Colorimetric estimation of inorganic phosphate in colored and/or turbid biological samples: assay of phosphohydrolases

A simple method of inorganic phosphate determination for colored and/or turbid biological samples is described. The procedure is mild, and so is suitable for routine phosphohydrolase assays. Following deproteinization by ice-cold trichloroacetic (or silicotungstic) acid, the sample was treated with acid-washed charcoal to remove interference due to color. The phosphate in the colorless supernatant was assayed either by measuring the phosphomolybdate spectrophotometrically at 310 nm, following its extraction in organic solvents or by a modified Fiske and Subbarow method. The turbidity interference in the latter case was eliminated either by centrifugation, by sodium dodecyl sulfate treatment, or by extraction of reduced phosphomolybdate blue color by cyclohexanone. Though deproteinization by silicotungstic acid eliminated the turbidity problem, its use in conjunction with charcoal treatment was not convenient.     

A semiautomated hemolysis microassay for membrane lytic proteins

A simple, rapid, semiautomated microassay for hemolysis using a microtiter plate spectrophotometric system is described. The assay relies on the differences in light scattering (turbidity) properties of intact and of lysed erythrocytes. Lysis of erythrocyte suspensions in 96-well plates is determined by absorbance at 690 nm. A linear correlation between the percentage of hemolysis and the turbidity decrease is observed, indicating that this assay may be used for both rapid screening and quantitation of the hemolytic activity. This assay allows screening of 300 samples in less than 6 min. Small samples derived from protein fractionation columns (HPLC, for example) can be rapidly screened. This assay has been used in the successful isolation of a cytolytic membrane-lytic protein from the granules of cloned cytotoxic T lymphocytes and NK cells.     

An assay for heparin by decrease in color yield (DECOY) of a protein-dye-binding reaction

The interference by heparin and some related molecules with the well-known Bradford dye-binding assay for proteins is used as the basis of a rapid, sensitive method for the quantitation of these polysaccharides. Whereas the available methods for the assay of glycosaminoglycans have lacked specificity for sulfated polyanions in general and for heparin in particular, the procedure described here distinguishes among different uronic acid derivatives and, when performed in conjunction with heparinase digestion or cetylpyridinium chloride precipitation, can be used to determine heparin content of complex biological samples.     

Assay of proteins by Lowry's method in the presence of high concentrations of β-mercaptoethanol

β-Mercaptoethanol interferes in the determination of protein by the Lowry method (1–6). The interference can be overcome by the precipitation of proteins by trichloroacetic acid or acetone or by the use of H₂O₂ which oxidizes the sulfhydryl groups of β-mercaptoethanol (5). Both these methods have inherent disadvantages. Ross and Schatz (5) described a procedure for protein determination in the presence of high concentrations of β-mercaptoethanol where they removed the interference by the addition of iodoacetate. But addition of iodoacetate decreased the sensitivity of the reaction. The removal of interference by β-mercaptoethanol by heating has also been reported (3), but we observed that this procedure is not feasible when a large amount of β-mercaptoethanol is present in the protein samples. In the method reported in this communication, we made use of vacuum drying for the removal of interference by β-merceptoethanol. This method is simple, sensitive, takes less time, and can be used for the determination of protein in the presence of β-mercaptoethanol at levels as high as 10% in a sample volume of 1.0 ml (1.43 mmol) without using any additional chemical steps.     

Analysis of all protein amino acids as their tert.-butyldimethylsilyl derivatives by gas-liquid chromatography

A gas chromatographic method for the separation and quantitation of the 20 protein amino acids is described using N-methyl-N(tert.-butyldimethylsilyl)trifluoroacetamide, with 1% tert.-butyldimethylchlorosilane as catalyst, to prepare the tert.-butyldimethylsilyl amino acid derivatives. Alkylsilylation of amino acids proceeds at 140 degrees C in 20 min. The derivatives formed in the one-step reaction are used directly for gas-liquid chromatographic analysis, using a flame-ionization detector, without prior isolation or purification. Complete separation and quantitation of all protein amino acids are readily achieved using a 15-m DB-5 capillary column. Strict linearity extends from less than 15 to about 100 ng for all amino acids except Arg, which has a linear range from 50 to 300 ng. The limits of detection, however, range from one to several hundred nanograms. The method was used to analyze the free amino acid pool in carnation petals.     

