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Acetylation of prothrombin fragment 1 in acetate-borate buffer at pH 8.5 resulted in the appearance of increased light absorbance at about 250 nm. Protease digestions resulted in isolation of a single peptide (residues 94-99) with intense absorbance at about 250 nm (estimated extinction coefficient of 5000 M-1 cm-1). Amino acid analysis showed the expected composition except for the absence of His-96. Instead, an unidentified amino acid which had a ninhydrin product with absorption properties similar to those of proline eluted near aspartate. When sequenced, this peptide (YP?KPE containing epsilon-amino-acetyllysine) lacked histidine at the third position but gave a high yield of a PTH derivative that eluted near PTH-Gly from the HPLC column. Fast atom bombardment mass spectrometry of the derivatized 94-99 peptide showed a mass that was 74 units higher than expected. The histidine degradation product was identified as a di-N-acetylated side chain with an opened imidazole ring and loss of C2 of the ring. While a similar degradation pattern has previously been reported during acylation of histidine, the high chemical reactivity exhibited by His-96 was unusual. For example, under conditions sufficient for quantitative derivatization of His-96, His-105 of fragment 1 was not derivatized to a detectable level. Furthermore, His-96 in fragment 1 was at least an order of magnitude more susceptible to degradation than His-96 in the isolated 94-99 peptide. His-96 is therefore one of several neighboring amino acids of the kringle portion of fragment 1 that displays highly unusual chemistry (see also Asn-101 [Welsch, D.J., & Nelsestuen, G. L. (1988) Biochemistry 27 4946-4952] and Lys-97 [Pollock, J.S., Zapata, G.A., Weber, D.J., Berkowitz, P., Deerfield, D.W., II, Olson, D.L., Koehler, K.A., Pedersen, L.G., & Hiskey, R.G. (1988) in Current Advances in Vitamin K Research (Suttie, J.W., Ed.) pp 325-334, Elsevier Science, New York]).(ABSTRACT TRUNCATED AT 250 WORDS)

The chemical properties of the three amino groups of insulin were obtained at 10 and 37 degrees C using the competitive labelling technique with acetic anhydride as the labelling reagent. At 10 degrees C, pK values of 7.9, 7.2, and 7.8 were found for the glycyl A1, phenylalanyl B1, and lysyl B29 amino groups. When compared with standard amino compounds by means of a Brønsted plot, the two amino-termini were found to be 'super-reactive' and the lysyl epsilon-amino group buried. In the presence of carbon dioxide at physiological pH values, all three amino groups became much less reactive indicating that they had reacted to form carbamino derivatives. Above pH 8 the reactivities of the glycyl amino terminus and epsilon-amino group increase sharply indicating that insulin is undergoing a conformational change which is most likely a change in its association state. At 37 degrees C the amino groups do not titrate normally but exhibit sharp increases in reactivity over the physiological pH range with the midpoints in the pH reactivity profiles between pH values of 7.0 and 7.3. This behaviour is interpreted as a rapid disaggregation of insulin to form monomers as a result of the ionization of the amino groups. It is concluded that at physiological pH and temperature all three amino groups are deprotonated.

A gas chromatographic-mass spectrometric method for analysis of L-[2-15N]- and L-[5-15N]glutamine is described. The method is based on direct acylation of glutamine with trifluoroacetic anhydride and the formation of the N,N-bis-trifluoroacetyl-L-glutamine derivative. This simple and sensitive method is capable of detecting approximately 0.5 atom% excess 15N in as little as 10 microliter of plasma with a mean coefficient of variance of 11.6%. The method was applied to determine the appearance of 15N enrichment in plasma amino-N and amide-N of glutamine in a healthy adult volunteer during a constant infusion of 15NH4Cl. A plateau level of 3.7 and 2.6 atom% excess was observed in amide-N and amino-N, respectively, at 1 and 2 h after 15NH4Cl infusion was started.

Aliphatic, alicyclic and arylcarboxylic esters of norethisterone and levonorgestrel were prepared in a one-step synthesis and in near-quantitative yield using trifluoroacetic anhydride.

