A HISTOCHEMICAL METHOD FOR DISTINGUISHING BETWEEN SIDE-CHAIN AND TERMINAL (α-ACYLAMIDO) CARBOXYL GROUPS OF PROTEINS

The specificity of the Barrnett-Seligman method for the histochemical demonstration of α-acylamido carboxyl groups (C terminal) of proteins is dependent on the conversion of such groups to ketones by the action of acetic anhydride and absolute pyridine. Studies on model compounds show that the side-chain carboxyl groups also react in the method and that most of the final color developed can be attributed to these carboxyls, rather than to the C terminal carboxyl groups. It is postulated that the side-chain carboxyls react by formation of mixed anhydrides in the presence of acetic anhydride and pyridine. This mixed anhydride then could link with a hydrazide to form a dihydrazide, which is capable of coupling with a diazo dye. Acetic anhydride treatment alone, without pyridine, also yields mixed anhydride. The mixed anhydride derived from the side-chain carboxyls can be destroyed by base, whereas the methyl ketone derived from the C terminal carboxyl is unaffected, and this treatment makes the method specific for C terminal carboxyl groups. Tissues treated in such a fashion demonstrate that all the color reaction obtained in the method is due to side-chain carboxyls, and that C terminal groups yield little or no staining as would be expected for "average" molecular weight proteins.     

Fluorescence of 2-hydroxy-3-naphthoic acid hydrazide derivatives of side-chain carboxyl groups of proteins

Summary Sections processed through the first part of the Barrnett and Seligman (1958) procedure, which is considered to be selective for side-chain carboxyl groups of proteins, are fluorescent when exposed to incident blue light. The results of a number of tests suggest that the fluorescence can be ascribed principally to binding of 2-hydroxy-3-naphthoic acid hydrazide (HNAH) by mixed acid anhydride derivatives of side-chain carboxyl groups of proteins. Thus, the procedure appears to be a fluorescent counterpart of the bright-field method of Barrnett and Seligman.     

Optimization of the histochemical demonstration of DNA using 3-hydroxy-2-naphthoic acid hydrazide and fast blue B

Previous methods for the histochemical demonstration of DNA were optimized. p-Toluene sulfonic acid as catalyst for hydrazone formation between the aldehydes generated after Feulgen hydrolysis and 3-hydroxy-2-naphthoic acid hydrazide (NAH) was used instead of acetic acid. Modifications of the conditions of the coupling reaction with Fast Blue B reduced the background staining. The optimized histochemical staining method for DNA (NAH-FB-DNA staining) can be performed easily and reproducibly. Without prior Feulgen hydrolysis the optimized method can also be used for the histochemical demonstration of reactive carbonyls undissolved under the given histochemical conditions.     

Quantification of the histochemical staining for carbonyles and DNA using 3-hydroxy-2-naphthoic acid hydrazide and fast blue B

Fixed cells and tissues pretreated with 4-hydroxynonenal were used as models for the histochemical demonstration of protein bound aldehydic groups. The aldehydes were stained with both a modification of the 2,4-dinitrophenylhydrazine method (2,4-DNPH) and the optimized staining using 3-hydroxy-2-naphthoic acid hydrazide and Fast blue B (NAH-FB). A correlation has been found between the specific microphotometric mean integrated maximum absorbance values of cells and tissues stained with 2,4-DNPH and with NAH-FB (cc = 0.999). The maximum absorbance measured after 2,4-DNPH-staining (epsilon 367 = 21,000) were 1.893 +/- 0.072 (P less than 0.01) times that of NAH-FB-staining at 550 nm. Microphotometrically determined DNA-values of different cells stained with the NAH-FB-DNA-method correlated with those determined with methods of analytical biochemistry and published by other authors.     

Studies in fluorescence histochemistry

Summary Tissue proteins believed to contain a relatively high concentration of C-terminal carboxyl groups emit an intense blue fluorescence after being treated with first a hot mixture of acetic anhydride and pyridine, second salicylhydrazide and last zinc acetate. Characteristically they do not fluoresce when the zinc treatment is omitted. Muscular tissues emit the strongest fluorescence, but normally neither mucosubstances nor loose connective tissues fluoresce at all.These and other results are consistent with Barrnett and Seligman's view that acetic anhydride in the presence of hot pyridine transforms the C-terminal carboxyl groups of proteins into methyl ketones. They do not support Karnovsky's more recent theory that hot acetic anhydride more or less exclusively converts side-chain carboxyl groups of proteins into mixed acid anhydrides instead.     

