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.     

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.     

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.     

N-acetylchitosan gel: A polyhydrate of chitin

N-Acetylchitosan gel, a polyhydrate of chitin, and partially O-acetylated N-acetylchitosan gel were produced by a facile acetylation of chitosan with acetic anhydride in 10% acetic acid and in aqueous acetic acid/methanol at room temperature. Under the same conditions, a series of N-acylchitosan gels was produced in reaction with the other carboxylic anhydrides. The gels thus produced were colorless, transparent and rigid, and stable on heating. The gels were insoluble in cold and boiling water, formic acid, aqueous acids, and the other solvents examined. Significant changes in specific rotation occurred in the acylation of chitosan and its aggregation.     

Dicarboxylic acid anhydrides as dissociating agents of protein-containing structures

Dissociation of protein-containing structures by modification of protein amino groups with dicarboxylic acid anhydrides is a mild procedure which, in some cases, offers advantages over treatment with alternative dissociating agents, such as urea, guanidine hydrochloride, detergents, high ionic strength, and extremes of pH: In addition to dissociating multimeric proteins and protein aggregates, dicarboxylic acid anhydrides are effective dissociating agents for membrane-bound proteins and nucleoprotein particles. With most dicarboxylic acid anhydrides reviewed, the introduced reagent residues can be eliminated under moderate acid conditions, which allows the purification of unmodified individual components, and the use of disassembly-reconstitution systems valuable for investigating the structural and functional roles played by the individual components of complex particles:Each reagent can be suitable for a particular purpose, depending on the required specificity of the modification and stability of the modified groups: The stability of the acylated amino groups ranges from the very stable succinylated amino groups to the very labile acylation obtained with dimethylmaleic anhydride: Between these extremes, the stability of the modified amino groups decreases stepwise in the following order: maleic, exo-cis-3,6-endoxo-⁴-tetrahydrophthalic, citraconic, and 3,4,5,6-tetrahydrophthalic anhydride. With respect to the selectivity of the produced modification, little or no modification of hydroxyamino acid and cysteine residues has been observed with dimethylmaleic, exo-cis-3,6-endoxo-⁴-tetrahydrophthalic, and 3,4,5,6-tetrahydrophthalic anhydrides: With the other reagents, the extent of modification of hydroxyamino acid residues increases in the order citraconic, maleic and succinic anhydride: Citraconic and maleic anhydrides can produce irreversible modification of cysteine residues, the reactivity of sulfhydryl groups being higher with maleic anhydride:     

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.     

A fluorescent histochemical method for the detection of tissue disulphide groups

Summary Bennett and Yphantis (1948) introduced into histochemistry the use of organic mercurials for the detection of sulphydryl groups. The new fluorescent method which is now reported is similarly based upon the reaction of mercaptans with an organic mercurial, fluorescein mercuric acetate (FMA). This method is sensitive; moreover it is not as elaborate as the dihydroxy-dinaphthyl-disulphide (DDD) technique of Barrnett and Seligman (1952).     

Chemical Modification of Lyophilized Proteins in Nonaqueous Environments

Lyophilized proteins were reacted in vacuo with a volatile reagent or dispersed in octane and reacted with dissolved reagent. Three novel derivatives were formed with iodomethane: (a) quaternized trimethyl amino groups, (b) N¹,N³-dimethylimidazolium cation, and (c) phenolic O-methyl ether. Acid anhydrides acylated amino groups and formed mixed anhydrides with side-chain carboxyl groups. Under nonaqueous conditions it was observed that: (i) The same derivatives are formed as under aqueous conditions. (ii) Hydrolytic breakdown of protein is prevented. (iii) Less reagent is required. (iv) Unreacted reagent can be recovered. (v) Water-labile derivatives can be isolated as stable intermediates. (vi) The yield of a derivatized functional group was directly related to its pK_a, its surface exposure, and the pH of the solution from which the protein was lyophilized. (vii) The physicochemical factors governing the reactivity of protein functional groups in nonaqueous environments appear to reflect the protein solution structure prior to lyophilization.     

Histochemical Demonstration of Protein-Bound Alpha-Acylamido Carboxyl Groups

A method has been developed to demonstrate the alpha-acylamido carboxyl groups of protein, taking advantage of the fact that acylamido carboxyl groups are converted to ketonic carbonyls by the action of acetic anhydride and absolute pyridine. The method utilizes deparaffinized sections of tissues fixed in a variety of fixatives. Following the conversion of carboxyls to the methyl ketones, the latter are stained with 2-hydroxy-3-naphthoic acid hydrazide. Control experiments have indicated that methylation of carboxyls prevented staining, as did carbonyl reagents after the carboxyls were transformed to methyl ketones. Leucofuchsin did not stain the ketonic carbonyls, and only elastic tissue stained with 2-hydroxy-3-naphthoic acid hydrazide without the previous use of the catalyzed reaction with anhydride. A brief survey of the reaction on various tissues of the albino rat was made, and the effects of various fixatives were assayed. Of particular interest were certain sites, such as acidophiles of the anterior pituitary gland, where an intense reaction occurred. The possibility exists that certain specific proteins rich in terminal acylamido carboxyl groups, by virtue of their protein side chains or low molecular weight, may be demonstrated by this method.     