A rapid gas chromatographic-mass spectrometric method for the determination of 15N in the guanidino nitrogens of arginine is described. The method is based on formation of the N-tetratrifluoroacetyl-arginine derivative. Approximately 0.15 mol% excess 15N can be detected in as little as 50 microliters of plasma with an average coefficient of variation of 8.8%. The possible fragmentation pattern of the N-tetra-trifluoroacetyl-arginine derivative is described. The method was applied to determine the appearance of 15N enrichment in plasma arginine in a healthy adult volunteer during a constant infusion of 15NH4Cl. A plateau level of 0.7 atom% excess was observed 2 h after the 15NH4Cl infusion was started.

The possible existence of intramolecular interactions involving the tyrosine and histidine residues in angiotensin II has been investigated by measuring the reactivities of the functional groups in the molecule. Angiotensin II catalyzed the hydrolysis of p-nitrophenylacetate in the pH range 6.6-8.2 at higher rates than were consistent with the reactivities of the free constituent functional groups, and had 2-4% of the activity of chymotrypsin between pH 6.6 and 7.5. Treatment of angiotensin II with acetic anhydride demonstrated that the tyrosine hydroxyl and the imidazole side-chain in angiotensin II acetylated and deacetylated at markedly higher rates than for the free amino acids, indicating increased nucleophilicities and the presence of intrinsic deacetylation mechanisms for these residues in angiotensin II. These findings are consistent with the presence of tyrosine hydroxyl-histidine-carboxylate charge relay system in ANG II in aqueous environments, and suggest that ANG II may act at membrane receptors by a mechanism which is analogous to that operating in serine proteases.

Modification with acetic anhydride of nucleosomes from chicken erythrocytes at low ionic strength (less than 0.1 M NaCl) is accompanied by the formation of residual particles and the release of free DNA. This DNA has been identified as single-stranded by thermal denaturation, digestion with nuclease S1, and elution from hydroxyapatite. In contrast, if modification takes place at 0.6 M NaCl, the liberated DNA is mainly double-stranded. The release of the free energy stored in folded nucleosomal DNA, triggered by the weakening of lysine-DNA interactions which takes place upon modification, might be responsible for the observed denaturation of DNA at low ionic strength.

Protein P, an anion-specific channel-forming protein from the outer membrane of Pseudomonas aeruginosa was chemically modified by acetylation and syccinylation of its accessible amino groups. The chemically modified protein retained its ability to form oligomers on sodium dodecyl sulfate polyacrylamide gels, whereas only the acetylated protein formed channels in reconstitution experiments with lipid bilayers. Acetylated protein P demonstrated a substantially reduced mean single channel conductance (25 pS at 1 M KCl) compared to the native protein P channels (250 pS at 1 M KCl) when reconstituted into black lipid bilayer membranes. The homogeneous size distribution of single-channel conductances suggested that all of the protein P molecules had been acetylated. Zero-current potential measurements demonstrated that the acetylated protein P channel was only weakly selective for anions and allowed the permeation of cations, in contrast to the native protein P channels, which were more than 100-fold selective for anions over cations. The dependence of conductance on salt concentration was changed upon acetylation, in that acetylated protein P demonstrated a linear concentration-conductance relationship, whereas native protein P channels became saturated at high salt concentrations. These data strongly suggested that the basis of anion selectivity for native protein P channels is fixed amino groups. In agreement with this, we could demonstrate a 2.5-fold decrease in single-channel conductance between pH 7 and pH 9, between which pH values the epsilon-amino groups of amino acids would start to become deprotonated. Two alternative schemes for the topography of the protein P channel and localization of the fixed amino groups are presented and discussed.

Oligosaccharides were prepared from alpha 1-antitrypsin (PiM) and human serum transferrin by hydrazinolysis and then reacetylated with radiolabelled acetic anhydride. Gel permeation chromatography revealed similarities between the two preparations. The antitrypsin oligosaccharides were resolved into 76% concanavalin A binding and 24% nonbinding sugar chains by lectin affinity chromatography; both fractions were further resolved by preparative paper electrophoresis. Small amounts of neutral oligosaccharides, as well as approximately 30% "monosialo" and 67% "disialo" biantennary oligosaccharides, were identified in the lectin-binding pool. The disialo oligosaccharide fraction was susceptible to sequential degradation with exoglycosidases. Upon incubation of the monosialo biantennary oligosaccharide fraction with CMP-[14C]sialic acid and a rat liver Golgi preparation as a source of transferase, a new doubly labelled peak was formed that eluted from the gel permeation chromatography column in the same volume as the disialo biantennary oligosaccharide fraction. The paper electrophoretogram of the lectin-nonbinding oligosaccharides revealed the presence of limited amounts of neutral oligosaccharides and oligosaccharides carrying two and three negative charges. The lectin-nonbinding oligosaccharide fraction with two charges was tentatively identified as disialo biantennary oligosaccharide containing a bisecting N-acetylglucosamine residue, and the fraction with three negative charges was tentatively identified as trisialo triantennary oligosaccharide.