Determination of plasma ziprasidone using liquid chromatography with fluorescence detection

A liquid chromatographic procedure was developed for the determination of a new antipsychotic agent ziprasidone in plasma using fluorescence detection. A one-step liquid-liquid extraction from 1 ml of alkalinized plasma containing an internal standard alpha-ergocryptine using methyl-t-butyl ether afforded a greater than 84% recovery of ziprasidone. Chromatography was performed using a reversed-phase trimethylsilyl bonded silica column with a mobile phase of 72:28 phosphate buffer:acetonitrile at a flow rate of 1.5 ml/min. Detection of the eluted peaks was observed using excitation and emission wavelengths of 320 and 410 nm, respectively. Chromatographic run time did not exceed 14 min with no interference from endogenous material. The calibration curve was linear over the concentration range of 0.5 to 200 ng/ml and the inter- and intra-assay imprecision (CV) was less than 10%. The lower limit of quantitation was assessed at 0.5 ng/ml. Specificity of the method is demonstrated by the lack of interference from a large number of commonly used drugs and their metabolites in clinical use. The utility of the method is exemplified with the presentation of clinical data from patients receiving ziprasidone.     

Adaptation of the Bradford protein assay to membrane-bound proteins by solubilizing in glucopyranoside detergents

A procedure was developed for the quantitation of solubilized proteins using the Bradford assay in the presence of glucopyranoside detergents. These detergents solubilized membrane-bound proteins with minimal background absorbance at 595 nm. Absorbance at 650 nm was also low, indicating that these detergents do not significantly stabilize the neutral species of Coomassie brilliant blue G-250 that produces interference in the presence of other detergents. Hexyl-beta-D-glucopyranoside produced less absorbance than did larger glucopyranosides, and the increase in its absorbance at 595 nm in the presence of dye reagent was related linearly to its concentration from 0 to 2%. Absorbance produced by membrane-bound protein was increased by the presence of up to 0.2% hexyl-beta-D-glucopyranoside (final concentration in dye reagent) and then remained stable up to 1%, indicating that these concentrations of this detergent allowed membrane-bound proteins to react completely with the dye reagent. Standard curves of several proteins were similar in the absence or presence of 0.1-0.5% hexyl-beta-D-glucopyranoside. The quantitation of both soluble and membrane-bound proteins by the Bradford assay was similar in the presence of 0.2% hexyl-, heptyl-, and octyl-beta-D-glucopyranoside. Estimates of membrane-bound protein by this assay agreed with estimates obtained with the Lowry assay and with quantitative amino acid analysis. This procedure requires no extra steps; thus, it is as rapid and convenient as the original Bradford protein assay.     

A colorimetric method for the determination of submicrogram quantities of protein

A simple procedure for the measurement of submicrogram quantities of protein is described which can be used without interference from most common reagents. Protein-containing solutions are spotted on glass fiber filters, washed with trichloroacetic acid, and stained with Coomassie blue. The filters are destained, and the protein-bound colorant is eluted and measured in a spectrophotometer at 590 nm. The response is linear to 5 μg of protein per filter, and as little as 0.1 μg per filter can be accurately determined.     

Quantitative determination of growth hormone by immunoblotting

Blotting techniques have been extensively used, not only analytically for protein identification, but also preparatively to isolate and purify specific proteins from a large variety of cellular extracts and biological fluids. The process involves the separation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transfer of proteins to nitrocellulose membranes, and immunostaining to identify proteins which often are at very low concentrations. Because of the quantitative interactions of the proteins with specific antibodies, we have coupled the immunoblot procedure with photographic and densitometric methods for the quantitative determination of bovine growth hormone (bGH). In this way, the method is suitable for bGH detection and quantitation for a small number of samples by use of a single Western blot analysis. The sensitivity of this method permits determinations of bGH to 0.5 ng. The method uses a comparative procedure in which purified bovine growth hormone is used as a standard.     

