Effects of different buffers on the thermostability and autolysis of a cold-adapted protease MCP-01

A cold-adapted protease MCP-01 was obtained from deep-sea psychrotrophic bacterium Pseudoaltermonas sp. SM9913. The effects of four different buffers, all at 50 mmol/l concentration, on its thermostability and autolysis were studied. The autolysis process of MCP-01 was studied by capillary electrophoresis. The thermostability of MCP-01 increased successively in the following order: carbonate < Tris < phosphate < borate. The optimum temperature for casein hydrolysis also increased in the same order. This suggested that the conformation of MCP-01 was flexible and its autolytic susceptibility was affected by some factors in the buffers such as charge and ionic species. The results also showed that different buffers, in addition to affecting the autolysis speed, gave different patterns of autolysis products. In carbonate buffer, Tris buffer, phosphate buffer and borate buffer, the autolysis patterns of MCP-01 were different. These results suggested that protease MCP-01 probably have different conformations in different buffers, thus exposing different autolysis sites on the enzyme surface. In addition, the loss of activity correlated with the speed of autolysis in the four different buffers, showing that autolysis may be a reason for the low thermostability of the enzyme.     

Chromatographic behavior of Bothrops erythromelas phospholipase and other venom constituents on Superdex 75

In a chromatographic method modification intended to preserve protease activity in Bothrops erythromelas venom, 2 mM CaCl2 was added to the gel filtration buffer [50mM Tris/HCl/150mM NaCl (pH 8.0)], in lieu of an equimolar portion of NaCl. This minor compositional change induced significant differences in the venom elution profile on Superdex 200. For this reason, the influence of buffer composition on chromatographic behavior was investigated using an analytical Superdex 75 HR 10/30 column. Phospholipase (PLA) was used as a marker because Naja atra PLA had previously been observed to interact hydrophobically with this resin. PLA elution volumes generally increased as buffer pH decreased. Addition of 20% acetonitrile to the Tris buffer with CaCl2, reduced hydrophobic interaction of the PLA so significantly that its elution was non-overlapping in the two buffers. Other venom constituents, including bradykinin-potentiating peptides and probable hemorrhagic metalloproteases, were similarly affected. Buffer calcium, bound by vicinal dextran hydroxyl groups, appears to retard elution of this acidic PLA.     

Simultaneous determination of ampicillin and metampicillin in biological fluids using high-performance liquid chromatography with column switching

A new high-performance liquid chromatographic method with column switching has been developed for the simultaneous determination of metampicillin and its metabolite ampicillin in biological fluids. The plasma, urine and bile samples were injected onto a precolumn packed with LiChrosorb RP-8 (25–40 μm) after simple dilution with an internal standard solution in 0.05 M phosphate buffer (pH 7.0). The polar plasma components were washed out using 0.05 M phosphate buffer (pH 7.0). After valve switching, the concentrated drugs were eluted in the back-flush mode and separated by an Ultracarb 5 ODS-30 column with a gradient system of acetonitrile-0.02 M phosphate buffer (pH 7.0) as the mobile phase. The method showed excellent precision, accuracy and speed with a detection limit of 0.1 μg/ml. The total analysis time per sample was less than 40 min and the coefficients of variation for intra- and inter-assay were less than 5.1%. This method has been successfully applied to plasma, urine and bile samples from rats after intravenous injection of metampicillin.     

On-line solid-phase extraction and high-performance liquid chromatographic determination of chlorthalidone in urine

A simple and rapid on-line method for the determination of chlorthalidone in urine is proposed. The sample containing the internal standard is injected in a CN precolumn. After a 2-ml water rinsing, the precolumn is coupled for 30 s to the HPLC column via a switching valve, allowing the on-line elution of the compounds of interest. Analysis is carried out by reversed-phase chromatography with an acetonitrile-0.01 M phosphate buffer pH 7 (20:80, v/v) eluent, using UV detection at 214 nm. While the LC separation is performed, the precolumn is regenerated and conditioned, and is ready to receive the next sample at the end of the run. Accurate (>95%) and precise (<10%) analyses, in the range of 0.1–20 μg/ml of chlorthalidone in urine, have been achieved using this method.     

