Glutathione localization by a novel o-phthalaldehyde histofluorescence method

Summary Glutathione in tissues forms an intense fluorophore with a solution ofo-phthalaldehyde at room temperature. We have studied the loss of glutathione from tissue sections and find that it is not measurable from thick sections. The fluorescence spectra of the induced fluorophore between glutathione ando-phthalaldehyde are identical in model and tissue sections, while depletion of hepatic glutathione by diethyl maleate produces a comparable fall in fluorescence measured biochemically or histochemically. This simple method is specific as interfering substances, such as spermine and spermidine, produce very weak fluorescence under the conditions employed.     

A simple and sensitive method of analysis for histamine,putrescine, and polyamines without the use of an amino acid analyzer

Histamine, putrescine, spermidine, and spermine in rat tissues and human urine were separated on a CM-cellulose column (0.6 × 10 cm). These amines in the chromatographic eluate were determined by the reactions with o-phthalaldehyde (for histamine), fluorescamine, o-phthalaldehyde-mercaptoethanol, or 2,4,6-trinitrobenzene sulfonate (for putrescine and polyamines). The procedures are rapid and simple when popular instruments are used. The limits of determination by the present method were of the order of 0.1 to 0.2 nmol for histamine and 2 to 4 nmol for putrescine and polyamines.     

S1 nuclease: Immunoaffinity purification and evidence for the proximity of cysteine 25 to the substrate binding site

A simple procedure, involving heat treatment, gel filtration on Sephadex G-100 followed by chromatography on anti-S1 nuclease antibodies bound to Sepharose, was developed for purification of S1 nuclease to homogeneity with an overall yield of 72%. S1 nuclease was rapidly inactivated, at pH 6.0 and 37°C, in presence of o-phthalaldehyde. Kinetic analysis of o-phthalaldehyde mediated inactivation showed that the reaction followed pseudo-first-order kinetics and the loss of enzyme activity was due to the formation of a single isoindole derivative per molecule of the enzyme. Absorbance and fluorescence spectrophotometric data also gave similar results. The isoindole derivative formation, as a result of o-phthalaldehyde treatment is known to occur through crosslinking of the thiol group of cysteine and the ε-amino group of lysine, situated in close proximity in the native enzyme. Since, modification of only available cysteine residue (Cys 25) did not affect the catalytic activity of the enzyme, the o-phthalaldehyde mediated inactivation of S1 nuclease is due to the modification of lysine. Substrates of S1 nuclease, namely ssDNA, RNA and 3′ AMP, could protect the enzyme against o-phthalaldehyde mediated inactivation. Moreover, the modified enzyme (having very little catalytic activity) showed a significant decrease in its ability to bind 5′ AMP, a competitive inhibitor of S1 nuclease, suggesting that the modification has occurred at the substrate binding site. The above results point towards the presence of cysteine 25 in close proximity to the substrate binding site.     

Fluorometric determination of spermidine using o-phthalaldehyde: optimum reaction conditions and tests of identity

Spermidine and histamine react with o-phthalaldehyde at alkaline pH, giving rise to fluorescent conjugation products that are stabilized by acidification to pH 2–4. The reaction has been used in the fluorometric assay of both these compounds. In the present study, the reaction conditions have been analyzed with the purpose of improving the sensitivity as well as the specificity of the spermidine assay. The sensitivity was improved by carrying out the condensation at pH 11.0–11.6 for 2 min at 100°C. Under these conditions, the lowest measurable amount of spermidine was 10 ng/ml, and the fluorescence of histamine (below 1–2 μg/ml) was nonmeasurable. Boiling the samples for 1 hr after the acidification did not affect the spermidine fluorescence but abolished residual histamine fluorescence. The spermidine fluorescence failed to develop in the presence of CdCl₂ or SrCl₂, whereas the histamine fluorescence was unaffected. Crude extracts of rat brain gave fluorescence readings similar to those of butanol extracts, suggesting that extensive purification of tissue sperimidine prior to assay is not always necessary.     

