A microassay for proteases using succinylcasein as a substrate.

A photometric assay for proteases has been developed. A chemically modified casein whose amino groups were succinylated was used as a substrate. After incubation with trypsin, chymotrypsin, thermolysin, and subtilisin, the extent of hydrolysis of the substrate was determined with trinitrobenzene sulfonate (TNBS). The whole procedure of the assay was performed in the microtiter plate wells and the increase in the absorbance resulting from the reaction between TNBS and newly formed amino groups in the substrate was able to be determined with a high sensitivity by a microtiter plate reader, enabling the simultaneous measurement of a number of samples. Application of this method to the measurement of proteolytic activity contained in the protein extract of Tapes philippinarum is demonstrated.     

Fluorometric quantitation of amino sugars in the picomole range.

A fluorometric method has been developed for the convenient and quantitative assay of amino sugars over the concentration range of 10 nm to 6 mm. Linear results are obtained for reaction mixtures containing 6 pmol to 60 nmol hexosamine. The procedure involves the condensation of amino sugars with the fluorogenic reagent o-phthalaldehyde, at alkaline pH in the presence of 2-mercaptoethanol. Relative fluorescence intensities are then determined using excitation and emission wavelengths of 340 and 455 nm, respectively. The presence of 2-mercaptoethanol in reaction mixtures not only enhanced sensitivity of the assay, but also defined the excitation/emission spectra. Under the conditions described, amino acids were also found to react with o-phthalaldehyde, yielding fluorescence intensities similar to those of amino sugars. These results suggest the applicability of fluorescence techniques in automated amino sugar analyses, as well as the potential interference of other compounds containing primary amines.     

A colorimetric assay for a pyridoxal phosphate-dependent beta-replacement reaction with L-cysteine: application to studies of wild-type and mutant tryptophan synthase alpha 2 beta 2 complexes

We present an improved and simple direct assay for formation of inorganic sulfide from L-cysteine in a beta-replacement reaction catalyzed by tryptophan synthase. This method provides a useful enzymatic assay for pyridoxal phosphate-dependent beta-replacement reactions in which the amino acid substrate is L-cysteine and the cosubstrate is 2-mercaptoethanol. The assay should be applicable to similar reactions with L-cysteine and other cosubstrates. The method has several advantages over other methods which have been used to assay similar beta-replacement reactions. The assay is highly reproducible and sensitive and is conveniently carried out in disposable 1.5-ml centrifuge tubes. The color remains stable for several hours. The thiol compounds L-cysteine and 2-mercaptoethanol do not interfere at the concentrations used. The method has useful applications to studies of the rates and reaction specificities of several other pyridoxal phosphate enzymes which catalyze beta-replacement reactions. We demonstrate the use of the method to study the effects of site-directed mutagenesis on the reaction specificity and mechanism of the tryptophan synthase alpha 2 beta 2 complex.     

Use of water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for the fluorescent determination of uronic acids and carboxylic acids

Reaction between glucuronic acid and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was monitored by the o-phthalaldehyde (OPA) method, which was developed for the fluorescent assay of compounds containing an amino group. About 1 nmol of glucuronic acid was detected by this method. This EDC-OPA method was effective in detecting not only acidic sugar but also carboxylic acid. Although the sensitivity of the EDC-OPA method was somewhat lower than that of amino acid determination by OPA, a very simple and convenient assay was attained for compounds containing a carboxyl group.     

