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PubMed1993

Influence of dietary buffer value index on the ruminal milieu of lactating dairy cows fed sorghum silage and grain.

The objective of this study was to evaluate the influence of dietary buffer value index on ruminal fluid pH, buffering capacity, and buffer value index in lactating cows. Three Holstein cows averaging 18 +/- 10 DIM were used in a 3 x 3 Latin square with 3-wk experimental periods. Diets contained grain:sorghum silage DM ratios of 50:50, 60:40, and 70:30. By analysis, these diets had buffer value indexes of -74, -41, and -7. The analyzed dietary buffer value index of the total diets was higher than the index that was calculated from the buffer value index of individual ingredients. Although milk fat content tended to be highest for milk from cows fed the 50:50 concentrate to forage diet and although milk protein production was highest for cows fed the 70:30 concentrate to forage diet, milk yield, 4% FCM, milk fat yield, protein content, and milk fat content were not significantly affected by dietary buffer value index. Dietary buffer equivalents were calculated to be 11% of total buffering equivalents available to the cow, and dietary acid equivalents were 15% of total acid production in the rumen. Compared with ruminal acid production and salivary buffering in the rumen, dietary acid and dietary buffer contributions to the acid-base balance of the cow are minor quantitatively. Ruminal fluid pH, hydrogen ion concentration, buffer value index, buffering capacity, and total VFA were not affected significantly by dietary buffer value index. Hence, dietary acid-base status alone is inadequate as a predictor of the need for buffers in the diet of lactating cows.

Animal Feed↗

PubMed1996

Buffers differentially alter the binding of [3H]rauwolscine and [3H]RX821002 to the alpha-2 adrenergic receptor subtypes.

3H-antagonists are known to bind to the alpha-2A adrenergic receptor with higher affinity in glycylglycine buffer than in Tris buffer. The purpose of this study was to examine the effect of buffers on the binding of antagonists to all four subtypes of the alpha-2 adrenergic receptor. Our approach was to examine the effects of glycylglycine, Tris, sodium phosphate (NaPO4) and potassium phosphate buffers on the binding of [3H]rauwolscine, [3H]RX821002, prazosin and oxymetazoline. We found that the affinities for the different subtypes varied with the buffer and the ligands used. Although the Bmax values varied somewhat with the buffers, they were similar for both radioligands for a specific subtype. The highest affinities and Bmax values for both radioligands were generally obtained with NaPO4 buffer. The affinities of antagonists in Tris buffer were always significantly lower than in either NaPO4 or glycylglycine buffer, and the affinities decreased as the concentration of Tris increased. In contrast, the affinity of norepinephrine for the alpha-2B subtype was higher in Tris than in NaPO4 buffer. The buffer effects did not appear to be dependent on the cell membrane composition. There appeared to be some species differences in the effects of buffers on the alpha-2C subtype. These results indicate that buffers affect the binding of antagonists to alpha-2 adrenergic receptors, that not all subtypes are altered in the same manner and that buffers alter the binding of different antagonists differently. It is generally recommended that NaPO4 buffer be used, and that Tris be avoided, when measuring the binding of antagonists to the alpha-2 adrenergic receptor.

Animals↗

PubMed2001

Toward quantifying water pollution abatement in response to installing buffers on crop land.

The scientific research literature is reviewed (i) for evidence of how much reduction in nonpoint source pollution can be achieved by installing buffers on crop land, (ii) to summarize important factors that can affect this response, and (iii) to identify remaining major information gaps that limit our ability to make probable estimates. This review is intended to clarify the current scientific foundation of the USDA and similar buffer programs designed in part for water pollution abatement and to highlight important research needs. At this time, research reports are lacking that quantify a change in pollutant amounts (concentration and/or load) in streams or lakes in response to converting portions of cropped land to buffers. Most evidence that such a change should occur is indirect, coming from site-scale studies of individual functions of buffers that act to retain pollutants from runoff: (1) reduce surface runoff from fields, (2) filter surface runoff from fields, (3) filter groundwater runoff from fields, (4) reduce bank erosion, and (5) filter stream water. The term filter is used here to encompass the range of specific processes that act to reduce pollutant amounts in runoff flow. A consensus of experimental research on functions of buffers clearly shows that they can substantially limit sediment runoff from fields, retain sediment and sediment-bound pollutants from surface runoff, and remove nitrate N from groundwater runoff. Less certain is the magnitude of these functions compared to the cultivated crop condition that buffers would replace within the context of buffer installation programs. Other evidence suggests that buffer installation can substantially reduce bank erosion sources of sediment under certain circumstances. Studies have yet to address the degree to which buffer installation can enhance channel processes that remove pollutants from stream flow. Mathematical models offer an alternative way to develop estimates for water quality changes in response to buffer installation. Numerous site conditions and buffer design factors have been identified that can determine the magnitude of each buffer function. Accurate models must be able to account for and integrate these functions and factors over whole watersheds. At this time, only pollutant runoff and surface filtration functions have been modeled to this extent. Capability is increasing as research data is produced, models become more comprehensive, and new techniques provide means to describe variable conditions across watersheds. A great deal of professional judgment is still required to extrapolate current knowledge of buffer functions into broadly accurate estimates of water pollution abatement in response to buffer installation on crop land. Much important research remains to be done to improve this capability. The greatest need is to produce direct quantitative evidence of this response. Such data would confirm the hypothesis and enable direct testing of watershed-scale prediction models as they become available. Further study of individual pollution control functions is also needed, particularly to generate comparative evidence for how much they can be manipulated through buffer installation and management.

