New aspects on factors determining the sensitivity of the formaldehyde and glyoxylic acid fluorescence histochemical methods for monoamines

Summary The fluorophore and fluorescence yield from tryptamine and 3-methoxytyramine in histochemical protein models have been compared in the standard formaldehyde reaction, the acid-catalyzed formaldehyde reaction, the formaldehyde-ozone reaction, and the aluminum-formaldehyde reaction. In the standard formaldehyde reaction both the fluorophore and fluorescence yields are low. However, the other reactions give a dramatic increase in fluorescence intensity (18–20 times) from tryptamine and 3-methoxytyramine whereas only minor changes (up to 100% increase) in fluorophore yield are observed. It is concluded that the relative fluorescence intensity of each fluorophore molecule formed in the three modifications of the formaldehyde reaction is much higher than that of the molecules formed in the standard formaldehyde reaction. It has previously been demonstrated that the fluorophores formed from dopamine in the gaseous formaldehyde and glyoxylic acid reactions have a much higher (10 times) relative fluorescence intensity than the synthetic fluorophores. The present experiments show that if the histochemical models are dissolved in buffer after the reaction and new models are made from this solution, the fluorescence intensity of the fluorophores formed in the reaction is drastically reduced and becomes comparable to that of the synthetic ones. The results of this and our previous studies indicate that hitherto unknown fluorescence enhancing mechanisms play a major role for the fluorescence yield, i.e. the sensitivity, in the various formaldehyde and glyoxylic acid methods. One possible explanation to the high relative fluorescence intensity of the fluorophores formed in the histochemical reactions could be an energy transfer between, e.g. the non-fluorescent intermediary reaction products (the tetrahydro derivatives) and the fluorophores (the dihydroisoquinolines and dihydro--carbolines). Such an energy transfer is probably attenuated in the dissolved models, where the distances between and orientations of the various molecules have been changed.Abbreviations DA dopamine - FA formaldehyde - GA glyoxylic acid - 3-MT 3-methoxytyramine - 4-MT 3-hydroxy-4-methoxyphenylethylamine - T tryptamine - DHC dihydro--carbolines - THC tetrahydro--carboline - 2-Carb.Me-DHIQ 2-carboxymethyl-3,4-dihydroisoquinolinium compound - THIQ-1-COOH tetrahydroisoquinoline-1 carboxylic acid     

Photo and thermal reactions of ferrous hydroxide

The reactions of ferrous ion near neutral pH are of interest because of its known presence in the Archaean oceans. We have confirmed the long wavelength ultraviolet photochemical and the thermal reactions of ferrous hydroxide to form hydrogen. We have shown that a claim of the reduction of carbon dioxide to formaldehyde at neutral pH is mistaken. By the use of¹⁴C labelled compounds, we have found that less than 1 ppm of carbon dioxide is reduced to formaldehyde and less than 10 ppm of formate ion is so reduced. The thermal reaction to form hydrogen has a small activation energy of 7 kcal mole^–1. We conclude that thermal and photochemical formation of hydrogen from ferrous ion in the Archaean ocean could be comparable at pH 8–9. At lower pH, toward its limit at pH 5, the photochemical reaction would predominate. Both the thermal and photochemical reactions are specific for ferrous hydroxide, being far slower for the phosphate (>50- and 7-fold) and the bicarbonate (2- and 30-fold) complexes.     

Heteropolypeptides on Titan?

Since hydrogen cyanide is a component of Titan's hazy atmosphere, HCN polymers might also be present by way of a low energy pathway leading initially to the synthesis of polyaminomalonitrile. Subsequent reactions of HCN with the activated nitrile groups of this HCN homopolymer would then yield heteropolyamidines, readily converted to heteropolypeptides following contact with frozen water on the surface of Titan.Similar HCN polymers in the reducing atmospheres of Jupiter and Saturn could be major contributors to the yellow-brown-orange appearance of these giant planets.Any detection of such HCN chemistry by the Voyager missions or the pending Galileo probe would constitute evidence for the hypothesis that heteropolypeptides on the primitive Earth were synthesized directly from hydrogen cyanide and water without the intervening formation of -amino acids.Paper presented at the 6th College Park Colloquium, October 1981.     