Rapid and sensitive ion-exchange fluorometric measurement of γ-aminobutyric acid in physiological fluids

Improvements of the ion-exchange/fluorometric method for measuring γ-aminobutyric acid (GABA) have resulted in an automatic three-column analyzer which produces an analysis every 15 min using 200-μl samples to a sensitivity of 1 pmol. This high-performance liquid chromatographic procedure utilizes three stainless-steel microbore columns containing cation-exchange resin. GABA is eluted using a lithium citrate buffer and then detected in the flow stream after reaction with the fluorogenic reagent orthophthalaldehyde. Each column utilizes a timer-controlled, pneumatically actuated two-way 10-port valve containing a 200-μl loop for sample injection and a 1-ml loop for regeneration. Similar 4-port valves sequentially direct the output from one of the columns to the reaction manifold. Measurement of GABA levels in sequential aliquets of the first 20 ml of human lumbar cerebrospinal fluid revealed an increasing gradient of GABA level in cerebrospinal fluid from six patients and no gradient in a seventh patient, the mean rate of increase being 2% per milliliter.     

Rapid down regulation of insulin receptors in adipocytes artifact of the incubation buffer

Rat adipocytes were incubated with 15 nM insulin in different buffers at 37°C. The cells were washed and reincubated at 16°C in the presence of 18 pM A14-[¹²⁵I]monoiodoinsulin to determine the insulin receptor concentration. After incubation for 2 h in Tris buffer the binding decreased to about 30 %, whereas no decrease was found after incubation in Hepes, phosphate or bicarbonate buffers. Binding of tracer insulin reached a constant level by 45 min in Hepes buffer at 37°C, whereas it continued to increase in Tris buffer. Washout of tracer insulin after incubation in Tris buffer at 37°C showed a large, slowly dissociable fraction. It is suggested that the rapid down regulation of insulin receptors $\text{in}\text{vitro}$ is an artifact of the Tris buffer and that the phenomenon is due to a slowly reversible occupancy of a receptor pool with unlabelled insulin.     

A procedure for anaerobic column chromatography employing an anaerobic freter-type chamber

A technique is described for the anaerobic fractionation of oxygen-sensitive material. A Freter-type chamber in which the oxygen concentration is maintained at 2 to 5 μl/liter is used in conjunction with anaerobic chromatographic columns that are exterior to the chamber. The column inlet and outlet are connected via thick-walled polyethylene tubing to access ports in the chamber wall. Anaerobic buffer inside the chamber is pumped from the chamber to equilibrate the column. The oxygen-labile sample then is pumped onto the anaerobic column followed by the elution gradient buffer. Column eluate is returned to a fraction collector inside the chamber. At no stage is the sample exposed to air. This technique has been used effectively for fractionation of highly oxygen-sensitive enzymes from methanogenic bacteria where use of other methods failed.     

Optimized dipicolinate activation for viable counting of Bacillus stearothermophilus spores

Spores of Bacillus stearothermophilus were exposed to calcium and sodium salts of dipicolinic acid (DPA) in phosphate and Tris acid maleate buffers over the range pH 4.5–10.0. The exposed spores were enumerated using a standard plate counting technique from which the kinetics of colony formation were determined and maximum colony counts were obtained for each condition examined. Exposure of the spores to calcium-DPA (50-40 mmol/l) in Tris acid maleate buffer pH 9.0 maintained at 10°C was found to produce an optimal response. Following this method the total viable population of a spore suspension was enumerated. This was demonstrated statistically using the Wilcoxon rank-sum test for significance. Calcium-DPA was found to produce activation in spores but further germinants and nutrients were required for colony formation. The Ca-DPA treatment was found to be effective in enumerating both naturally dormant spores and heat injured spores.     

