subunits of R-phycocyanin from Polysiphonia urceolata were isolated and renatured. The renatured subunits were characterized by electrophoresis, molecular weights and spectra. The blue-shifted spectra, fluorescence recovery and restoring of the energy transfer suggested correct refolding of the subunits. The molecular properties of the subunits in potassium phosphate buffer (KPB) were investigated in detail. The total fluorescence yields (Q(T)) of the beta subunit declined while the energy transfer efficiency (E(T)) in the beta subunit was promoted with the increase of KPB concentration. On the other hand, both Q(T) and E(T) were enhanced with the increasing of the subunit concentrations. Based on the structural information, the fluorescence quenching in high concentrations of KPB was ascribed to less rigid chromophores caused by the weakening of the hydrogen-bond interaction network, while the enhancement of the fluorescence and E(T) was due to the aggregation of the subunits in the ionic solvent. Aggregation was confirmed by cysteine-assisted promotion of renaturation yield and stability, as well as equilibrium unfolding tests. Optimal conditions were proposed for the refolding/unfolding studies, under which the subunits were mainly monomeric. Compared to that in C-PC, the blue-shifted spectrum of PCB in R-PC is suggested to bring larger energy transfer efficiency, probably due to the necessity of the light harvesting for P. urceolata living in deep water.     

In-gel precipitation of enzymatically released phosphate

The phosphate precipitation reaction using ammonium molybdate and triethylamine under low pH has been applied to gel-based assays for detecting phosphate-releasing enzymes. The sensitivity of the assay is 10 pmol Pi/mm2 of 1.5-mm-thick gel. The assay is applicable to enzymes with a wide range of optimal pH, from acid (pH 4.5) to alkaline phosphatase (pH 9.7), and to enzymes that use acid-labile substrates such as apyrase and glutamine synthetase. Using a negative staining approach, maltose phosphorylase, a phosphate-consuming enzyme, can also be detected. The assay was used to detect glutamine synthetase isoforms, separated by nondenaturing polyacrylamide gel electrophoresis from crude maize extracts. For downstream applications such as staining gels for proteins, the gels with precipitate should be incubated in 10 mM dithiothreitol or beta-mercaptoethanol until the precipitate is dissolved and then thoroughly washed in water. In comparison to calcium phosphate precipitation or the phosphomolybdate-malachite green method, this method is more sensitive. It is a very simple, rapid, versatile, reproducible, and inexpensive method that could be a useful tool in enzymological studies.     

Comparative inactivation of poliovirus type 3 and MS2 coliphage in demand-free phosphate buffer by using ozone.

MS2 coliphage (ATCC 15597-B1) has been proposed by the U.S. Environmental Protection Agency as a surrogate for enteric viruses to determine the engineering requirements of chemical disinfection systems on the basis of previous experience with chlorine. The objective of this study was to determine whether MS2 coliphage was a suitable indicator for the inactivation of enteric viruses when ozone disinfection systems were used. Bench-scale experiments were conducted in 2-liter-batch shrinking reactors containing ozone demand-free 0.05 M phosphate buffer (pH 6.9) at 22 degrees C. Ozone was added as a side stream from a concentrated stock solution. It was found that an ozone residual of less than 40 micrograms/liter at the end of 20 s inactivated greater than 99.99% of MS2 coliphage in the demand-free buffer. When MS2 was compared directly with poliovirus type 3 in paired experiments, 1.6 log units more inactivation was observed with MS2 coliphage than with poliovirus type 3. It was concluded that the use of MS2 coliphage as a surrogate organism for studies of enteric virus with ozone disinfection systems overestimated the inactivation of enteric viruses. It is recommended that the regulatory agencies evaluate their recommendations for using MS2 coliphage as an indicator of enteric viruses.     

Comparative inactivation of poliovirus type 3 and MS2 coliphage in demand-free phosphate buffer by using ozone.

MS2 coliphage (ATCC 15597-B1) has been proposed by the U.S. Environmental Protection Agency as a surrogate for enteric viruses to determine the engineering requirements of chemical disinfection systems on the basis of previous experience with chlorine. The objective of this study was to determine whether MS2 coliphage was a suitable indicator for the inactivation of enteric viruses when ozone disinfection systems were used. Bench-scale experiments were conducted in 2-liter-batch shrinking reactors containing ozone demand-free 0.05 M phosphate buffer (pH 6.9) at 22 degrees C. Ozone was added as a side stream from a concentrated stock solution. It was found that an ozone residual of less than 40 micrograms/liter at the end of 20 s inactivated greater than 99.99% of MS2 coliphage in the demand-free buffer. When MS2 was compared directly with poliovirus type 3 in paired experiments, 1.6 log units more inactivation was observed with MS2 coliphage than with poliovirus type 3. It was concluded that the use of MS2 coliphage as a surrogate organism for studies of enteric virus with ozone disinfection systems overestimated the inactivation of enteric viruses. It is recommended that the regulatory agencies evaluate their recommendations for using MS2 coliphage as an indicator of enteric viruses.