邻苯二甲醛(OPA)法检测铝吸附疫苗中抗原含量

目的:建立快速测定铝胶吸附的疫苗中抗原含量的OPA荧光检测法。 方法:利用邻苯二甲醛(OPA)在2-巯基乙醇存在下与氨基酸的N端或L-谷氨酸侧链反应,在460nm处生成荧光衍生物的原理,建立了无需进行抗原蛋白提取的检测方法,并对该方法的特异性、线性、精密度、回收率、重复性进行考察。同时,配合钠十二烷基的硫酸盐-聚丙烯酰胺凝胶电泳法(SDS-PAGE),研究在配制过程中抗原蛋白与佐剂是否分离。结果: OPA荧光法,在抗原蛋白含量为0.02~0.16mg/ml时,线性良好,准确度达90%~115%,批内和批间相对标准偏差≤15%。电泳结果显示,CpG的加入可能会导致少量抗原蛋白从铝胶中解离。结论:该方法快速、准确、灵敏度较高,重复性好,可以应用于含铝胶疫苗制剂的实验室甚至生产质控过程。     

Theoretical possibilities and limitations of protease primary specificity determination by statistical analysis of MALDI mass-spectra of proteolysis products

Possibilities and limitations of method examination of proteolytic enzymes' primary specificity by statistical analysis of MALDI (matrix-assisted laser desorption/ionization) mass spectra of products obtained by protein substrates proteolysis without direct determination of their amino acid sequences were investigated theoretically. The optimum ranges given by the errors of the peptides masses measuring for the fabrication of statistical set of the events and the form of statistical data presentation were chosen. It was shown that the proposed method can be applied only for proteases with a relatively narrow primary specificity (two or three amino acids). The influence of protein substrate molecular weight and amino acid composition on the efficiency of specific to a particular protease amino acids display under statistical treatment of the set of proteolysis products masses was studied on the model of trypsin, chymotrypsin, glutamylendopeptidase, pepsin (pH 1.3).     

Theoretical possibilities and limitations of protease primary specificity determination by statistical analysis of MALDI mass-spectra of proteolysis products

Possibilities and limitations of the method of examination of proteolytic enzymes’ primary specificity by statistical analysis of MALDI (matrix-assisted laser desorption/ionization) mass spectra of products obtained by protein substrate proteolysis, without direct determination of their amino acid sequences, were investigated theoretically. The optimum range given by the measurement errors of the peptides’ masses for obtaining a statistical set of the events, and the form of statistical data presentation, were chosen. It was shown that the proposed method can be applied only for proteases with a relatively narrow primary specificity (two or three amino acids). The influence of protein substrate molecular weight and amino acid composition on the efficiency of specifics for a particular protease amino acid, revealed under statistical treatment of the set of proteolysis product masses, was studied on the model of trypsin, chymotrypsin, glutamylendopeptidase, pepsin (pH 1.3).     

Liquid chromatography method for detecting native fluorescent bioamines in urine using post-column derivatization and intramolecular FRET detection

Liquid chromatography (LC) with fluorescence detection is described for simultaneous determination of native fluorescent bioamines (indoleamines and catecholamines). This is based on intramolecular fluorescence resonance energy transfer (FRET) in an LC system following post-column derivatization of native fluorescent bioamines' amino groups with o-phthalaldehyde (OPA). OPA fluorescence was achieved through an intramolecular FRET process when the molecules were excited at maximum excitation wavelength of the native fluorescent bioamines. Bioamines separated by reversed-phase LC on ODS column were derivatized with OPA and 2-mercaptoethanol. This method provides sufficient selectivity and sensitivity for the determination of normetanephrine, dopamine, tyrosine, 5-hydroxytryptamine, tryptamine, and tryptophan in healthy human urine without prior sample purification.     

Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis.

A rapid and convenient method for peptide mapping of proteins has been developed. The technique, which is especially suitable for analysis of proteins that have been isolated from gels containg sodium dodecyl sulfate, involves partial enzymatic proteolysis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by polyacrylamide gel electrophoresis. The pattern of peptide fragments produced is characteristic of the protein substrate and the proteolytic enzyme and is highly reproducible. Several common proteases have been used including chymotrypsin, Staphylococcus aureus protease, and papain.     