The title linker was synthesized in the manner of dimer-units introduction through liquid-phase and phosphoroamidate approaches involving deamidation with tetrabutylammonium nitrite-acetic anhydride.

The effectiveness of acetolysis as a tool in structural characterization of mono- and di-O-alkyl phosphoglycerides was investigated. Surprisingly, it was found that the di-O-alkyl phosphoglycerides were resistant to attack during acetolysis, whereas the mono-ether types, with a free hydroxyl function or an ester on carbon-2, were easily attacked at the glycerol-phosphate bond. On the other hand, Vitride reduction occurred readily with the mono-ether or di-ether phosphoglycerides. The implications of these findings as they relate to identification of ether phospholipids in tissues are discussed.

The relative reactivities of lysine residues of tropomyosin complexed with troponin have been measured in order to locate the binding site of troponin on tropomyosin in a complex between the two native proteins. The lysines were labeled with acetic anhydride using a competitive labeling procedure and the relative reactivities of tropomyosin lysine containing peptides were compared to those from tropomyosin labeled in the absence of troponin (S. E. Hitchcock-DeGregori, S. F. Lewis, and T. M.-T. Chou, (1985) Biochemistry 24, 3305-3314). Analysis of about two-thirds of the lysines indicates that troponin affects the reactivities of lysines along the length of the tropomyosin, indicating long-range effects. The inferred binding site is more extensive than previously reported, about 25 nm, extending from res. 136 to the carboxy-terminus and to res. 30 beyond the end-to-end overlap in the amino-terminal region of the next tropomyosin molecule.

Human plasma low-density lipoprotein (LDL) was modified chemically with either the monocarboxylic acid derivative, acetic anhydride, or the dicarboxylic acid derivative, citraconic anhydride, reagents which react principally with the lysine residues of protein. The modifications increased the net negative charge on the LDL particles, with citraconyl-LDL displaying a greater negative charge than acetylated LDL. Neither the antigenic reactivity nor the overall gross protein/lipid composition of the LDL were affected by the modification procedures, although a small reduction in the total cholesterol content was observed. The altered LDL species lost the ability to bind to the high-affinity cell surface B/E receptor but both bound to mouse peritoneal macrophages with saturable high-affinity kinetics. At 4 degrees C, the macrophages bound 125I-labelled citraconyl-LDL more avidly (K = 21 X 10(-3) ml/ng) than they bound labelled acetyl-LDL (K = 2 X 10(-3) ml/ng). Competitive inhibition studies indicated that acetyl-LDL and citraconyl-LDL were bound to non-identical sites on the macrophage monolayer surface and that the binding site for citraconyl-LDL was also different from that recognized by hypercholesterolaemic rabbit plasma VLDL (beta VLDL).

Purothionin from wheat flour was chemically modified by acetic or succinic anhydride under specific conditions. The complete modification of all amino groups of purothionin caused a large change in the net charge of the molecule, leading to the loss of the toxicity to mice and yeast. The sole tyrosyl residue in purothionin was nitrated by tetranitromethane at neutral pH or iodinated by the lactoperoxidase method. The nitro- and diiodo-derivatives of purothionin showed considerably reduced toxicity. Based on these modification studies we conclude that the positive charges of lysyl residues have an important role in the interaction with the negatively charged cell surface, and that the emergence of the toxicity of purothionin depends on a certain state of the tyrosyl residue.

The C-17 fatty acid esters of estradiol, known as the lipoidal derivatives of estradiol, LE2, are metabolites of estradiol that were originally isolated from various tissues after in vitro incubations with estradiol. These steroidal esters are active estrogens with extremely prolonged potencies. The present study investigates the existence of LE2 in human blood using a radiochemical isotope dilution technique. The LE2 fraction from blood was isolated, saponified, and the hydrolyzed estradiol was then acetylated with [3H]acetic anhydride. It was found that 3H was incorporated into estradiol diacetate, demonstrating that LE2 is present in human blood. Thus these steroidal esters represent a new class of endogenous estrogens which have not been previously considered in the physiology of the female sex steroids.