Conformational changes accompany the oxidative inactivation of rhodanese by a variety of reagents

Rhodanese is oxidatively inactivated by several reagents, some of which are not normally considered oxidants. Rhodanese, in a form not containing persulfide sulfur (E), was inactivated by phenylglyoxal under conditions where disulfides are formed. There was the concomitant increase in the fluorescence of the apolar probe 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bisANS). At 0.2 mg/ml protein, there was no turbidity, while at 1 mg/ml, turbidity formed after an induction period of 23 min. Phenylglyoxal-inactivated E was extensively digested by endoproteinase glutamate C (V8 protease) to give two discrete high molecular weight fragments (Mr = 29,500 and 16,000). Enzymatically active E or ES, the form of rhodanese containing transferred sulfur (Mr = 33,000) was totally refractory to V8 protease and gave only small fluorescent enhancement of bisANS. Phenylglyoxal inactivated ES (reaction at arginine) gave very little fluorescence enhancement of bisANS and was not digested by V8. Hydrogen peroxide rapidly inactivated E (t1/2 less than 2 min) giving a slow increase in bisANS fluorescence (t1/2 greater than 10 min) identical to that observed with phenylglyoxal. The turbidity also increased after an induction period of approximately 30 min. Inactivation of E by hydrogen peroxide gave the same digestion pattern as that observed with phenylglyoxal inactivation. The turbidity was associated with the formation of disulfide-bonded structures that formed with the stoichiometry of E, 2E, 4E, 6E, 8E, etc. relative to the native enzyme, E. E was inactivated with several other reagents that lead to oxidatively inactivated rhodanese including NADH, dithiothreitol, mercaptoethanol, and m-dinitrobenzene. Enzyme inactivated with dithiothreitol or NADH gave an identical digestion pattern as above. In addition, with the exception of NADH which could not be used due to optical interference, each of the reagents gave rise to increased fluorescence of bisANS after inactivation. The results are consistent with a model in which the oxidized rhodanese resulting from diverse treatments is in a new conformation that has extensive exposed apolar surfaces and can form both noncovalent and disulfide-bonded aggregates.     

Measurement of protein by dye-binding assay in the presence of metrizamide

Quantitative determination of protein using the binding of Coomassie Brilliant Blue G-250 was investigated with respect to interference with the density gradient material metrizamide, and compared with the corresponding interference using the Lowry method. The background absorption obtained with metrizamide in the absence of protein was less than 10% of that obtained with the Lowry method. In the presence of 0–4% metrizamide, parallel standard curves were obtained with 0–67 μg of protein in the samples. The curves overlapped in the range 0–40 μg of protein when metrizamide was included in the blanks. With up to 2% final concentration of metrizamide in the assay, the curves overlapped at all protein concentrations tested (0–67 μg). Correction for metrizamide interference is thus a simple procedure and a precise estimation of the metrizamide concentration is less critical than when the Lowry assay is used. The method is well suited for quantitation of protein in samples collected from metrizamide grandients.     

Amino acid analysis: determination of cysteine plus half-cystine in proteins after hydrochloric acid hydrolysis with a disulfide compound as additive

All cysteine and cystine in a protein are derivatized during hydrolysis in hydrochloric acid containing 3,3'-dithiodipropionic acid. The resulting derivative can be separated from other amino acids and used for quantitation of cysteine plus half-cystine. A procedure is presented for accurate determination by ion exchange chromatography and postcolumn derivatization of all amino acids from acid hydrolysis of a protein, including the Cys-derivative.     

Simple and sensitive high-performance liquid chromatography method for the determination of docetaxel in human plasma or urine

Several methods for quantification of docetaxel have been described mainly using HPLC. We have developed a new isocratic HPLC method that is as sensitive and simpler than previous methods, and applicable to use in clinical pharmacokinetic analysis. Plasma samples are spiked with paclitaxel as internal standard and extracted manually on activated cyanopropyl end-capped solid-phase extraction columns followed by isocratic reversed-phase HPLC and UV detection at 227 nm. Using this system, the retention times for docetaxel and paclitaxel are 8.5 min and 10.5 min, respectively, with good resolution and without any interference from endogenous plasma constituents or docetaxel metabolites at these retention times. The total run time needed is only 13 min. The lower limit of quantification is 5 ng/ml using 1 ml of plasma. The validated quantitation range of the method is 5–1000 ng/ml with RSDs≤10%, but plasma concentrations up to 5000 ng/ml can be accurately measured using smaller aliquots. This method is also suitable for the determination of docetaxel in urine samples under the same conditions. The method has been used to assess the pharmacokinetics of docetaxel during a phase I/II study of docetaxel in combination with epirubicin and cyclophosphamide in patients with advanced cancer.     