A rapid phospholipase D assay using zirconium precipitation of anionic substrate phospholipids: application to n-acylethanolamine formation in vitro

Activation of phospholipase D (PLD) is involved in a number of signal transduction pathways in eukaryotic cells. The most common method for determination of PLD activity in vitro involves incubation with a radiolabeled substrate and lipid extraction followed by thin-layer chromatography in order to separate and quantify substrate and product(s). A more rapid assay can be used when utilizing phosphatidylcholine as a substrate because one of the products, choline, is water soluble and therefore easily separated from the substrate. However, this separation principle is not applicable in evaluating N-acylphosphatidylethanolamine (NAPE)-hydrolyzing PLD activity, which produces two lipophilic products, N-acylethanolamine (NAE) and phosphatidic acid. Therefore, we developed a rapid assay for the routine detection of NAPE-hydrolyzing PLD activity. This assay is based on precipitation of radiolabeled substrate (NAPE) in the presence of ZrOCl(2), followed by quantification of radiolabeled NAE released into a methanolic supernatant. The precipitation involves a chemical reaction of the zirconyl cation with the phosphate anion. Conditions were optimized for the complete precipitation of NAPE, whereas N-acyllysophosphatidylethanolamine and glycerophospho(N-acyl)ethanolamine were precipitated at least 95%. Furthermore, this precipitation method can be extended to assays of other anionic phospholipid-hydrolyzing PLD activities by selecting an optimal pH of the precipitation solution. For example, 98;-99% precipitation of phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylserine was achieved.Consequently, this new assay allows for a convenient examination of PLD activities toward a variety of phospholipid substrates, and in particular allows for the analysis of NAE formation from NAPE in vitro, a feature that will facilitate a more complete biochemical characterization of this anandamide-generating enzyme.     

Purification of B-50 by 2-mercaptoethanol extraction from rat brain synaptosomal plasma membranes

Several methods have been described previously for the purification of the nervous-tissue specific protein kinase C substrate B-50 (GAP-43). In this paper we present a new purification method for B-50 from rat brain which employs 2-mercaptoethanol to release the protein from isolated synaptosomal plasma membranes. Most likely, 2-mercaptoethanol reduces disulfide bonds involved in the linkage of B-50 to the membrane. After washing the membranes with 100 mM NaCl to detach loosely bound proteins, B-50 is the major protein (and the only protein kinase C substrate) released by 0.5% 2-mercaptoethanol treatment. Further purification to apparent homogeneity is achieved by affinity chromatography on calmodulin sepharose. B-50 binds to calmodulin in the absence of calcium and specifically elutes from the column with 3 mM calcium. The procedures described is simple, rapid and highly suitable for large scale purification of B-50 from rat brain.     

An assay for glycosyltransferases using phenel partition and gas-liquid chromatography

A method applicable to the assay of glycosyltransferases is described, Features include stopping the reaction using phenol, which results in the partition of the protein into the phenol phase and of the carbohydrates into the aqueous phase. Substrates and products are then assayed using gas-liquid chromatography. The method is shown to be convenient and rapid by application to the assay of invertase and fructosyltransferase.     

A convenient manual trinitrobenzenesulfonic acid method for monitoring amino acids and peptides in chromatographic column effluents.

The trinitrobenzenesulfonic acid (TNBS) method of R. Fields (1971, Biochem. J., 124, 581–590) has been modified for the manual detection of amino acids and peptides in chromatographic column effluent by changing the reaction conditions to 1 mm TNBS in 0.4 m potassium borate buffer, pH 9.2, at room temperature for 30 to 50 min. The reaction with amines and the spontaneous hydrolysis of TNBS are stopped by neutralization to pH 6.25 with sodium monobasic phosphate (0.33 m). Sodium sulfite (3 mm) is added to increase the absorptivity of the product. The TNBS reagent blank is less than 0.100 A₄₂₀ after 50 min of reaction. Since the ΔA₄₂₀ of the reagent blank is ～0.002/min before quenching the reaction, and zero afterward, the time required for reaction and for absorbance measurements need not be controlled precisely. Alkaline hydrolysis of peptides is carried out prior to detection to increase the sensitivity of the method. This procedure is convenient for the manual determination of 5 to 100 nmol of amino acids in the 50–100 samples required to define a chromatographic elution profile.     