Optimization of the histochemical demonstration of DNA using 3-hydroxy-2-naphthoic acid hydrazide and Fast Blue B

Summary Previous methods for the histochemical demonstration of DNA were optimized. p-Toluene sulfonic acid as catalyst for hydrazone formation between the aldehydes generated after Feulgen hydrolysis and 3-hydroxy-2-naphthoic acid hydrazide (NAH) was used instead of acetic acid. Modifications of the conditions of the coupling reaction with Fast Blue B reduced the background staining. The optimized histochemical staining method for DNA (NAH-FB-DNA staining) can be performed easily and reproducibly. Without prior Feulgen hydrolysis the optimized method can also be used for the histochemical demonstration of reactive carbonyls undissolved under the given histochemical conditions.Dedicated to Prof. Dr. E. Schauenstein on the occasion of his 70th birthday     

Some evidence confirming the specificity of Barrnett and Seligman's technique for demonstrating side-chain carboxyl groups in proteins

Summary There is some confusion in the literature as to whether the protein carboxyl groups demonstrable with Barrnett and Seligman's histochemical technique are C-terminal or side-chain. It seems that in the first step of this technique, hot mixtures of acetic anhydride and pyridine convert the commonly-occurring side-chain carboxyls into mixed acid anhydrides. That they do has now been confirmed; proteins in tissue sections treated with such acetic anhydride mixtures do not react with hydroxynaphthoic acid hydrazide after being left for a long time in either cold methanol or a cold solution of aniline in xylene. In this they resemble the acid anhydrides used for synthezising peptides in vitro. Thus all the lilac-reddish colours observable in tissue proteins on which the Barrnett and Seligman technique proper has been carried out can be ascribed to side-chain carboxyl groups.     

Quantification of the histochemical staining for carbonyles and DNA using 3-hydroxy-2-naphthoic acid hydrazide and Fast blue B

Summary Fixed cells and tissues pretreated with 4-hydroxynonenal were used as models for the histochemical demonstration of protein bound aldehydic groups. The aldehydes were stained with both a modification of the 2,4-dinitrophenylhydrazine method (2,4-DNPH) and the optimized staining using 3-hydroxy-2-naphthoic acid hydrazide and Fast blue B (NAH-FB). A correlation has been found between the specific microphotometric mean integrated maximum absorbance values of cells and tissues stained with 2,4-DNPH and with NAH-FB (cc=0.999). The maximum absorbance measured after 2,4-DNPH-staining (₃₆₇=21 000) were 1.893±0.072 (P<0.01) times that of NAH-FB-staining at 550 nm. Microphotometrically determined DNA-values of different cells stained with the NAH-FB-DNA-method correlated with those determined with methods of analytical biochemistry and published by other authors.     

Studies in fluorescence histochemistry. VII. The mechanism of the complex reactions that may take place between protein carboxyl groups and hot mixtures of acetic anhydride and pyridine in the acetic anhydride-salicylhydrazide-zinc (or fluorescent ketone) method for localizing protein C-terminal carboxyl groups

Synopsis Theoretical arguments are marshalled with experimental evidence to support claims made previously (Stoward & Burns, 1967, 1968) that at 60°C acetic anhydride in the presence of pyridine transforms C-terminal carboxyl groups of proteins to methyl ketones and converts side-chain carboxyl groups to acid anhydrides. On balance the experimental evidence also supports another claim, namely that the methyl ketones thus formed from C-terminal carboxyl groups may be demonstrated specificallyin situ by the intense fluorescence they emit after treatment successively with aqueous solutions of salicylhydrazide and zinc acetate.The experiments carried out included ones favouring the exclusive formation of acid anhydrides, blocking of possible anhydrides with aromatic amines or alcohols, hydrolysis of anhydrides with alkalis, and prior methylation of carboxyl groups.     