The structure and function of ribonuclease T1. XXIII. Inactivation of ribonuclease T1 by reversible blocking of amino groups with cis-aconitic anhydride and related dicarboxylic acid anhydrides.

Ribonuclease T1 [EC 3.1.4.8] was inactivated rapidly by treatment at pH 8.0 and 0 degrees C with cis-aconitic anhydride and related dicabroxylic acid anhydrides, including citraconic, maleic, and succinic anhydrides. Under reaction conditions used, roughly 90% inactivation occurred within 30 min. Analyses of the inactivated enzymes indicated that the reaction took place fairly specifically at the alpha-amino group of the N-terminal alanine and the epsilon-amino group of lysine-41. Upon incubation of these inactivated enzymes at pH 3.6 and 37 degreeC, the activity was regenerated to various extents, depending on the nature of the introduced acyl groups. Under these conditions, the enzyme modified with cis-aconitc anhydride or citraconic anhydride recovered much of the origninal activity after 48 h whereas the enzyme modified with maleic anhydride recovered its activity only partially. Practically no activity was regenerated in the case of the enzyme modified with succinic anhydride under these conditions. The inactivation appears to be due mainly to the effect of the carboxyl group introduced at the epsilon-amino group of lysine-41. The results suggest the usefulness of cis-aconitic anhydride as a reversible blocking reagent for amino groups in proteins.     

Calcium binding by human erythrocyte membranes. Significance of carboxyl, amino and thiol groups

1. The role of the ionized carboxyl groups of proteins of the erythrocyte membrane as Ca(2+) receptor sites was investigated. A water-soluble carbodi-imide [1-cyclohexyl-3-(2-morpholinoethyl)carbodi-imide methotoluene-p-sulphonate], referred to as carbodi-imide reagent, and glycine methyl ester were used to modify the free carboxyl groups of the membrane. The degree of modification was estimated from amino acid analyses, which showed the amount of glycine incorporated. As the concentration of carbodi-imide reagent was raised (0.1-0.4m) incorporation of glycine increased and Ca(2+) binding decreased by about 77%. At 0.4m-carbodi-imide reagent all of the binding of Ca(2+) to protein was abolished and it was estimated that about 37% of the side-chain carboxyl groups of aspartic acid plus glutamic acid had been blocked by glycine. 2. Acetylation of all of the free amino groups was achieved by incubating the erythrocyte ;ghosts' at pH10.3 with acetic anhydride (10-15mg/10mg of ;ghost' protein). Acetylation increased by 1.5-fold the capacity of the ;ghost' to bind Ca(2+), indicating that the remaining carboxyl groups of aspartic acid and glutamic acid were made available for Ca(2+) binding by this procedure. These findings support the concept that in normal ;ghosts', at pH7.4, Ca(2+) binding to free carboxyl groups is partially hindered by the presence of charged amino groups. 3. Treatment of ;ghosts' with N-acetylneuraminidase, which removed 94% of sialic acid residues, and treatment with 1mm-p-chloromercuribenzoate did not alter Ca(2+) binding. The major effect of 5.8mm-p-chloromercuribenzoate upon ;ghosts' was to cause a solubilization of a calcium-membrane complex, which included about one-third of the ;ghost' protein. The molar ratio of Ca(2+): protein in the solubilized material was the same as that in the intact (untreated) ;ghosts'.     

Amino Acid Homochirality may be Linked to the Origin of Phosphate-Based Life

Phosphorylation has to have been one of the key events in prebiotic evolution on earth. In this article, the emergence of phosphoryl amino acid 5′-nucleosides having a P–N bond is described as a model of the origin of amino acid homochirality and Genetic Code. It is proposed that the intramolecular interaction between the nucleotide base and the amino acid side-chain influences the stability of particular amino acid 5′-nucleotides, and the interaction also selects for the chirality of amino acids. The differences between l- and d-conformation energies (ΔE _conf) are evaluated by DFT methods at the B3LYP/6-31G(d) level. Although, as expected, these ΔE _conf values are not large, they do give differences in energy that can distinguish the chirality of amino acids. Based on our calculations, the chiral selection of the earliest amino acids for l-enantiomers seems to be determined by a clear stereochemical/physicochemical relationship. As later amino acids developed from the earliest amino acids, we deduce that the chirality of these late amino acids was inherited from that of the early amino acids. This idea reaches far back into evolution, and we hope that it will guide further experiments in this area.     