The buffering power of plasma in brown bullhead (Ameiurus nebulosus)

Brown bullhead (Ameiurus nebulosus) blood plasma was found to exhibit an unusually high non-bicarbonate buffer capacity (beta) in relation to that of other teleost fish. In brown bullhead, the non-bicarbonate buffer capacity of plasma (beta(plasma)), at -5.72 +/- 0.34 mmol l(-1) pH unit(-1) (mean +/- S.E.M., N=30), constituted 37% of whole blood beta and was 2.5 times higher than beta(plasma) in rainbow trout (-2.33 +/- 0.42 mmol l(+/-1) pH unit(-1); N=7). The strong buffering power of bullhead plasma was not the result of unusually high plasma protein levels. Size separation chromatography in conjunction with a spectrophotometric assay for buffering capacity were used to isolate a plasma fraction of high buffering power. SDS-polyacrylamide gel electrophoresis revealed that this fraction contained four proteins, but was dominated by a protein of approximately 68-70 kDa molecular mass. On the basis of the amino acid composition of this fraction, the dominant protein was identified as albumin. In comparison to other fish albumins, bullhead albumin appears to be histidine-rich (6.7%). Thus, the unusually high non-bicarbonate buffer capacity of bullhead plasma appears to stem from the presence in the plasma of a histidine-rich albumin.     

Coating efficiency of a 1 -acid glycoprotein in competitive enzyme immunosorbent assays with antigen immobilized on the solid phase

Coating efficiency of rat and human serum a 1 -acid glycoprotein (a 1 -AGP) was investigated for competitive enzyme immunosorbent assays with antigen immobilized on the solid phase by using different pHs and buffers. Blocking materials and pH of coating buffer had a marked influence on the amount of a 1 -AGP that binds to plate. Usually, carbonate buffer is used at pH 9.6 or 9.0, but phosphate buffered saline (PBS) at pH 7.2 can be used for an effective coating. At pH 7.2, coating of a 1 -AGP in Tris buffered saline was five - tenfold as effective as in PBS and phosphate buffer. Blocking of uncoated surface with casein was ten - twenty times as effective as with fetal bovine serum albumin for coating of a 1 -AGP.     

Purification from goat antiserum of immunoglobulins against G6PD from rabbits]

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.     

Isolation and purification of iridoid glycosides from Gardenia jasminoides Ellis by isocratic reversed-phase two-dimensional preparative high-performance liquid chromatography with column switch technology

A two-dimensional column-switching system without sample loop trapping, where two columns were switched directly via a six-port two-position switching valve, was successfully applied for the first time to the isolation and purification of six iridoid glycosides including geniposide, gardenoside, shanzhiside, scandoside methyl ester, deacetyl-asperulosidic acid methyl ester and genipin-1-beta-D-gentiobioside from Gardenia jasminoides Ellis, a plant used in the traditional Chinese medicine. The introduction of the six-port switching valve instead of sample loop assured 100% recovery from the first dimension to the second, and the injection volumes of the second dimension could reach 20 ml. In this mode of operation, the sample size of the two-dimensional approach was more than 1.3 times that of conventional gradient methods with even less solvent consumption. And the simultaneous operations of the two dimensions allowed the cycle time to be less than 19 min, compared with that (90 min) in the gradient elution single-dimension mode of operation. All of the six isolated iridoid glycosides were isolated at high purities of over 99% with approximately 96% recoveries.     

Experimental determination of net protein charge, [A]tot, and Ka of nonvolatile buffers in bird plasma.