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.     

Determination of serum protein concentration in nanoliter blood samples using fluorescamine or 9-phthalaldehyde.

Fluorometric procedures were developed to permit measurement of total protein concentration in nanoliter serum samples, using either fluorescamine or o-phthalaldehyde. The sensitivities of assays using these two reagents were similar, but the o-phthalaldehyde method was found to be somewhat simpler and more reproducible. Accurate measurements could be obtained on serum samples of 4 to 5 nl with either reagent, by using a serum standard. Fluorescence differed considerably among individual serum proteins, albumin generally showing greater fluorescence than globulins. Small molecular weight species in serum did not contribute appreciably to total serum fluorescence with either reagent.     

Polyamine cytochemistry: Comparisons between cytochemical, autoradiographic, immunocytochemical and chemical results in the prostate

Summary Results obtained with two newly developed fluorescence cytochemical methods for detecting the polyamines spermidine and spermine have been compared to autoradiographic localization of biosynthetically labelled polyamines, to immunocytochemical results obtained with antibodies directed against spermidine and spermine, and to chemical polyamine determinations using the rat prostate as a model tissue. Complete agreement between all five methods was obtained. Application of perchloric acid to formaldehyde-fixed sections of rat prostate strongly reduced theo-phthalaldehyde inducible and formaldehyde-fluorescamine inducible fluorescence characteristic of spermidine and spermine. Perchloric acid extracted 40% of tissue-bound polyamines from formaldehyde-fixed tissue sections, and molecules with the physicochemical characteristics of polyamines constituted 80–90% of all fluorescamine reactive molecules extracted. Our results therefore confirm the specificity of theo-phthalaldehyde and formaldehyde-fluorescamine methods for polyamine cytochemistry. As polyamines are strongly implicated in cellular growth regulation and cancer, simple and inexpensive techniques for polyamine histochemistry may be useful for interpreting the biological and pathophysiological roles of these molecules.     

High-performance liquid chromatographic procedure for the simultaneous determination of the natural polyamines and their monoacetyl derivatives

The separation of the natural polyamines and their monoacetyl derivatives by high-performance reversed-phase liquid chromatography is reported. Octane sulfonate was used to form ion pairs with the polycations and the o-phthalaldehyde method for post-column derivatization. The method allows polyamine and acetylspermidine determinations directly from tissue extracts and body fluids without pre-purification.     

A simple quantitative method for the determination of 3-fluorotyrosine substitution in proteins

A rapid quantitative method is described for determining 3-fluorotyrosine incorporation into proteins. Derivatives of tyrosine and 3-fluorotyrosine with o-phthalaldehyde are well separated from one another by a reverse-phase high-performance liquid chromatography system used for routine analyses of o-phthalaldehyde-amino acid derivatives. Since both amino acids are well resolved from all other derivatized amino acids, the method is useful for amino acid analyses of proteins. Determination of the fluorotyrosine content of proteins by this method involves a single separation step, is reproducible, and requires no corrections for stability or yield. Further, the o-phthalaldehyde derivatives of 5-fluorotryptophan, 2-fluorophenylalanine, 3-fluorophenylalanine, and 4-fluorophenylalanine can also be resolved. The method may be generally applicable to fluorinated aromatic amino acid-labeled proteins that are studied structurally and dynamically by nuclear magnetic resonance.     

Automated fluorometric amino acid analysis: the determination of proline and hydroxyproline.

A fluorometric method for the automated determination of the imino acids proline and hydroxyproline has been developed. The assay is based on the postcolumn reaction of the imino acids with alkaline sodium hypochlorite, which yields oxidation products amenable to detection with fluorogenic amine reagents. The method is simple and can be adapted readily to high-sensitivity amino acid analyzers which use o-phthalaldehyde for detection. As little as 10 pmol proline and 20 pmol hydroxyproline can be determined accurately. Thus the full array of natural imino and amino acids can now be determined on a high-sensitivity amino acid analyzer using o-phthalaldehyde.     