Binding of Ricinus communis agglutinin (RCA 120) to carbohydrate receptors of human lymphocytes and erythrocytes is enthalpically driven. As in the case of simple saccharides, the delta S contribution is always unfavorable to the interaction. This result is different from that observed for other lectins and might indicate that hydrophobic interactions do not play a dominant role in binding of RCA 120 to cell surfaces.

Structural analysis of the phosphomannan isolated from yeast-form cells of a pathogenic yeast, Candida albicans J-1012 strain, was conducted. Treatment of this phosphomannan (Fr. J) with 10 mM HCl at 100 degrees C for 60 min gave a mixture of beta-1,2-linked manno-oligosaccharides, from tetraose to biose plus mannose, and an acid-stable mannan moiety (Fr. J-a), which was then acetolyzed by means of an acetolysis medium, 100:100:1 (v/v) mixture of (CH3CO)2O, CH3COOH, and H2SO4, at 40 degrees C for 36 h in order to avoid cleavage of the beta-1,2 linkage. The resultant manno-oligosaccharide mixture was fractionated on a column of Bio-Gel P-2 to yield insufficiently resolved manno-oligosaccharide fractions higher than pentaose and lower manno-oligosaccharides ranging from tetraose to biose plus mannose. The higher manno-oligosaccharide fraction was then digested with the Arthrobacter GJM-1 alpha-mannosidase in order to cleave the enzyme-susceptible alpha-1,2 and alpha-1,3 linkages, leaving manno-oligosaccharides containing the beta-1,2 linkage at their nonreducing terminal sites, Manp beta 1----2Manp alpha 1----2Manp alpha 1----2Manp alpha 1----2Man, Manp beta 1----2Manp beta 1----2Manp alpha 1----2Manp alpha 1---- 2Manp alpha 1----2Man, and Manp beta 1----2Manp beta 1----2Manp beta 1----2Manp alpha 1---- 2Manp alpha 1----2Manp alpha 1----2Man. However, the result of acetolysis of Fr. J-a by means of a 10:10:1 (v/v) mixture of (CH3CO)2O, CH3COOH, and H2SO4 at 40 degrees C for 13 h was significantly different from that obtained by the mild acetolysis method; i.e., the amount of mannose was apparently larger than that formed by the mild acetolysis method. In summary, a chemical structure for Fr. J as a highly branched mannan containing 14 different branching moieties was proposed.

A sensitive and convenient electron-capture gas chromatographic procedure for analysis of MHPG in small volumes of urine is described. The method involves acetylation under aqueous conditions, extraction with an organic solvent and reaction with trifluoroacetic anhydride under anhydrous conditions. The resultant derivative is separated and quantitated on a gas chromatograph equipped with a fused silica capillary column and an electron-capture detector.

A procedure for the determination of the proportions of diacyl, alkenylacyl and alkylacyl subclasses of glycerophospholipids was developed. The procedure involves: (1) acid methanolysis of the phospholipid followed by Bligh/Dyer extraction of fatty acid methyl esters (FAME) derived from acyl chain types, dimethylacetals (DMA) derived from alkenyl ether chain types, and lysoalkyl phosphatidic acids (lysoalkyl-PA) derived from alkyl ether chain types; and (2) subsequent acetolysis to convert the lysoalkyl-PA to monoalkyl glycerol diacetates (MAGD). GLC analysis and quantitation (using internal standard, 21:0 FAME) of FAME, DMA and MAGD allowed calculation of the proportions of the three molecular subclasses. The methanolysis/acetolysis procedure gave an overall mean phospholipid recovery of 95 +/- 3%. Analysis of the major phospholipids in four separate preparations of fresh resting human platelets by this procedure showed the following range of molecular subclasses: phosphatidylcholine (PC), 86-92 mol % diacyl, 6-10 mol % alkylacyl and 2-3 mol % alkenylacyl; and phosphatidylethanoline (PE), 39-60 mol % diacyl, 5-8 mol % alkylacyl and 34-55 mol % alkenylacyl. The results of these subclass analyses were in general agreement with those reported in the literature.