The quantitative mechanistic acid-base approach to clinical assessment of acid-base status requires species-specific values for [A]tot (the total concentration of nonvolatile buffers in plasma) and Ka (the effective dissociation constant for weak acids in plasma). The aim of this study was to determine [A]tot and Ka values for plasma in domestic pigeons. Plasma from 12 healthy commercial domestic pigeons was tonometered with 20% CO2 at 37 degrees C. Plasma pH, Pco2, and plasma concentrations of strong cations (Na, K, Ca), strong anions (Cl, L-lactate), and nonvolatile buffer ions (total protein, albumin, phosphate) were measured over a pH range of 6.8-7.7. Strong ion difference (SID) (SID5=Na+K+Ca-Cl-lactate) was used to calculate [A]tot and Ka from the measured pH and Pco2 and SID5. Mean (+/-SD) values for bird plasma were as follows: [A]tot=7.76+/-2.15 mmol/l (equivalent to 0.32 mmol/g of total protein, 0.51 mmol/g of albumin, 0.23 mmol/g of total solids); Ka=2.15+/-1.15x10(-7); and pKa=6.67. The net protein charge at normal pH (7.43) was estimated to be 6 meq/l; this value indicates that pigeon plasma has a much lower anion gap value than mammals after adjusting for high mean L-lactate concentrations induced by restraint during blood sampling. This finding indicates that plasma proteins in pigeons have a much lower net anion charge than mammalian plasma protein. An incidental finding was that total protein concentration measured by a multianalyzer system was consistently lower than the value for total solids measured by refractometer.     

Resolution of human mercapt- and nonmercaptalbumin by high-performance liquid chromatography

Gel-exclusion high-performance liquid chromatographic (HPLC) analysis of human serum albumin (HSA) on PGP 2000 column (0.10 M sodium phosphate buffer, 0.30 M NaCl, pH 6.86) showed at least two peaks, the principal component corresponding to human mercaptalbumin (HMA) and the second one to human nonmercaptalbumin (HNA). Mechanism for the separation of HMA and HNA might be due to weak resin-HSA interaction. HPLC analysis of bovine plasma albumin (BPA) showed a single peak on PGP 2000 column. The elution volume of HSA was larger than that of BPA, resulting in a clear resolution of HSA and BPA.     

Plasma carnitine and acetyl-carnitine levels at different times of the day

An interest in both biochemical and clinical carnitine investigation has recently developed. A more complete and extensive study is obtained if acetyl-carnitine as well as carnitine are investigated. This research, using an improved and simplified method for carnitine and acetyl-carnitine determination in the same sample (1 ml) without radioisotopic tracer use, investigates if there are the same differences in their plasma levels at different times of the day. The sample was eluted in a chromatographic column (55 X 15 mm) containing Sephadex G-25M with phosphate buffer (25 mmol/l, pH 7.4). The fraction containing acetyl and free carnitine was divided and employed separately for two assays. The carnitine assay uses an enzymatic reaction catalyzed by carnitine acetyl-transferase (CAT) and measurements are carried out spectrophotometrically. The calibration curve shows r = 0.987 and sensitivity at 5 mumol/l (reference plasma values: 38 +/- 3 mumol/l in 9 subjects). The acetyl-carnitine assay is carried out concentrating the sample by lyophilization and then measuring the enzymatic coupled reactions catalyzed by CAT, malate dehydrogenase and citrate synthase fluorimetrically. The calibration curve gives r = 0.991 and sensitivity at 1.4 mumol/l (reference plasma values: 2.8 +/- 0.3 mumol/l in 9 subjects). Both assay methods are measured at the end point. The carnitine and acetyl-carnitine measured in the plasma of 6 normal subjects at different times of the day vary respectively from 28 to 37 mumol/l and from 1.1 to 5.2 mumol/l in agreement with plasma free fatty acid (FFA) variation from 230 to 779 microEq/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

High performance liquid chromatographic method for the determination of cetirizine and ambroxol in human plasma and urine--a boxcar approach