NADPH-dependent lipid peroxidation capacity in unfixed tissue sections: characterization of the pro-oxidizing conditions and optimization of the histochemical detection

Summary Factors which influence the iron-stimulated lipid peroxidation in rat liver have been studied by incubating unfixed cryostat sections with a pro-oxidant system and using an optimized histochemical detection method for lipid peroxidation products with 3-hydroxy-2-naphthoic acid hydrazide and Fast Blue B. We used a method that was slightly different from the one described previously. The final reaction product was exclusively localized in the cytoplasm of liver parenchymal cells with a homogeneous distribution within the liver lobule. The absorbance maximum, as measured cytophotometrically, was found to be 550 nm. Maximum lipid peroxidation was observed when the pro-oxidant system contained 0.2 mm NADPH, 1 mm ADP and 15 μm FeCl₂. Some reaction product was found when NADPH was omitted. Iron concentrations higher than 180 μm prevented the formation of lipid peroxidation products in certain areas of the sections, whereas ADP concentrations higher than 1 mm inhibited the reaction in the whole section. A pH dependency was also observed, with the highest lipid peroxidation at pH 7.2. Optimum lipid peroxidation was induced by incubating for 30 min at 37°C with the pro-oxidant system. A linear relationship was found between the thickness of the sections (up to 20 μm) and the amount of lipid peroxidation products. The addition of scavengers of O2- (superoxide dismutase), hydrogen peroxide (catalase) and OH · (mannitol) to the first step medium did not affect the amount of final reaction product. These findings appear to confirm the hypothesis proposed for events occurring in isolated microsomes, leading to the formation of hydroperoxides and ultimately lipid peroxidation-derived carbonyls. The present method is a useful tool for studying the capacity of lipid peroxidation in tissues under different (patho)physiological conditions.     

Transformation of 1- and 2-methylnaphthalene by Cunninghamella elegans.

Cunninghamella elegans metabolized 1- and 2-methylnaphthalene primarily at the methyl group to form 1- and 2-hydroxymethylnaphthalene, respectively. Other compounds isolated and identified were 1- and 2-naphthoic acids, 5-hydroxy-1-naphthoic acid, 5-hydroxy-2-naphthoic acid, 6-hydroxy-2-naphthoic acid, and phenolic derivatives of 1- and 2-methylnaphthalene. The metabolites were isolated by thin-layer and reverse-phase high-pressure liquid chromatography and characterized by the application of UV-visible absorption, 1H nuclear magnetic resonance, and mass spectral techniques. Experiments with [8-14C]2-methylnaphthalene indicated that over a 72-h period, 9.8% of 2-methylnaphthalene was oxidized to metabolic products. The ratio of organic-soluble in water-soluble metabolites at 2 h was 92:8, and at 72 h it was 41:59. Enzymatic treatment of the 48-h aqueous phase with either beta-glucuronidase or arylsulfatase released 60% of the metabolites of 2-methylnaphthalene that were extractable with ethyl acetate. In both cases, the major conjugates released were 5-hydroxy-2-naphthoic acid and 6-hydroxy-2-naphthoic acid. The ratio of the water-soluble glucuronide conjugates to sulfate conjugates was 1:1. Incubation of C. elegans with 2-methylnaphthalene under an 18O2 atmosphere and subsequent mass spectral analysis of 2-hydroxymethylnaphthalene indicated that hydroxylation of the methyl group is catalyzed by a monooxygenase.     

Native Enzyme Mobility Shift Assay (NEMSA): a new method for monitoring carboxyl group modification of carboxymethylcellulase from Aspergillus niger

A simple, sensitive, accurate and more informative assay for determining the number of modified groups during the course of carboxyl group modification is described. Monomeric carboxymethylcellulase (CMCase) from Aspergillus niger was modified by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide. The different time-course aliquots were subjected to non-denaturing PAGE and the gel stained for CMCase activity. The number of carboxyl groups modified are directly read from the ladder of the enzyme bands developed at given time. This method showed that after 75 min of modification reaction there were five major species of modified CMCases in which 6 to 10 carboxyls were modified.     

A fluorescent water soluable carbodiimide-2-hydroxy-3-naphthoic acid hydrozide reaction for the demonstration of carboxyl groups in proteins and mucosubstances.

The carbodiimide-2-hydroxy-3-haphthoic acid hydrazide reaction as developed by Geyer (1964) was used without subsequent diazonium coupling as a fluorescent method for the demonstration of carboxyl groups in both proteins and mucosubstances. The topological distribution of the fluorophore was similar to that reported by Geyer. Quantitative microfluorometric studies on cartilage sections revealed differences in detail between emissions in cartilage matrix mucoprotein as compared to the dense connective tissue associated with the perichondrium which consists principally of protein. It would also appear that the primary fluorescent emission of unstained preparations at 450 mm should be useful in microfluorometric determinations of proteins.     