Agriculture↗

PubMed2006

A bifurcation analysis of calcium buffering.

Many mathematical models of calcium oscillations model buffering implicitly by using a rapid buffering approximation. This approximation assumes that separate time scales can be distinguished, with the buffer reactions occurring on a faster time scale than the other calcium fluxes. The rapid buffering approximation is convenient as it reduces the model to a single transport equation for calcium, but buffering is not always so fast. We investigated what happens if such an assumption is made for slower buffers for parameter values typical of both endogenous and exogenous buffers. We found that no qualitative differences are introduced to the bifurcation structure, i.e. there are no anomalous behaviour or artifacts in the dynamics when a rapid buffering approximation is used compared with including buffering explicitly in the model. We found that there exist distinct buffer parameter regions in which either the rapid buffering approximation or an assumption of no buffering could be used. Separating the two regions was a small transition region of buffer parameters for which care needs to be taken in modelling buffers. However, overall, the qualitative behaviour in all three regions was similar.

Animals↗

PubMed1998

New zwitterionic butanesulfonic acids that extend the alkaline range of four families of Good buffers: evaluation for use in biological systems.

Four new zwitterionic butanesulfonic acid buffers that are structurally related to four families of Good buffers were evaluated for use in biological systems. These buffers, with pKa values from 7.6 to 10.7, were compared with a variety of other buffers from the same family and with unrelated buffers to determine their effect on enzyme activity and on microbial growth. The activity of four enzymes with optimum pH values in the alkaline range were tested: beta-galactosidase, esterase, phosphodiesterase and alkaline phosphatase. In general, all the Good buffers, including the new butanesulfonic acid buffers, gave good activity; however, there was variation in activity of certain enzymes with certain buffers. Tris, glycine, and phosphate buffers typically showed variation in activity compared to the family of Good buffers. beta-Galactosidase, in particular, showed greater activity with Good buffers than with phosphate or Tris buffers. Similarly, growth of seven bacterial strains was consistent, with a few exceptions, for all the Good family of buffers with Tris often inhibiting growth. Quantitation of alkaline phosphatase conjugated to antibodies is an important tool in many applications in molecular biology. Several Good buffers gave good signals when compared with Tris at pH 9.5 for detection of proteins using alkaline phosphatase-conjugated antibodies.

Alkaline Phosphatase↗

PubMed2006

Comparison of four nuclear isolation buffers for plant DNA flow cytometry.