Über intrazelluläre und extrazelluläre Lokalisierung der alkalischen Phosphatase

Zusammenfassung Nierenblöckchen von Mäusen wurden für 2–3 min in einer OsO₄-Lösung vorfixiert und nach drei Methoden (Gomori-Molnar, Mizutani und Barrnett, Mölbert, Duspiva und v. Deimling) für die histochemische Lokalisierung der alkalischen Phosphatase-Aktivität in toto inkubiert. In den Tubuli, die an der Blockoberfläche liegen, befindet sich das Reaktionsprodukt nach sämtlichen Methoden, intrazellulär. Nach Vorfixierung in einer Glutaraldehyd-Lösung bildet sich das Reaktionsprodukt bei der nichtsimultanen Methode von Gomori-Molnar an den Außenflächen der Zellmembran; bei den simultanen Methoden von Mizutani und Barrnett und Mölbert, Duspiva und v. Deimling teils intrazellulär teils extrazellulär.Das Fixierungsmittel und die Art der histochemischen Reaktion beeinflussen also die Lokalisierung der alkalischen Phosphatase-Aktivität.

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Summary For the demonstration of alkaline phosphatase activity, blocks of mouse kidney were fixed 2–3 min in a OsO₄ solution and in toto incubated. The methods of Gomori-Molnar, Mizutani and Barrnett, and Mölbert, Duspiva and v. Deimling were applied.In the proximal convolute tubules of the outer zone of the blocks, in every case, threaction product is localized along the inside of the cell membrane. After fixation in a glutaraldehyde solution, with the non-simultaneous method of Gomori-Molnar the reaction product occurs always outside of the cell membrane; with the simultaneous methods of Mizutani and Barrnett and Mölbert, Duspiva and v. Deimling it is formed irregularly both intracellular and extracellular.These observations show that the localization of the alkaline phosphatase activity depends on the fixative and on the histochemical method used.

     

Efficient kinetic resolution of dl-menthol by lipase-catalyzed enantioselective esterification with acid anhydride in fed-batch reactor

Acid anhydrides were used as highly reactive and non-water-producing acyl donors for hydrolase-catalyzed enantioselective esterification. Efficient kinetic resolution of dl-menthol has been achieved via lipase-catalyzed enantioselective esterification in cyclohexane when propionic anhydride as an acyl donor was continuously fed into a reactor containing dl-menthol and Candida cylindracea lipase OF 360, while a high concentration of the acid anhydride in a batch reaction system with a dehydrated organic solvent did not facilitate the reaction, because water necessary for the enzyme function was consumed by the competing hydrolysis of the anhydride catalyzed by the same enzyme. The efficiency of this fed-batch reaction system using acid anhydride was higher and the enzyme stability in repeated use was much better than those of conventional batch and fed-batch reaction systems using propionic acid as an acyl donor. The optical purity (more than 98% e.e.) of the l-menthyl ester produced in the fed-batch system using the anhydride was comparable to that in the system using the corresponding acid. *** DIRECT SUPPORT *** AG903062 00002     

Studies in fluorescence histochemistry

Summary Pretreatment of sections of fixed tissue with selective blocking reagents for indigenous thiol groups did not, it was found, impair the fluorescence subsequently obtainable with the acetic anhydridesalicylhydrazide-zinc technique (Stoward and Burns, 1967) for localizing C-terminal carboxyl groups of proteins. This suggests that thiol and S-acetyl groups do not participate in the complex reactions involved in the method. In further support of this suggestion it was found that artificially introduced thiol groups also do not take part.Sites containing either, glycogen or neutral periodate-reactive mucosubstances did not fluoresce in sections subjected to the technique. This indicates that O-acetyl groups, although probably formed, are not involved in the reactions giving rise to the fluorescence reaction in proteins.     

Femtomole sensitive radioimmunoassay for cyclic AMP and cyclic GMP after 2'0 acetylation by acetic anhydride in aqueous solution.

The sensitivity of radioimmunoassays for cyclic AMP and cyclic GMP has been markedly improved to readily detect femtomole (10-15) amounts in tissue extracts by acetylating the cyclic nucleotides at the 2'0 position with acetic anhydride. Acetylation of cyclic nucleotides by acetic anhydride in aqueous solution proceeds more rapidly than the hydrolysis of acetic anhydride to acetic acid thus yielding 100% acetylated cyclic nucleotide. 2'0 substituted cyclic nucleotides have greater affinity for the antibody than the parent cyclic nucleotides because the antibody has been made to a protein conjugate coupled at the 2'0 position. This simple acetylation technique makes it possible to measure cyclic AMP and cyclic GMP in minute quantities of tissue without purification or concentration of the sample.     