The chymotryptic fragment of bacteriorhodopsin, C-2 (residues 1-71), has been acetylated completely at its three lysines (residues 30, 40, and 41) by treatment with acetic anhydride. The triacetylated C-2 fragment is able to reassociate with fragment C-1 (residues 72-248) and the complex binds all-trans-retinal to form a native bacteriorhodopsin-like chromophore, which is essentially identical with that formed from fragments C-2 and C-1. Further, the kinetics and pH dependence of chromophore regeneration and the proton pumping of the reconstituted triacetylated C-2 and C-1 complex are indistinguishable from that of the unmodified C-2 and C-1 complex. However, the extent of regeneration of the chromophore from triacetylated C-2 and C-1 is less than that from fragments C-2 and C-1, suggesting that the acetylated C-2 fragment is less stable than unacetylated C-2 in the reconstitution medium. We conclude that the amino groups in Lys-30, -40, and -41 do not contribute to the stabilization of the folded bacteriorhodopsin structure and are not required for proton translocation.

The effect of a "bisecting" 2-acetamido-2-deoxy-beta-D-glucopyranosyl group, linked (1----4) to the beta-D-mannopyranosyl group of asparagine-linked complex and hybrid oligosaccharides, on the binding of [14C]acetylated glycopeptides to columns of immobilized concanavalin A (Con A), Phaseolus vulgaris erythroagglutinin (E-PHA), and Ricinus communis agglutinin-120 (RCA-120) was investigated. The presence of this "bisecting" GlcNAc group caused significant inhibition of the binding to ConA-agarose of biantennary complex glycopeptides in which the two branches are terminated at their nonreducing ends by two GlcNAc groups, or by a Gal and a GlcNAc group, or by two Gal groups, or by a Man and a GlcNAc group. Binding of biantennary, complex glycopeptides to E-PHA-agarose required a "bisecting" GlcNAc group, a Gal group at the nonreducing terminus of the alpha-D-Man-p-(1----6) branch, and a terminal or internal GlcNAc residue linked beta-(1----2) to the alpha-D-Manp-(1----3) branch. Binding to RCA-120-agarose occurred only when at least one nonreducing terminal Gal group was present, and increased as the proportion of terminal Gal groups increased; the presence of a "bisecting" GlcNAc group caused either enhancement or inhibition of these binding patterns. It is concluded that a "bisecting" GlcNAc group affects the binding of glycopeptides to all three lectin columns.

A modified procedure in developed for acetylation by means of 14C-acetic anhydride of highly purified collagen I preparations. The acetylated collagen exhibited high specific radioactivity and was effectively hydrolyzed by bacterial collagenase. The 14C-acetylated collagen was used as a radioactive marker in electrophoretic analysis of labelled proteins.

Capping is a useful technique to facilitate purification of a crude deprotected peptide in solid-phase peptide synthesis. However, we observed a serious side reaction caused by the Ac2O capping procedure, when it was applied to a synthesis of a peptide containing His. The mechanism of the side reaction was studied.

The kinetics of acetylation of alcohols by acetyl chloride and acetic anhydride, with N-methylimidazole as the catalyst, were studied in acetonitrile solution at 25 degrees; some measurements were also made with 4-dimethylaminopyridine as the catalyst. The acetic anhydride-N-methylimidazole system proceeds entirely by a general base catalysis, whereas the acetyl chloride-N-methylimidazole system reacts entirely via a nucleophilic route, with the intermediate formation of the N-acylated catalyst. The reaction of this intermediate with the alcohol is general base catalyzed. The acetyl chloride-4-dimethylaminopyridine system also reacts via the nucleophilic route. In the acetic anhydride-4-dimethylaminopyridine system a small fraction of the intermediate was detected. The acetic anhydride-N-methylimidazole system was studied in n-propanol-acetonitrile solvent mixtures; no spectral evidence for intermediate formation was seen. However, the hydrolysis reaction in acetic anhydride-N-methylimidazole, studied over a wide range of water-acetonitrile mixtures, revealed a change in mechanism from general base in dry acetonitrile to a solely nucleophilic route at high water concentrations.