Studies on the mechanisms of the antibacterial action of ortho-phthalaldehyde

The reaction of ortho-phthalaldehyde (OPA) with amino acids and proteins was investigated as a possible mode of action. Bacterial pellets (obtained by centrifugation) changed colour after exposure to OPA. These colours were more intense at alkaline than acidic pH. Acidic and alkaline OPA reacted with primary amino acids to form coloured products. The reaction rate accelerated with increasing pH. OPA increased the optical density of bacterial cell suspensions (an indication of protein coagulation or microbial surface or other changes in the opacity of cell constituents). The inhibition of ethylenediaminetetraacetic acid- and sodium lauryl sulphate-induced lysis was not as great as for glutaraldehyde (GTA), possibly indicating less cross-linking of amines. Interactions with primary amino groups of the outer envelope or cell wall probably play a part in the action of OPA but the level of cross-linking associated with the outer membrane does not appear to be as extensive as that of GTA. The aromatic component might allow OPA to penetrate the outer layers of cells, thus helping to explain the very high activity of OPA against Gram-negative vegetative organisms even though the degree of cross-linking seems to be less than that seen with GTA. Thus, OPA reacts strongly with primary amines and stabilizes, to some extent, the outer membrane and cell walls of vegetative organisms and this probably accounts for part, but not necessarily all, of its lethal action.     

A fast and sensitive method for measuring picomole levels of total free amino acids in very small amounts of biological tissues

Summary. In the present study we describe a simple and fast method to measure the concentration of total free amino acids in very small amounts of biological tissues. The procedure described here is based on the reaction of free amino acids with o-phthaldialdehyde (OPA) in the presence of a reducing agent, β-mercaptoethanol (MET), to give a complex which can be measured by fluorescence. It is a very rapid process and has the same reliability as the conventional ninhydrin method of Moore and Stein but is about 500 times more sensitive. The sensitivity of the new protocol is such to permit the determination with high reliability of very small amounts of free amino acids at picomole levels, either in a standard amino acid mixture or in biological tissues, without chromatographic separation of the amino acids. It is particularly useful when the amount of the sample is very low, e.g. on a single pituitary or pineal gland of small animals or on single cells, such as oocytes or eggs, as well as single ganglions or axons of marine invertebrates. Received September 22, 1999 Accepted July 5, 2000     

A continuous spectrophotometric assay for mitogen-activated protein kinase kinases

We describe a convenient and simple continuous spectrophotometric method for the determination of mitogen-activated protein kinase (MAPK) kinase activity with its protein substrate. The assay relies on the measurement of phosphoprotein product generated in the first step of the MAPK kinase reaction. Dephosphorylation of the phosphoprotein is coupled to a MAPK phosphatase to generate phosphate, which is then used as the substrate of purine nucleoside phosphorylase to catalyze the N-glycosidic cleavage of 2-amino 6-mercapto 7-methyl purine ribonucleoside. Of the reaction products ribose 1-phosphate and 2-amino 6-mercapto 7-methylpurine, the latter has a high absorbance at 360nm relative to the nucleoside and, hence, provides a spectrophotometric signal that can be continuously followed. In the presence of excess phosphatase, the phosphorylated protein substrate molecules undergo dephosphorylation almost immediately after their formation; the steady-state use of the resultant inorganic phosphate is a reflection of the constant initial velocity of the exchange reaction. The validity of this method has been confirmed by using it to measure the activities of MEK1 (MAPK/ERK kinase 1) and MKK6 (MAPK kinase 6) toward their physiological substrates. Our findings of the MAPK kinases in the current study provide evidence that the substrate binding affinities of this subfamily of protein kinases are at the submicromolar concentration.     