BACKGROUND AND AIMS: DNA flow cytometry requires preparation of suspensions of intact nuclei, which are stained using a DNA-specific fluorochrome prior to analysis. Various buffer formulas were developed to preserve nuclear integrity, protect DNA from degradation and facilitate its stoichiometric staining. Although nuclear isolation buffers differ considerably in chemical composition, no systematic comparison of their performance has been made until now. This knowledge is required to select the appropriate buffer for a given species and tissue. METHODS: Four common lysis buffers (Galbraith's, LB01, Otto's and Tris.MgCl2) were used to prepare samples from leaf tissues of seven plant species (Sedum burrito, Oxalis pes-caprae, Lycopersicon esculentum, Celtis australis, Pisum sativum, Festuca rothmaleri and Vicia faba). The species were selected to cover a wide range of genome sizes (1.30-26.90 pg per 2C DNA) and a variety of leaf tissue types. The following parameters were assessed: forward (FS) and side (SS) light scatters, fluorescence of propidium iodide-stained nuclei, coefficient of variation of DNA peaks, presence of debris background and the number of nuclei released from sample tissue. The experiments were performed independently by two operators and repeated on three different days. KEY RESULTS: Clear differences among buffers were observed. With the exception of O. pes-caprae, any buffer provided acceptable results for all species. LB01 and Otto's were generally the best buffers, with Otto's buffer providing better results in species with low DNA content. Galbraith's buffer led to satisfactory results and Tris.MgCl2 was generally the worst, although it yielded the best histograms in C. australis. A combined analysis of FS and SS provided a 'fingerprint' for each buffer. The variation between days was more significant than the variation between operators. CONCLUSIONS: Each lysis buffer tested responded to a specific problem differently and none of the buffers worked best with all species. These results expand our knowledge on nuclear isolation buffers and will facilitate selection of the most appropriate buffer depending on species, tissue type and the presence of cytosolic compounds interfering with DNA staining.

Buffers↗

PubMed1992

Cerebral acid buffering capacity at different ages measured in vivo by 31P and 1H nuclear magnetic resonance spectroscopy.

Cerebral acidosis occurring during ischemia has been proposed as one determinant of tissue damage. Newborn animals appear to be less susceptible to ischemic tissue damage than adults. One possible component of ischemic tolerance could derive from maturational differences in the extent of acid production and buffering in newborns compared to adults. The purpose of this study was to measure the dependency of acid production on the blood plasma glucose concentrations and acid buffering capacity of piglets at different stages of development. Complete ischemia was induced in 29 piglets ranging in postconceptual age from 111 to 156 days (normal term conception, 115 days). Brain buffering capacity during the first 30 min of ischemia was quantified in vivo, via 31P and 1H nuclear magnetic resonance (NMR) spectroscopy, by measuring the change in intracellular brain pH for a given change in the concentration of compounds that contribute to the production of hydrogen ions. Animals from all four age groups showed a similar linear correlation between preischemia blood glucose concentration and intracellular pH after 30 min of ischemia. For each animal the slope of the plot of intracellular pH versus cerebral buffer base deficit was used to calculate the buffer capacity. Using data obtained over the entire 30 min of ischemia, there was no difference in the mean buffer capacity of the different age groups, nor was there a significant correlation between buffer capacity and age. However, there was a significant increase in buffer capacity for the intracellular pH range 6.6-6.0, compared to 7.0-6.6, for all age groups. No significant differences in buffer capacity for these two pH ranges were observed between any of the age groups. Acid buffering capacity was also measured by performing pH titrations on brain tissue homogenized in the presence of inhibitors of glycolysis and creatine kinase. Plots of homogenate pH versus buffer base deficit showed a nonlinear trend similar to that seen in vivo, indicating an increase in buffer capacity as intracellular pH decreases. A comparison of newborn and 1-month-old brain tissue frozen under control conditions or after 45 min of ischemia revealed no differences that could be attributed to age and a slight decrease in buffer capacity of ischemic brain compared to control brain tissue homogenates. There was no difference between the brain buffering capacity measured in vivo using 31P and 1H NMR and that measured in vitro using brain homogenates.

Acids↗

PubMed2002

[Effect of the pretreatment with organic acid buffer solutions on the recovery of Legionella species from environmental water samples].

Anti-microbial effect of the pretreatment with various organic acid buffer solutions against co-existing microorganisms which were isolated from cooling-tower water samples along with Legionella spp. was examined. Among several buffer solutions, a 0.1 M potassium citrate-citric acid buffer solution (hereafter, citrate buffer solution, pH 2.2) hardly affected the recovery of Legionella spp., but effectively inhibited the growth of co-existing microorganisms. To evaluate the buffer action of these buffer solutions, pHs of 9 cooling-tower water samples were evaluated after addition of an equal volume of each buffer solution. When a citrate buffer solution. pH 2.2 was combined to a 200-fold concentrated solution of each cooling-tower water sample, the pH of the combined solution became 2.5 to 2.7 and maintained a stably low pH value than that (pH 3.0 to 7.4) obtained after mixture of a 0.2 M HCl-KCl buffer solution (hereafter, HCl buffer solution, pH 2.2), suggesting strong buffer action of the citrate buffer solution, pH 2.2 in the combined solutions. Furthermore, when cooling-tower water samples were pretreated with a citrate buffer solution, pH 2.2, the recovery of Legionella spp. was successful in 7 out of 9 cooling-tower water samples, suggesting 3 times higher recovery rate than that obtained by addition of a HCl buffer solution, pH 2.2 (3 out of 9 cooling-tower water samples).