Improved method for the measurement of large neutral amino acids in biological matrices

A high-performance liquid chromatographic method for measuring neutral amino acids in rat sera, brain tissues, and perfusates was developed by using o-phthalaldehyde sulfite as a pre-column derivatization reagent. With the present method, it was possible to separate the neutral amino acids within a single run in 25 min, while the acidic amino acids were eluted near or at the solvent front. The recovery was above 88.8% with a relative standard deviation (RSD) below 4.2%. The within- and between-day assay reproducibility for the determination of rat serum amino acids showed RSDs below 1.35 and 7.61%, respectively. In the present study, the neutral amino acids were assayed with high sensitivity, accuracy and good reproducibility in a relatively short time and on a small sample size.     

High-performance liquid chromatographic analysis of biogenic amines in biological materials as o-phthalaldehyde derivatives

A remarkably sensitive, simple and selective reversed-phase high-performance liquid chromatographic (HPLC) method has been developed, allowing, for the first time, the direct measurement of histamine, norepinephrine, octopamine, normetanephrine, dopamine, serotonin and tyramine in a single sample of plasma (2 ml), tissue (0.2 g), or urine. The biogenic amines were modified by pre-column derivatization with o-phthalaldehyde which stabilizes the molecules, aids in extraction, and improves HPLC detection at the nanogram level. To minimize losses during the sampling procedure a careful collection procedure was designed.     

An automated assay for brain serotonin

This communication describes an automated assay for brain serotonin. The assay is an adaptation of a commonly used manual assay which utilizes the reaction of o-phthalaldehyde with serotonin to develop fluorescence (2). In the automated assay the samples to be analyzed were mixed with 7.5 N HCl and o-phthalaldehyde in the presence of reduced glutathione, heated at 80°C, cooled and their fluorescence recorded. Linearity with respect to serotonin was demonstrated from 0 to at least 5 μM, and the lower limit of sensitivity was 0.7 ng serotonin (0.4 ml of 0.01 μM). The % error (standard deviation times 100 divided by the mean) was always 2% or less. The specificity was studied, and the assay was applied to the measurement of rat brain serotonin levels by demonstrating an increase in brain serotonin levels after pargyline treatment and a decrease after reserpine treatment.     

Chromatographic analysis of amino acids and primary amines with o-phthalaldehyde detection

The applicability of the o-phthalaldehyde reaction with fluorometric detection to the simultaneous chromatographic analysis of amines and nonprotein amino acids is demonstrated. The majority of the compounds tested could be quantitatively determined with high sensitivity. The method is particularly well suited for the analysis of β-amino acids. Amino compounds lacking an α-hydrogen atom are detected with lower sensitivity, due to a lower relative fluorescence. Secondary amino compounds are undetectable with this system.     

Liquid Chromatographic-Fluorescence Determination of Ammonia from Nitrogenase Reactions: A 2-Min Assay

The analytical potential of the reaction of ammonia with o-phthalaldehyde mercaptoethanol reagent at pH 7 (an atypical fluorescence) has already been demonstrated. This, coupled with additional findings reported here, has led to an ammonia determination well suited to nitrogenase studies. As a result, large numbers of samples can be rapidly analyzed by high-pressure liquid chromatrography methods under mild conditions and without prior microdiffusion. Neither sodium dithionite (or other components of the usual nitrogenase assay), nor alternative substrates (cyanide, azide, methyl isonitrile), nor their products (methylamine, dimethylamine, hydrazine) interfere. High-pressure liquid chromatography showed that the fluorescent “product” of the o-phthalaldehyde mercaptoethanol reagent-ammonia reaction was, in fact, more than just a single compound. Despite this, once the proper solvent composition was found, high-pressure liquid chromatography with a small inexpensive C₁₈ “guard” column proved quite fast and reproducible for this measurement. Fluorescence response to ammonia was linear to at least 40 nmol/ml. A previous problem, long-term stability of the fluorescence, was solved by running the reactions in the dark. Background ammonia in the buffer could be substantially reduced by an analogous o-phthalaldehyde mercaptoethanol reagent reaction, using t-butyl mercaptan, and solvent extraction.     