A new non-natural arginine-like amino acid derivative with a sulfamoyl group in the side-chain

Sulfamoylation of the l-ornithine methyl ester side-chain generates a non-natural arginine isostere which can be coupled with N-Fmoc-l-proline to synthesize analogues which maintain the structural characteristics of the biologically important Pro-Arg dipeptide sequence. As a probe of its biological importance, the sulfamoylated amino acid derivative was also incorporated as P1 residue in tripeptide structures matching the C-terminal subsequence of fibrinogen. The reported results demonstrate that the functionalization of l-ornithine side-chain with a neutral sulfamoyl group can generate an arginine bioisostere which can be used for the synthesis of prototypes of a new class of human thrombin inhibitors.     

The reversible reaction of protein amino groups with exo-cis-3,6-endoxo-Δ4-tetrahydrophthalic anhydride. The reaction with lysozyme

1. The reaction of exo-cis-3,6-endoxo-Delta(4)-tetrahydrophthalic anhydride with amino groups of model compounds and lysozyme is described. 2. Reaction with the in-amino group of N(alpha)-acetyl-l-lysine amide gives rise to two diastereoisomeric products; at acid pH the free amino group is liberated with anchimeric assistance by the neighbouring protonated carboxyl group with a half-time of 4-5h at pH3.0 and 25 degrees C. 3. The amino groups of lysozyme can be completely blocked, with total loss of enzymic activity. Dialysis at pH3.0 results in complete recovery of the native primary and tertiary structure of lysozyme and complete return of catalytic activity. 4. The specificity of reaction of this and other anhydrides with amino groups in proteins is discussed.     

13C NMR studies of fatty acid-protein interactions: comparison of homologous fatty acid-binding proteins produced in the intestinal epithelium

Summary A high-resolution, solution-state NMR method for characterizing and comparing the interactions between carboxyl ¹³C-enriched fatty acids (FA) and individual binding sites on proteins has been developed. The utility of this method results from the high degree of resolution of carboxyl from other carbon resonances and the high sensitivity of FA carboxyl chemical shifts to intermolecular environmental factors such as degree of hydrogen-bonding or hydration, degree of ionization (pH), and proximity to positively-charged or aromatic side-chain moieties in proteins. Information can be obtained regarding binding heterogeneity (structural as well as thermodynamic), binding stoichiometries, relative binding affinities, the ionization behavior of bound FA and protein side-chain moieties, the physical and ionization states of unbound FA, and the exchange rates of FA between protein binding sites and between protein and non-protein acceptors of FA, such as model membranes.Cytosolic fatty acid binding proteins represent an excellent model system for studying and comparing fatty acid-protein interactions. Prokaryotic expression vectors have been used to direct efficient synthesis of several mammalian intestinal FABPs in E. coli. This has enabled us to isolate gram-quantities of purified FABPs, to introduce NMR-observable isotopes, and to generate FABP mutants.The intestine is the only tissue known to contain abundant quantities of more than one FABP homologue in a single cell type. It is likely that these homologous FABPs serve distinct functional roles in intestinal lipid transport. This paper presents comparative ¹³C NMR results for FA interactions with FABP homologues from intestine, and the functional implications of these analyses are discussed.Abbreviations FA Fatty Acid(s) - FABP Fatty Acid Binding Protein(s) - I-FABP_c Cytosolic rat intestinal Fatty Acid Binding Protein - L-FABP_c Cytosolic rat liver Fatty Acid Binding Protein - CD Circular Dichroic spectroscopy Established Investigator of the American Heart Association     

The utilization of the mixtures of monosaccharides by some mucorales

Summary The utilization of mixtures of monosaccharides byBlakeslea trispora Thaxter,Choanephora circinans (Naganish &Kawakami)Hesseltine &Benjamin,Gilbertella persicaria var.indica Mehrotra &Mehrotra andHelicostylum piriforme Bainier was studied. The effect of sorbose on the utilization of other sugars present in the mixtures was also studied. It was found that all the mixtures of sugars in combination with asparagine or ammonium chloride were valueless for all the organisms exceptHelicostylum piriforme. Growth ofHelicostylum piriforme on the mixtures with asparagine as the nitrogen source was better than on the mixtures with ammonium chloride as the source of nitrogen. Asparagine being a favourable source counteractecd sorbose inhibition, while ammonium chloride failed to do so. On the other hand, both of the nitrogen sources failed to counteract sorbose inhibition in the rest of the organisms. None of the organisms could finish sorbose and rhamnose from any of the mixtures within the specified period.