Acids↗

PubMed1992

Influence of dietary fiber and buffer value index on the ruminal milieu of lactating dairy cows.

The influence of dietary buffer value index and dietary ADF content on ruminal fluid pH, buffering capacity, and buffer value index was measured. Four lactating Holstein cows (two primiparous) averaging 72 +/- 60 DIM were used in a 4 x 4 Latin square with 3-wk experimental periods. Treatments were a 2 x 2 factorial arrangement of TMR containing two ADF concentrations (16 and 21% of DM) and two buffer value indexes (calculated from analysis of individual dietary ingredients to be -200 and 0). Milk fat content and milk fat yield tended to be increased by high ADF, and protein yield tended to increase with low buffer value index and low ADF. Although the high ADF diets increased ruminal fluid pH, they reduced buffering capacity; because the magnitude of the pH increase was greater than the reduction in buffering capacity, ruminal fluid buffer value index was increased by added ADF. The high buffer value index diets reduced ruminal fluid pH and increased ruminal fluid buffering capacity; effects on pH outweighed those on buffering capacity so that the ruminal fluid index paradoxically decreased as the dietary index increased. Ruminal fluid acetate increased and propionate decreased as ADF increased. We conclude that ruminal fluid buffer value index increases with dietary ADF, likely because of reduced ruminal concentrations of fermentation acids. Because diets with the highest index produced the lowest ruminal indexes, dietary buffer value index must be studied further before it can be included in any model purporting to predict the need for supplemental dietary buffers.

Acetates↗

PubMed2006

Reduction of stream sediment concentration by a riparian buffer: filtering of road runoff in disturbed headwater basins of montane mainland southeast Asia.

We determined the extent that a riparian buffer reduces stream suspended sediment concentrations by filtering road runoff during 18 rain events in a 2.5-ha, multi-use watershed in northern Thailand. The dominant buffer species was the perennial sedge Fimbristylis aphylla Zoll. ex Steud. (Cyperaceae). We monitored stream sediment concentration for situations where road runoff either flowed into the riparian buffer or was diverted directly into the stream (buffer and no buffer scenarios). These data were used to develop the following relationships between instantaneous stream sediment concentration (Ci) and discharge (Qi): Ci= 28.329Qi(0.851) (buffer scenario) and Ci= 22.265Qi(1.579) (no buffer scenario). Using these functions to calculate total event suspended concentrations, we determined that the buffer reduced suspended sediment concentration by 34 to 87%, for the range of events monitored. Removal of sediment from runoff generated on a 2.4-m-wide, 165-m-long unpaved road section was achieved principally via ponding, which reduced the transport capacity as flow entered the relatively flat, saturated buffer. Sediment deposition occurred primarily within the first 10 m of the buffer. Some sediment was also deposited on the fillslope leading to the buffer. Maximum road sediment concentration during the largest buffer event approached 100,000 mg L(-1). Meanwhile, the corresponding maximum stream suspended sediment concentration was <4000 mg L(-1). In contrast, maximum stream concentrations when flow bypassed the buffer during smaller events were commonly 4000 to 7000 mg L(-1). Naturally occurring buffers represent an economical means of mitigating road-related impacts in upland basins in Southeast Asia, particularly if combined with measures limiting sediment and runoff production on contributing road sections.

Asia, Southeastern↗

PubMed1981

Amino acid uptake by isolated renal brush border membrane vesicles in various buffers.

The uptake of amino acids by isolated rat renal brush border membrane vesicles in a modified Krebs-Ringer bicarbonate buffer and a phosphate buffer was compared to the uptake in the standard membrane vesicle buffer, Tris-Hepes-mannitol. The uptake in the modified Krebs-Ringer bicarbonate buffer was similar to that in the Tris-Hepes-mannitol buffer. Removal of the ionic constituents other than NaCl and NaHCO3 in the modified Krebs-Ringer bicarbonate buffer (KCl, CaCl2, KH2PO4 and MgSO4) did not affect the amino acid uptake by the isolated membrane vesicles. The timed uptake of proline under sodium gradient conditions in a phosphate buffer had a markedly dampened overshoot. Kinetic analysis of the initial rate of proline uptake in a phosphate buffer compared to a Tris-Herpes-mannitol buffer showed two entry systems for proline in each buffer with similar Km values, but the maximal rate of transport (V) for each system in the phosphate buffer was much lower than that in the Tris-Hepes-mannitol buffer. From these data, phosphate buffer does not appear to be a suitable medium for the study of amino acid uptake by isolated brush border membrane vesicles.