A column switching high performance liquid chromatographic method with estimable sensitivity and accuracy was developed for the determination of cetirizine and ambroxol in human plasma using nebivolol as the internal standard. Plasma samples were prepared by liquid-liquid extraction in methylene chloride and a mixture of diethylether (80:20, v/v). The extracted samples were injected into a multifunctional clean-up column Supelcosil LCABZ (50 mm × 4.6 mm, 5 μm particle size) using mobile phase 1 comprising acetonitrile-phosphate buffer (pH 3.5; 20 mM) (20:80, v/v). The eluate of cetirizine and ambroxol were separated to an analytical Kromasil C(8) micro bore column (50 mm × 0.3 mm, 5 μm particle size) via a column switching device. A Kromasil C(18) analytical column (250 mm × 2.1 mm, 5 μm particle size) was used as a separation column. Mobile phase 2 consisting acetonitrile-triethylamine (0.5%) in phosphate buffer (pH 3.5; 20mM) (55:45, v/v) was used for the compound elution. The eluents were detected at 230 nm with photodiode array detector. An aliquot of 150 μl of plasma sample was introduced into the pretreatment column via the auto sampler using mobile phase 1 at a flow rate of 0.5 ml/min, column switching valve being positioned at A. The pretreatment column retained cetirizine, ambroxol and nebivolol (IS) in the column leaving the residual proteins of plasma eluted in void volume and drained out. The switching valve was shifted to position B at 7.5 min. Cetirizine, ambroxol and IS were eluted from the pretreatment column between 7. 5 and 11.5 min and introduced to the concentration column. Finally, cetirizine, ambroxol and IS were introduced to the separation column by switching valve using mobile phase 2 at a flow rate of 0.4 ml/min. During the analysis the pretreatment column was washed for the next analysis and resume to the position A. The total run time was 25 min for a sample. The procedure was repeated for urine analysis also. The method was linear from 2 to 450 ng/ml and 7-300 ng/ml for cetirizine and ambroxol respectively in plasma and 1-500 ng/ml and 5-400 ng/ml, respectively for cetirizine and ambroxol in urine. Intra-day and inter-day precision of cetirizine and ambroxol was below 15% in terms of coefficient of variation and accuracy of cetirizine and ambroxol was ranged from 94 to 101.6% and 91.1 to 100.2%, respectively. The method demonstrated high sensitivity and selectivity and therefore, applied to evaluate pharmacokinetics of cetirizine and ambroxol in healthy human volunteer after a single oral administration. Urine samples obtained from healthy human volunteers and clinical subjects with renal impairment have also been analyzed by the method to compare the elimination pattern. The method was precise and accurate for the estimation of cetirizine and ambroxol both in blood and in urine.     

Determination of non-protein bound phenylbutazone in bovine plasma using ultrafiltration and liquid chromatography with ultraviolet detection

A liquid chromatographic procedure using UV detection was coupled with ultrafiltration for the quantitation of free phenylbutazone in bovine plasma, in the range of 20 ng/ml to 2.0 μg/ml. Whole plasma samples (0.5 to 1 ml) were placed in a 2-ml centrifugal concentrator with a molecular-mass cut-off membrane of 10 000 and centrifuged at 4500 g for 2 h at 4°C using a fixed angle rotor. The ultrafiltrate was transferred to an LC vial with a 200-μl insert and 100 μl was injected into an LC system. The chromatographic system used a C₁₈ reversed-phase column connected to a UV detector set at 264 nm. The mobile phase was 0.2 M sodium phosphate buffer (pH 7)–methanol (1:1). Recoveries of phenylbutazone from protein-free plasma water fortified at levels of 20 ng/ml to 2 μg/ml ranged from 91 to 93%, with relative standard deviations (R.S.D.s) ranging from 1 to 4%. The concentration of incurred non-protein bound phenylbutazone obtained from a cow intravenously dosed twice with 2 g phenylbutazone, 8 h apart, was 111, 26 and 11 ng/ml for 2, 72 and 104 h post first phenylbutazone dose, respectively.     