Determination of (+)-catechin in plasma by high-performance liquid chromatography using fluorescence detection

A high-performance liquid chromatographic method, using fluorescence detection, was developed for the determination of (+)-catechin in rabbit plasma. The procedure involved the precipitation of plasma protein using acetonitrile, followed by solid-phase adsorption onto alumina. After washing with water and methanol, the residue was vortex-mixed with perchloric acid solution to release the adsorbed (+)-catechin. Separation was performed on a reversed-phase column using an eluent consisting of phosphoric acid solution with 12% acetonitrile. The excitation and emission wavelengths were set at 280 and 310 nm, respectively. The retention times for (+)-catechin and the internal standard (deoxyhigenamine) were 6.87 and 8.47 min respectively, without any interference. Validations of accuracy and precision were satisfactory in both within- and between-run assays. All coefficients of variance were less than 6% and mean relative errors were within ± 3.75%. The average recovery was 73.77%. The limit of detection and quantitation were 1 ng and 0.02 μg/ml, respectively. Application of this method was successfully assessed by intravenous administration of a 15 mg/kg dose of (+)-catechin in rabbits. This new method provides a simple, specific and sensitive determination for (+)-catechin in rabbit plasma and is suitable for pharmacokinetic studies.     

Protein complexation with acrylic polyampholytes

The interaction of dilute mixtures of proteins and ABC triblock methacrylic polyampholytes at different values of pH was investigated turbidimetrically. The onset of interaction was manifested by large changes in turbidity at certain critical pHs which lie close to the isoelectric points of the two interacting components. Protein precipitation yields in protein-polyampholyte binary mixtures followed the corresponding turbidity profiles and varied from 10% to 90%. The synthetic polyampholytes self-aggregate around their isoelectric point. The kinetics of precipitation of one of the same polymer with soybean trypsin inhibitor were studied, with turbidity-based characteristic times (exponential fit) of 2-3 min. The kinetics of precipitation of the protein-polymer mixture are slower than that of pure polymer because a small, but steady, long-term increase in turbidity is observed in the former case. The pH-dependence of the turbidity of binary mixtures of one protein and one synthetic polyampholyte, as well as a tertiary mixture of two proteins and one polyampholyte, were measured 30 min after the pH adjustment. The observations in these experiments along with the measured protein precipitation yields in the binary mixtures and the polyampholyte self-aggregation can be used for polymer removal and recycling. The latter constitutes a significant advantage over the use of homopolyelectrolytes which cannot easily be recycled. (c) 1994 John Wiley & Sons, Inc.     

Measurement of picomoles of phosphoamino acids by high-performance liquid chromatography.

A reverse-phase, high-performance liquid chromatographic system (HPLC) is described that makes possible optimal resolution and quantitation of picomole levels of phosphoamino acids, both with or without the presence of a large excess of nonphosphorylated amino acids. The assay involves precolumn derivatization of an amino acid mixture with phenyl isothiocyanate (PITC) at room temperature, followed by separation of phosphoamino acids from other amino acids by HPLC. The liquid chromatography was carried out on a C18 reverse-phase column at pH 7.4 and 30 degrees C using gradient elution with eluent A as 157 mM sodium acetate containing 2% acetonitrile and eluent B as 60% acetonitrile in water. A uv absorption at 254 nm is employed for detection of the PITC-derivatized amino acids eluting from the column. Amino acids are eluted with baseline resolution in the following order: phosphoserine, phosphothreonine, aspartic acid, glutamic acid, and phosphotyrosine followed by other amino acids. The sensitivity is in the picomole range, and the separation time, injection to injection, is 36 min. Phosphoserine, phosphothreonine, and phosphotyrosine are resolved within the first 8 min. This procedure enables determination of as low as 5 pmol of nonradioactive phosphoamino acids in a 100-fold excess of amino acids, as is usually present in most phosphoproteins in the natural state. Phosphoamino acids in polypeptides separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane, or protein samples directly blotted on the membrane, can also be analyzed by this procedure after acid hydrolysis of the proteins bound to the PVDF membrane.     