Amino Acids↗

PubMed1985

Buffering capacity of bacilli that grow at different pH ranges.

Cytoplasmic buffering capacities and buffering by whole cells were examined in six bacterial species: Bacillus acidocaldarius, Bacillus stearothermophilus, Escherichia coli, Bacillus subtilis, Bacillus alcalophilus, and Bacillus firmus RAB. Acid-base titrations were conducted on whole cells and cells permeabilized with Triton X-100 or n-butanol. In all of the species examined, the buffering capacity of intact cells was generally a significant proportion of the total buffering capacity, but the magnitude of the buffering capacity varied from species to species. Over the entire range of pH values from 4 to 9.5, B. subtilis exhibited a cytoplasmic buffering capacity that was much higher than that of B. stearothermophilus, B. acidocaldarius, or E. coli. The latter three species had comparable cytoplasmic buffering capacities at pH 4 to 9.5, as long as optimal conditions for cell permeabilization were employed. All of the nonalkalophiles exhibited a decrease in cytoplasmic buffering capacity as the external pH increased from pH 5 to 7. At alkaline pH values, the two thermophiles in the study had particularly low cytoplasmic buffering capacities, and the two alkalophilic bacteria had appreciably higher cytoplasmic buffering capacities than any of the other species studied. Cytoplasmic buffering capacities as high as 1,100 nmol of H+ per pH unit per mg of protein were observed in alkalophilic B. firmus RAB. Since previous studies have shown that immediate cytoplasmic alkalinization occurs upon loss of the active mechanisms for pH homeostasis in the alkalophiles, the very high buffering capacities apparently offer no global protection of internal pH. Perhaps, the high buffering capacities reflect protective mechanisms for specific macromolecules or process rather than part of the mechanisms for bulk pH homeostasis.

Bacillus↗

PubMed1991

Intracellular pH in the OK cell. I. Identification of H+ conductance and observations on buffering capacity.

The regulation of intracellular pH (pHi) in the opossum kidney (OK) cell line was studied in vitro using the pH-sensitive excitation ratio of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Recovery from an NH4Cl acid load disclosed a Na-dependent component blocked by amiloride and a smaller Na-independent component. The Na-independent recovery rate was proportional to the H+ gradient from cell to buffer and was zero in the absence of an electrochemical gradient. The Na-independent recovery was not affected by N-ethylmaleimide, dicyclohexylcarbodiimide, HCO3, phloretin, or ZnCl2 but was accelerated in depolarized cells and by membrane-fluidizing drugs and was inhibited by glutaraldehyde. The apparent cellular buffering capacity changed in proportion to this H+ conductance. Consistent with an electrogenic H+ leak, steady-state cell pH alkalinized with depolarization and acidified with hyperpolarization. Removal of buffer Na+ produced a profound acidification, as did amiloride. In 0-Na+ buffers, extremely large cell-to-buffer H+ gradients were present and proportional to buffer pH. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid had no effect on steady-state pHi. Measurements of intracellular buffering capacity were derived from the change of cell pH induced by withdrawing NH4Cl. This buffering capacity was increased threefold in Na-free buffers, whereas the value measured by direct titration of cell lysate was the same or less than that of control cells. The NH4Cl-derived buffering capacity varied in direct proportion to the magnitude of the H+ leak. Drugs that changed H+ permeability produced the apparent changes of the measured buffering capacity within a few minutes. We conclude that, in HCO3-free buffer, the OK cell uses two membrane acid-base transport pathways: a Na-H antiporter active at physiological pH and a substantial passive H+ conductance. The results also reveal that the NH4Cl-derived buffering capacity is subject to artifacts, possibly due to a finite leak of ionic NH4+.

Amiloride↗

PubMed2005

Surface runoff water quality in a managed three zone riparian buffer.