High-pressure liquid chromatography: I. Quantitative separation of purine and pyrimidine nucleosides and bases

A high-pressure liquid column chromatographic method has been developed for the separation and identification of ribonucleosides, deoxyribonucleosides, and bases. This method is capable of detecting these components at 1.0 to 5.0 ng applied; is reproducible under conditions of constant pressure, temperature, and pH; and is rapid, requiring about 30 min for a complete chromatographic separation of ribonucleosides from deoxyribonucleosides and from bases. These separations were carried out under different pH values and buffers, namely, phosphate buffer containing 2.5% methanol at pH 6.9 and 3.0 or 50 mm sodium borate buffer, pH 9.0. These different conditions were utilized to obtain more definitive identification and quantitation of normal metabolites and their counterparts, the antimetabolites. The advantage of this method is that the 20 naturally occurring components are separated from each other by an isocratic elution method, alleviating the need for a gradient elution system, which produces a drift in the baseline with increasing concentration of the eluting buffer, especially when the instrument is operating at maximum sensitivity, thus hindering the quantitation of the separated components. The potential application of this method for the quantitation of plasma metabolites and antimetabolites such as Ara-C², Ara-U, and F-pyrimidine is describe.     

Remarkable effect of mobile phase buffer on the SEC-ICP-AES derived Cu, Fe and Zn-metalloproteome pattern of rabbit blood plasma

The development of an analytical method to quantify the major Cu, Fe and Zn-containing metalloproteins in mammalian plasma has been recently reported. This method is based on the separation of plasma proteins by size exclusion chromatography (SEC) followed by the on-line detection of the metalloproteins by an inductively coupled plasma atomic emission spectrometer (ICP-AES). To assess whether the mobile phase buffer can affect the SEC-ICP-AES-derived metalloproteome pattern, thawed rabbit plasma was analyzed using phosphate buffered saline (PBS)-buffer (0.15 M, pH 7.4), Tris-buffer (0.1 and 0.05 M, pH 7.4), Hepes-buffer (0.1 M, pH 7.4) or Mops-buffer (0.1 M, pH 7.4). In contrast to the Cu-specific chromatograms, the Fe and Zn-specific chromatograms that were obtained with Tris, Hepes and Mops-buffer were considerably different from those attained with PBS-buffer. The Tris, Hepes and Mops-buffer mediated redistribution of ~25% plasma Zn(2+) from <100 kDa to >100-600 kDa plasma proteins and to a smaller extent to a <10 kDa (Tris)(2)Zn(2+)-complex can be rationalized in terms of the abstraction of Zn(2+) from the weak binding site on albumin. In contrast, only Hepes and Mops-buffer redistributed ~20% of plasma Fe(3+) from the <100 kDa to the >600 kDa elution range. Based on these results and considering that the utilization of PBS-buffer has previously resulted in the detection of a number of Cu, Fe and Zn-containing metalloentities in rabbit plasma that was most consistent with literature data, this mobile phase buffer is recommended for metallomic studies regarding mammalian blood plasma.     

Experimental determination of net protein charge and A(tot) and K(a) of nonvolatile buffers in human plasma.

The mechanism for an acid-base disturbance can be determined by using the strong ion approach, which requires species-specific values for the total concentration of plasma nonvolatile buffers (Atot) and the effective dissociation constant for plasma weak acids (Ka). The aim of this study was to experimentally determine Atot and Ka values for human plasma by using in vitro CO2 tonometry. Plasma Pco2 was systematically varied from 25 to 145 Torr at 37 degrees C, thereby altering plasma pH over the physiological range of 6.90-7.55, and plasma pH, Pco2, and concentrations of quantitatively important strong ions (Na+, K+, Ca2+, Mg2+, Cl-, lactate) and buffer ions (total protein, albumin, phosphate) were measured. Strong ion difference was estimated, and nonlinear regression was used to calculate Atot and Ka from the measured pH and Pco2 and estimated strong ion difference; the Atot and Ka values were then validated by using a published data set (Figge J, Rossing TH, and Fencl V, J Lab Clin Med 117: 453-467, 1991). The values (mean +/- SD) were as follows: Atot = 17.2 +/- 3.5 mmol/l (equivalent to 0.224 mmol/g of protein or 0.378 mmol/g of albumin); Ka = 0.80 +/- 0.60 x 10-7; negative log of Ka = 7.10. Mean estimates were obtained for strong ion difference (37 meq/l) and net protein charge (13+.0 meq/l). The experimentally determined values for Atot, Ka, and net protein charge should facilitate the diagnosis and treatment of acid-base disturbances in critically ill humans.