Thylakoid membrane proteins are organized so as to shield 30-50 nmol H+ (mg Chl)-1 from freely equilibrating with either the external or the lumen aqueous phases. Amine groups provide binding sites for this metastable buffering array and can be quantitatively measured by acetylation using [3H]acetic anhydride. The principle of the assay is that a metastable acidic domain will have relatively less of the reactive neutral form of the amine compared to the amount present after addition of an uncoupler. The extent of the acetylation reaction is strongly influenced by whether the lumen pH comes to complete equilibrium with the external pH prior to adding the acetic anhydride. Determination of the lumen pH by [14C]methylamine distribution after the standard 3 or 5 min equilibration in pH 8.6 buffer indicated that the lumen may have been 0.2 to 0.3 pH more acidic than the external phase. This effect was taken into account by determining the pH dependence, in the pH 8.2-8.6 range, of acetylation of the membrane proteins studied, and the labeling data were conservatively corrected for this possible contribution. Experiments were carried out to identify the thylakoid proteins that contribute such metastable domain amine groups, using the above conservative correction. Surprisingly, plastocyanin contributes buried amine groups, but cytochrome f did not give evidence for such a contribution, if the conservative correction in the labeling was applied. If the correction was too conservative, cytochrome f may contribute amines to the sequestered domains. The new methodology verified earlier results suggesting that three Tris-releasable photosystem II-associated proteins also contribute significantly to the sequestered amine-buffering array.

Calmodulin was trace labeled by acetylation with [3H]acetic anhydride in the presence and absence of a 30% molar excess of the phosphatase calcineurin; phenylalanine was included in the reaction mixtures as an internal standard. The level of 3H acetylation of each of the 7 lysines was determined and corrected for differences arising from reaction conditions using the labeling of the internal standard, following procedures that are closely similar to those used in a previous study of the interaction of calmodulin with myosin light chain kinase (Jackson, A. E., Carraway, K. L., III, Puett, D., and Brew, K. (1986) J. Biol. Chem. 261, 12226-12232). The interaction with calcineurin was found to produce a 10-fold reduction in the acetylation of lysine 75, with lesser but significant effects on lysines 21 and 148. A small but reproducible perturbation of lysine 77 was also observed. The results are similar to those that are produced by the interaction with myosin light chain kinase. However, when they are compared with two recent reports between which there are major discrepancies (Manalan, A. S., and Klee, C. B. (1987) Biochemistry 26, 1382-1390; Winkler, M. A., Fried, V. A., Merat, D. L., and Cheung, W. Y. (1987) J. Biol. Chem. 262, 15466-15471), our results are in good agreement with those obtained in the former study. From the location of the perturbed groups in the three-dimensional structure of calmodulin, it appears that the interaction site on calmodulin for calcineurin, as well as for myosin light chain kinase, is very extended and may include hydrophobic pockets at homologous sites near the carboxyl-terminal ends of the two halves of the molecule.

The synthesis of the tripeptide D-Phe-Pro-Arg with the nitrile group instead of the carboxylgroup is described. Initially, the corresponding peptide amide was synthesized by conventional methods in solution using Boc and Fmoc as the protecting group for D-Phe. The dehydration in order to create the nitrile moiety was achieved by treating the peptide amide with phosphorus oxichloride or trifluoroacetic anhydride. Best results were obtained by the use of phosphorus oxichloride in pyridine as the solvent in the presence of imidazole. After deprotection of the N-terminal amino acid the crude product was purified by chromatography on Butyl-Fractogel HW-40 (S). The purity of the final product was checked on a RP18 phase by hplc. The existence of the nitrile group was demonstrated by i.r. and 13C-n.m.r. spectra. The peptide nitrile exhibited a strong inhibition of thrombin compared to the tripeptide amide.

Upon modification of neurotoxin RTX-III at amino groups with [3H]acetic anhydride, four monoacetyl and four diacetyl derivatives have been obtained. Acetylation of the N-terminal amino group led to 12-fold decrease of toxicity in mice. Monoderivatives of the toxin with either Lys or two out of three C-terminal Lys residues modified showed a 2-fold drop in toxicity as compared with the native RTX-III. Diacetylation caused a 30 to 35-fold decrease in toxicity, the N-terminal amino group being modified in all the derivatives. As assessed by circular dichroism method, the modification of amino groups, except for N-terminal one, affected secondary structure of the toxin. The data suggest the N-terminal amino group to be essential for toxicity of RTX-III.

The specificity of the Barrnett-Seligman method for the histochemical demonstration of alpha-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.