Managed riparian forest buffers are an important conservation practice but there are little data on the water quality effects of buffer management. We measured surface runoff volumes and nutrient concentrations and loads in a riparian buffer system consisting of (moving down slope from the field) a grass strip, a managed forest, and an unmanaged forest. The managed forest consisted of sections of clear-cut, thinned, and mature forest. The mature forest had significantly lower flow-weighted concentrations of nitrate, ammonium, total Kjeldahl N (TKN), sediment TKN, total N (nitrate + TKN), dissolved molybdate reactive P (DMRP), total P, and chloride. The average buffer represented the conditions along a stream reach with a buffer system in different stages of growth. Compared with the field output, flow-weighted concentrations of nitrate, ammonium, DMRP, and total P decreased significantly within the buffer and flow-weighted concentrations of TKN, total N, and chloride increased significantly within the buffer. All loads decreased significantly from the field to the middle of the buffer, but most loads increased from the middle of the buffer to the sampling point nearest the stream because surface runoff volume increased near the stream. The largest percentage reduction of the incoming nutrient load (at least 65% for all nutrient forms) took place in the grass buffer zone because of the large decrease (68%) in flow. The average buffer reduced loadings for all nutrient species, from 27% for TKN to 63% for sediment P. The managed forest and grass buffer combined was an effective buffer system.

Chlorides↗

PubMed1996

The influence of buffer composition on separation efficiency and resolution in capillary electrophoresis of 8-aminonaphthalene-1,3,6-trisulfonic acid labeled monosaccharides and complex carbohydrates.

The effect of buffer conditions -- varying in salt type, pH, and concentration -- on the separation of 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS)-labeled monosaccharides and complex-type carbohydrates was investigated. Different buffer systems for high and low electroosmotic flow conditions were chosen: a phosphate and a citrate background electrolyte, each at pH 2.5, a phosphate buffer, pH 9.0, and a borate buffer at pH 9.5. All buffer systems displayed differences in resolution and selectivity. Phosphate and borate buffer demonstrated the greatest selectivity changes for ANTS-labeled carbohydrates. While separation in the phosphate system relies mainly on differences in the charge-to-mass-ratio, additional selectivity can be achieved with borate complexation of glycoconjugates. The use of borate buffers improved monosaccharide separations whereas complex carbohydrates showed a loss in resolution. The citrate background electrolyte at low pH caused no significant changes in the separation performance. The pH 9.0 phosphate buffer showed a reversed migration order of the ANTS conjugates with a decreased resolution, compared to the pH 2.5 phosphate buffer, due to the strong electroosmotic flow generated under high pH conditions. An ovalbumin-derived oligosaccharide library demonstrates the significance of buffer selectivity for complex carbohydrate separations. The separation in the acidic phosphate and the alkaline borate buffer generates a different pattern and only the combination of both buffer systems allows an appropriate assessment of sample complexity.

Acids↗

PubMed1986

Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies.

Two modifications to Western blots which enhance immunochemical recognition have been developed. The first is transfer in carbonate buffer at pH 9.9, rather than the more commonly used Tris-glycine buffer at pH 8.3. This alteration improved the recognition of four of the five subunits of Escherichia coli F1-ATPase by monoclonal antibodies, the smaller subunits showing the greatest effects. Recognition of dinitrophenyl groups attached to the subunits by polyclonal antibodies was improved by the carbonate buffer only for the smallest ATPase subunit, epsilon. The second modification was incubation of the gel in mild buffers, designed to promote the renaturation of proteins, before the electrophoretic transfer step. The most effective buffer was 20% glycerol in 50 mM Tris-HCl, pH 7.4. Improvements in the signal obtained with monoclonal antibodies to all the subunits of ATPase were obtained by this procedure. As the subunits vary markedly in size, isoelectric point, and other properties, this method should be useful for most proteins. The fate of the 15,000-Da epsilon subunit, labeled with 125I, was followed through a blotting experiment. As long as no sodium dodecyl sulfate was added to the transfer buffer, epsilon was bound to nitrocellulose efficiently in either Tris-glycine or carbonate buffer. However, the epsilon was retained much more strongly during the subsequent incubation steps if the transfer was done in the carbonate buffer. The binding of epsilon to the nitrocellulose was even more stable when the gel had been treated with the buffered glycerol solution before transfer. These results indicate that the conditions under which epsilon subunit first encounters the nitrocellulose markedly affect the stability of binding during subsequent steps. The F1-ATPase was partially fragmented by treatment with proteases and then run on a gel and either transferred immediately in Tris-glycine buffer or else treated with the buffered glycerol solution and transferred in the carbonate buffer. The second blot gave stronger recognition of residual alpha subunit and fragments by an anti-alpha monoclonal antibody, with the largest improvement for the smaller fragments. This result suggests that the modified procedure may be particularly useful in enhancing the detection of small proteins.