Ketonic rancidity in coconut due to xerophilic fungi

A type of deterioration called ketonic rancidity occurred in coconut after inoculation with four xerophilic fungi, Eurotium amstelodami, E. chevalieri, E. herbariorum and Penicillium citrinum. The fungi were incubated at low water activity and oxygen tension. A homologous series of aliphatic methyl ketones and secondary alcohols C₅C₁₁ were isolated and identified in the rancid samples after fungal growth. Evidence is presented that odd numbered methyl ketones (C₅C₁₁) are derived from even numbered short chain fatty acids with one more carbon atoms than the ketones via a modified β-oxidation of the parent fatty acid. Heptan-2-one and heptan-2-ol are the main reaction products except in the case of E. herbariorum where even numbered hexan-2-one, hexan-2-ol, octan-2-one and octan-2-ol were produced. Other moulds grown on coconut under similar conditions—Aspergillus flavus Link and Chrysosporium farinicola (Burnside) Skou (C. fastidium Pitt)—did not cause ketonic deterioration.     

Histochemische Untersuchungen zur Spezifität und Anwendung der Carbodiimidreaktion für den Nachweis von Carboxylgruppen

Zusammenfassung Mittels vergleichender Untersuchungen und Blockierungs-bzw. Abbauverfahren wurde der Reaktionsumfang für die Säureanhydridreaktion und für die Carbodiimidreaktion zum histochemischen Nachweis für Carboxylgruppen bestimmt. Die Carbodiimidreaktion erfaßt COOH-Gruppen von Proteinen und von einigen sialinsauren Kohlenhydraten. Durch den zusätzlichen Nachweis saurer Kohlenhydrate unterscheidet sie sich von der Säureanhydridreaktion. Außerdem wurde der Fixierungseinfluß auf das Ergebnis der Carbodiimidreaktion ermittelt.

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Summary The reactive range of the acid anhydride reaction and the carbodiimide reaction for the histochemical demonstration of carboxyl groups was investigated by comparative methods, blocking tests and analytical methods. The carbodiimide reaction reveals COOH-groups of proteins and several sialinic acid carbohydrates. The additional demonstration of acid carbohydrates distinguishes the carbodiimide reaction from the acid anhydride reaction. The influence of fixatives on the outcome of the carbodiimide reaction is also investigated.

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Mit Unterstützung durch einen Forschungsauftrag des Ministeriums für das Hoch-und Fachschulwesen der DDR.     

Derivatization and gas chromatographic determination of some biologically important acids in cerebrospinal fluid

Halogenated derivatives of phenolic acids have been prepared by a convenient procedure. The method uses a combination of pentafluoropropionic anhydride and a halogenated alcohol to derivatize the carboxyl group, followed by reaction with pentafluoropropionic anhydride to derivatize the phenol and indole groups. The halogenated derivatives are extremely sensitive to electron capture detection and can be detected in amounts as low as 5 pg. The structures of the derivatives have been confirmed by mass spectrometry. Procedures have been developed using these derivatives for the determination of spinal fluid levels of vanillylmandelic acid, homovanillic acid, probenecid, and 2-pyrrolidone-5-carboxylic acid and for the identification of 2-pyrrolidone-5-carboxylic acid as a natural constituent of body fluids and tissues.     

On the action of carboxy groups in the citrate synthase reaction

The aza analogue (RS)-3-hydroxy-2,5-pyrrolidinedione-3-acetic acid (6) of the five-membered citric anhydride (2) was prepared in the sequence citric acid----2-phenyl-1,3-dioxolan-4-one-5,5-diacetic acid (1)----citric acid beta-amide (3)----6 and used to resolve ambiguities in the mechanism of the citrate synthase reaction. The results yield no indication for the formation of anhydride 2 on the enzyme and favour the direct hydrolysis of the intermediate (3S)-citryl-CoA. Ammonolysis of the dioxolanone 1 in the reaction sequence described above produced not only citric acid beta-amide but also the alpha-isomer. This is shown to originate in the transient formation of anhydride 2. Hydrolysis of the dioxolanone 1 under "physiological conditions" occurs via anhydride 2, generated in intramolecular bifunctional catalysis by a protonated and a deprotonated carboxyl group. The catalytic residue Asp375 of citrate synthase is considered to operate on the enzyme as does the protonated carboxyl group in the chemical reaction and to generate enolic acetyl-CoA in cooperative catalysis with His274. This reaction of Asp375 may also facilitate the hydrolysis of citryl-CoA.     