Quantitative separation of 4-hydroxyproline from skeletal muscle collagen by micellar electrokinetic capillary electrophoresis

The phenylthiohydantoin (PTH) derivatives of 3- and 4-hydroxyproline (Hyp) were separated using micellar electrokinetic capillary electrophoresis (MEKC). The separation protocol was also used to determine Hyp content of bovine skeletal perimysial collagen preparations and whole muscle samples. Amino acids from hydrolyzed tissues were labeled using a two step procedure that involved initial reaction with o-phthalaldehyde (OPA) to modify primary amines followed by their precipitation under acidic conditions. In the second step, imino acids were reacted with phenyl isothiocyanate (PITC). This labeling method was rapid and the Hyp values determined in these biological samples were found to be in close agreement with conventional methods and other published reports.     

Determination of agmatine in brain and plasma using high-performance liquid chromatography with fluorescence detection

Decarboxylated arginine, agmatine, is a neurotransmitter candidate for imidazoline receptors. A method is described to measure agmatine in rat brain and human plasma by isocratic high-performance liquid chromatography (HPLC) with flourescence detection and o-phthalaldehyde derivatization. Quantitation is based on the method of additions of internal agmatine spikes. This assay has sensitivity in the low picomole range and a detection limitof 100 fmol. The correlation coefficient for the agmatine standard curve was 0.999±0.001 S.D., and intra- and inter-assay C.V.s were less than 8%. The accuracy of our isocratic method compared favorably with a gradient HPLC protocol, originally developed for bacterial agmatine, which we modified for use with tissues. Agmatine concentrations in rat brain were proportioned similarly to the regional distribution of imidazoline-1 receptors. These methods can be used as reliable research tools in various biological samples.     

The use of the o-phthalaldehyde reaction as a sensitive assay for protein and to determine protein in bacterial cells and dental plaque

Alkaline hydrolysis of protein followed by reaction with o-phthalaldehyde has permitted the determination of less than 10 ng of protein fluorometrically. When intact proteins were analyzed with o-phthalaldehyde the reaction was less sensitive. This reaction has been applied to analysis of the protein content of dental plaque and bacterial cells.     

A manual subtractive end-group determination of oligopeptides using fluorescamine or o-phthalaldehyde

A new method for the end-group determination of peptides using the fluorogenic reagents fluorescamine or o-phthalaldehyde is described. The method is based on the property that the derivatives of the N-terminal amino group of peptides formed in solution after reaction with either reagent are resistant to acid hydrolysis. The N-terminal amino acid can be determined by simply comparing the amino acid analysis of the original peptide with the fluorescent derivative of the peptide. In general, the decrease of the N-terminal residue in the reacted peptides in 80–90% with fluorescamine and more than 90% with o-phthalaldehyde. Any N-terminal amino acid, with the exception of proline, can thus be determined.     

An improved method for the determination of γ-carboxyglutamic acid in proteins,bone, and urine

A method of high-performance liquid chromatographic separation of the fluorescence derivative of γ-carboxyglutamic acid (Gla) is presented. Alkaline hydrolysates of protein samples were reacted with o-phthalaldehyde in the presence of ethanethiol for 2 min, and the fluorescence derivative of γ-carboxyglutamic acid was resolved from the other amino acids by a short column packed with silica-based anion exchanger under isocratic conditions. By this method, as low as 200 fmol of γ-carboxyglutamic acid can be quantitatively analyzed within 10 min. The method presented here shortened the analysis time for Gla and was at least 10 times more sensitive than the method we described previously (Anal. Biochem.117, 259–265, 1981). The application of this method to the formic acid-soluble or insoluble γ-carboxyglutamic acid-containing proteins in chicken bone and the concomitant increase of γ-carboxyglutamic acid content in chicken bone with age are reported.