Antibodies, Monoclonal↗

PubMed2004

Effect of the pH, the concentration and the nature of the buffer on the adsorption mechanism of an ionic compound in reversed-phase liquid chromatography. II. Analytical and overloaded band profiles on Symmetry-C18 and Xterra-C18.

In a previous report, the influence of the pH, the concentration, and the nature of the buffer on the retention and overloading behavior of propranolol (pKa = 9.45) was studied on Kromasil-C18 at 2.75 < pH < 6.75, using four buffers (phosphate, acetate, phthalate, and succinate), at three concentrations, 6, 20, and 60 mM. The results showed that the propranolol cation was eluted as an ion-pair with the buffer counter-anion. A similar study was carried out with Symmetry-C18 and Xterra-C18. Two additional buffers, formate and citrate, were also used. Propranolol elution band profiles were recorded for a small (less than 1 microg) and a large (375 microg) sample size. The results are similar to those obtained with Kromasil and confirm earlier conclusions. The buffer concentration, not its pH, controls the retention time of propranolol, in agreement with the chaotropic model. The retention factor depends also on the nature of the buffer, particularly on its valence, and on the hydrophobicity of the basic anion. With the monovalent anions HCOO- (pH 3.75), H2PO4- (pH 2.75), HOOC-Ph-COO- (pH 2.75), HOOC-CH2-CH2-COO- (pH 4.16), CH3COO- (pH 4.75) and HOOC-CHCOOH-COO- (pH 3.14), at moderate loadings, and for the two larger buffer concentrations, the band profiles are well accounted for by a simple bi-Langmuir isotherm model (no adsorbate-adsorbate interactions). By contrast, these profiles are accounted for by a bi-Moreau isotherm model (i.e., with significant adsorbate-adsorbate interactions) with the bivalent anions -OOC-Ph-COO- (pH 4.75), -OOC-CH2-CH2-COO- (pH 5.61), HPO4(2-) (pH 6.75), and HOOC-CHCOO(-)-COO- (pH 4.77) and with the trivalent anion -OOC-CHCOO(-)-COO- (pH 6.39). The best values of the isotherm parameters were determined using the inverse method. The saturation capacity and the equilibrium constant on the low-energy sites increase with increasing buffer concentration, a result consistent with the formation in the mobile phase of a hydrophobic complex between the propranolol cation and the buffer anion. With bivalent and trivalent anions, adsorbate-adsorbate interactions are strong on the low-energy sites but they remain negligible on the high-energy sites. The density of the high energy sites is lower and the equilibrium constant on the low-energy sites are both higher with the bivalent and the trivalent buffer anions than with the univalent buffer anions. These results are consistent with the formation of a 2:1 and a 3:1 propranolol-buffer complex with the bivalent and the trivalent anions, respectively.

Adsorption↗

PubMed1994

Reducing the pain of local anesthetic infiltration: warming and buffering have a synergistic effect.

STUDY OBJECTIVE: To compare room-temperature unbuffered lidocaine, warm lidocaine, buffered lidocaine, and warm buffered lidocaine to determine which of the four solutions is least painful during infiltration. DESIGN: Randomized, controlled, double-blinded, volunteer study. TYPE OF PARTICIPANT: Thirty-two young healthy adults. MAIN RESULTS: Each subject received four subcutaneous injections of 1% lidocaine: room-temperature unbuffered, warm, buffered, and warm buffered. After each injection, participants recorded their perception of pain associated with infiltration of the solution on a visual analog scale. Mean pain scores for the four solutions were determined and analyzed. The mean perceived pain score for the warm buffered solution was significantly lower than for any of the other solutions (versus warm: P = .0005; versus buffered: P = .0028; versus room temperature: P = .0001). There was no statistically significant difference between either the warm solution or buffered solution and the room-temperature unbuffered lidocaine. The difference in mean pain score for the warm buffered solution, compared with those for the warm, buffered, and room-temperature solutions, suggests that warming and buffering have a synergistic effect. CONCLUSION: Skin infiltration with warm buffered lidocaine is significantly less painful than infiltration with room-temperature unbuffered lidocaine, warm lidocaine, or buffered lidocaine.

Adult↗