The intragranular location of carboxyl groups in neuromelanin and lipofuscin in human brain and in meningeal melanosomes in mouse brain

The intragranular location of carboxyl groups was tinctorially determined in human substantia nigra neuromelanin granules, human inferior olive lipofuscin granules, and mouse meningeal melanosomes. Soluble and insoluble lipid was stained with beta naphthol Sudans in unoxidized and oxidized frozen and paraffin sections containing neuromelanin or lipofuscin. Nile blue demonstrated carboxyls in unoxidized neuromelanin, lipofuscin, and melanin, and in oxidized neuromelanin and lipofuscin. Carbodiimide demonstrated carboxyls in unoxidized and oxidized lipofuscin and oxidized neuromelanin. In all instances, staining for carboxyls was inhibited by prior mild methylation, and proof of their presence was obtained by a pre-staining, stepwise, alternating, and repetitive mild demethylation, mild methylation sequence. Structurally, carboxyls were demonstrated in the neuromelanin granule's soluble lipid-free lipofuscin component, in the meningeal melanosome's melanin component, and virtually throughout the lipofuscin granule. The following structural and chemical basis was proposed for the different resistance of Nile blue staining of melanosomes and of neuromelanin and lipofuscin to acetone extraction. Nile blue forms an insoluble complex with melanosomal dopa-melanin's quinonoid, diphenolic, and undissociated carboxyl units. Such complex formation does not occur in neuromelanin's carboxyl-free dopamine-melanin component, however. Instead, Nile blue ionogenicly bonds with dissociated carboxyls belonging to the neuromelanin granule's lipofuscin component.     

Sulfanilation of lysozyme by carbodiimide reaction. Reactivities of individual carboxyl groups

The carboxyl groups of lysozyme were coupled with sulfanilic acid, a chromophoric nucleophile, using 1-ethyl-3-dimethylaminopropylcarbodiimide at pH 5. Other carbodiimides were less effective. Ninety percent of the carboxyl groups were sulfanilated through exhaustive reaction with 1.2 m nucleophile. Isolation and identification of the tryptic peptides from this material showed that all 10 of the carboxyls of lysozyme had reacted. In 0.05 m sulfanilic, Glu-35 and Asp-101 were most reactive while Glu-7, Asp-18, and Asp-66 were least. Change to high concentration of nucleophile (from 0.05 to 1.2 m sulfanilic) altered carboxyl reactivity. Addition of inhibitor reduced reactivity of Asp-101 and Glu-35. Side reactions were not important.     

Degradation of phenanthrene by <Emphasis Type="Italic">Burkholderia</Emphasis> sp. C3: initial 1,2- and 3,4-dioxygenation and <Emphasis Type="Italic">meta</Emphasis>- and <Emphasis Type="Italic">ortho</Emphasis>-cleavage of naphthalene-1,2-diol

Burkholderia sp. C3 was isolated from a polycyclic aromatic hydrocarbon (PAH)-contaminated site in Hilo, Hawaii, USA, and studied for its degradation of phenanthrene as a sole carbon source. The initial 3,4-C dioxygenation was faster than 1,2-C dioxygenation in the first 3-day culture. However, 1-hydroxy-2-naphthoic acid derived from 3,4-C dioxygenation degraded much slower than 2-hydroxy-1-naphthoic acid derived from 1,2-C dioxygenation. Slow degradation of 1-hydroxy-2-naphthoic acid relative to 2-hydroxy-1-naphthoic acid may trigger 1,2-C dioxygenation faster after 3 days of culture. High concentrations of 5,6-␣and 7,8-benzocoumarins indicated that meta-cleavage was the major degradation mechanism of phenanthrene-1,2- and -3,4-diols. Separate cultures with 2-hydroxy-1-naphthoic acid and 1-hydroxy-2-naphthoic acid showed that the degradation rate of the former to naphthalene-1,2-diol was much faster than that of the latter. The two upper metabolic pathways of phenanthrene are converged into naphthalene-1,2-diol that is further metabolized to 2-carboxycinnamic acid and 2-hydroxybenzalpyruvic acid by ortho- and meta-cleavages, respectively. Transformation of naphthalene-1,2-diol to 2-carboxycinnamic acid by this strain represents the first observation of ortho-cleavage of two rings-PAH-diols by a Gram-negative species.