Assay for aliphatic amino acid decarboxylases by high-performance liquid chromatography

A sensitive and rapid assay for aliphatic amino acid decarboxylases based on separation of the product from the substrate by ion-pairing reversed-phase high-performance liquid chromatography and subsequent fluorometric detection has been developed. The resolution of substrates and products of seven amino acid decarboxylases, namely, arginine, aspartate, 2,6-diaminopimelate, histidine, glutamate, lysine, and ornithine decarboxylase, is complete within 15 to 35 min of isocratic elution. The limit of detection for the product is 40 pmol. The applicability of the procedure was assessed with glutamate decarboxylase. The formation of the product 4-aminobutyrate proved to be linear with time and protein concentration. The method allows the time course of the reaction to be followed in a single assay and works well with crude extracts of bacteria or tissues.     

A microplate fluorescence assay for DAPA aminotransferase by detection of the vicinal diamine 7,8-diaminopelargonic acid

7,8-Diaminopelargonic acid (DAPA) aminotransferase is an enzyme of the biotin biosynthetic pathway that plays an essential role in Mycobacterium tuberculosis virulence. Inhibition of this enzyme is a potential strategy to combat this microorganism, the causative agent of tuberculosis. To identify new inhibitors as potential drugs, a simple enzymatic assay for high-throughput screening (HTS) is needed. Several methods for measuring DAPA aminotransferase activity are already available. However, requirements for their implementation for HTS are tedious. We describe here a microplate fluorescence assay for DAPA aminotransferase that is simple, cheap, and sensitive, allowing linear detection of DAPA in the range of 20 nM to 50 μM. The principle of the method is the direct detection in the enzymatic reaction mixture of the vicinal diamine DAPA derivatized with ortho-phthalaldehyde (OPA) and 2-mercaptoethanol (2ME). The assay was validated with the known inhibitor desmethyl-KAPA (8-amino-7-oxopelargonic acid) and adapted to microplate for HTS. The structure of the stable fluorescent adduct formed between a vicinal primary diamine and OPA in the presence of 2ME was characterized by mass spectrometry and nuclear magnetic resonance spectroscopy.     

Assay of tryptophan hydroxylase and aromatic L-amino acid decarboxylase based on rapid separation of the reaction product by high performance liquid chromatography

A rapid separation of 5-hydroxytryptophan by high performance liquid chromatography (HPLC) was achieved for the assay of tryptophan hydroxylase. "Bulk separation" of the product from all other components in the reaction mixture by HPLC was achieved by 1) the choice of a suitable column-solvent system so as to elute the reaction product ahead of other components in the sample mixture, 2) the use of a monitor selective for the reaction product, 3) minimization of the column length so as to achieve rapid separation of the product from the substrate. The method finally employed a reversed phase column of 5 cm length, relatively rapid elution at 2 ml/min and fluorescence detection at 350 nm with an excitation at 302 nm. The assay is convenient and as sensitive as the radioisotope method. The advantages of the method are 1) almost no pretreatment of samples, 2) repeatability every 2 min, 3) wide latitude of product determination from picomole to nanomole amounts per assay. The method was extended to the assay of 5-hydroxytryptophan decarboxylase by essentially the same procedures.     

An improved 2,4,6-trinitrobenzenesulfonic acid method for the determination of amines.

The use of 2,4,6-trinitrobenzenesulfonic acid (TNBS) as a reagent for determining the concentrations of amines has been widely accepted (1–3) since its introduction in 1960 by Satakeet al. (4). The original procedure has since been modified by Mokrasch (5) to permit the determination of amines, amino acids, and proteins in mixtures. In both procedures the trinitrophenylation reaction is followed by a quenching step, after which the amino content is related to the increase in absorbance at 340 nm (4) or 420 nm (5). We have studied the trinitrophenylation reaction and have found that amino content can be related directly to the absorbance of the trinitrophenylation reaction mixture after a relatively short incubation period (15–30 min). Therefore, it is unnecessary to quench this reaction. We describe herein an extremely convenient procedure for the determination of amines, amino acids, and proteins where the quenching step employed by previous investigators has been eliminated. The proposed method has a greater sensitivity than previously described techniques employing TNBS.