Protein measurement using bicinchoninic acid: elimination of interfering substances

Protein quantitation based on bicinchoninic acid (BCA) is simple, sensitive, and tolerant to many detergents and substances known to interfere with the Lowry method. However, certain compounds often used during protein purification do interfere with the BCA protein assay. The response of the BCA chromophore to various interfering substances has provided insight into the mechanism of protein quantitation by BCA. Certain substances (e.g., glucose, mercaptoethanol, and dithiothreitol) elicit a strong absorbance at 562 nm when combined with the BCA working reagent. The absorbance appears to be identical to the normal response elicited by protein. Other agents (e.g., ammonium sulfate and certain ampholytes) diminish the protein-induced color development and shift the wave-length of the color response. Both types of interference can be eliminated by selectively precipitating protein with deoxycholate and trichloroacetic acid (A. Bensadoun and D. Weinstein (1976) Anal. Biochem. 70,241-250) prior to reaction with bicinchoninic acid. The modifications described here permit quick, efficient removal of many interfering substances that are commonly utilized during protein purification.     

Single quadrupole mass spectrometry for pre-clinical pharmacokinetic analysis: quantitation of carvedilol in dog plasma

The pharmaceutical industry standard for bioanalysis is LC/MS/MS. There are, however, many instances where a single quadrupole detector could successfully be used to provide adequate sensitivity and selectivity for quantitation of drug substances in biological matrices. This paper presents one example of how a single quadrupole detector can be employed in a sensitive and selective analytical method for quantitation of carvedilol. A Synergi Hydro-RP (50 mm x 2 mm i.d.; 4 microm) column was used with acetonitile:water:formic acid mobile phase (32:68:0.01, v/v) at a flow rate of 200 microL/min into a single quadrupole mass spectrometer with an electrospray interface in the positive SIM mode. Using a 300 microL plasma aliquot and a liquid-liquid extraction procedure the limit of quantitation for the assay was 1 ng/mL. The assay utility was demonstrated in the analysis of carvedilol pharmacokinetic profiles in beagle dogs following oral carvedilol administration.     

Quantitation of proteins bound to polyvinylidene difluoride membranes by elution of coomassie brilliant blue R-250

A rapid and simple method for the quantitation of stained proteins bound to polyvinylidene difluoride (PVDF) membranes via the elution of Coomassie brilliant blue R-250 is described. A mixture of standard proteins was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto PVDF membranes. Spectrophotometric analysis of dye eluted from protein bands in the range of 0.5-10 micrograms gave a linear change in the absorbance at 595 nm. Maximal absorbance readings were attained following 5 min of dye elution, and the readings remained unchanged for elution times up to 60 min. The method requires no unusual reagents or equipment, is suitable for the analysis of multiple samples, and does not consume the protein in the process of quantitation. This technique provides a useful means for the quantitation of proteins bound to PVDF membranes prior to amino acid sequence determination, immunological analysis, or other biochemical characterizations.     

High-performance liquid chromatographic determination of fluconazole in plasma

A high-performance liquid chromatographic method with ultraviolet absorbance detection at 260 nm was developed for the analysis of fluconazole in plasma. The method involves sample clean-up by liquid-liquid extraction. The proposed technique is reproducible, selective, reliable and sensitive. Calibration standards were prepared in the range 1.25-20 mg/l. The limit of quantitation was 0.4 mg/l. The coefficients of variation were 5% between measurements of a single extract injected in duplicate, and 7% between two extractions of spiked samples at the same concentrations. The separation between fluconazole and endogenous substances was satisfactory. This method was designed in order to minimise the risk of interference from substances that could be co-administered to critically ill patients undergoing hemodiafiltration. With a run time below 5 min, the present method is rapid and easy to use for later clinical studies, as well as for routine monitoring.