Antagonistic reactions of arginine and lysine against formaldehyde and their relation to cell proliferation,apoptosis, folate cycle and photosynthesis

¹H, ¹³C NMR, ESMS and MS/MS investigations proved that there is an antagonism in the spontaneous reaction of formaldehyde with L-lysine and L-arginine. L-Arginine can only be hydroxymethylated on the guanidino group in a very fast reaction forming mono-, di-, and trihydroxymethyl arginines (HMA). L-Lysine can be methylated on the -amino group forming mono-, di-, and trimethyl lysine on physiological pH. Hydroxymethyl arginines are relative stable, isolable products, and can also be formed in biological systems, especially in plants. Significant amounts of hydroxymethyl arginines were identified in the aqueous extract of lyophilized kohlrabi, which can be formed in photosynthesis during CO₂ fixation. ¹⁴C-Formaldehyde formed in a short-term (10, 30 sec) ¹⁴CO₂ fixation reaction in Zea mays L. (early maturity variety: Szegedi TC 277) was captured by L-arginine, which occurs in leaves in large amount. Formaldehyde formed during photosynthesis can react not only with the arginine, but with ribulose-1,5-diphosphate present in leaves. In model reactions formaldehyde can react with the ene diole group of ribulose-1,5-diphosphate in the absence of Rubisco enzyme, which is a similar reaction to the addition of formaldehyde to L-ascorbic acid. Hydroxymethyl arginines (HMA) are endogenous formaldehyde carrier molecules transferring the bound formaldehyde to thymidylate synthase enzyme system incorporating it into the folate cycle. HMA can also carry the bound formaldehyde to the cells especially to the tumorous cells (HT29 adenocarcinoma), and cause significant inhibition of cell proliferation and causes apoptosis.     

Protein labeling by reductive methylation with sodium cyanoborohydride: Effect of cyanide and metal ions on the reaction

Previous studies (N. Jentoft and D. G. Dearborn, 1979, J. Biol. Chem.254, 4359) have demonstrated that reductive methylation with labeled formaldehyde and NaCNBH₃ provides a simple method for specifically labeling the amino groups of proteins using extremely mild reaction conditions. However, cyanide, which is one of the products of the reaction, reduces labeling effciency by reacting with formaldehyde to from the formaldehyde cyanohydrin addition product. Certain transition metal ions are able to prevent this secondary reaction by forming stable coordination complexes with cyanide. Inclusion of millimolar quantities of Ni(II) in reaction mixtures leads to a 20–30% increase in protein labeling so that maximal derivatization of amino groups can be realized with only a 3- to 4-fold ratio of formaldehyde to amine rather than the 5- to 10-fold excess necessary in the absence of metal ions.     

Theoretical interstellar and prebiotic organic chemistry: A tentative methodology

A theoretical methodology for the systematic study of the interstellar molecules is proposed.Some examples, dealing with formaldehyde excited states, formyl radical and ion, reactivity of the excited states of formic acid, methyl cyanide and methyl acetylene, as well as the reaction path of formaldehyde photodecomposition are presented.Quantum chemical methods appear to be a powerful tool to study the structure and behaviour of molecules related with interstellar space and the Origin of Life.     

Quantum-mechanical treatment of the electronic structure and geometry of hydrogen cyanide dimer

Experiments used to develop theories of chemical evolution seem to indicate that hydrogen cyanide, HCN, was an important molecule in prebiotic synthesis. In particular, polymerization products of hydrogen cyanide have been found to yield polypeptides upon hydrolysis. The proposed key intermediate in prebiotic synthesis is the aminocyanocarbene isomer of the dimer, either as a 1,3 biradical or as a dipolar singlet structure. Since this molecule has never been successfully isolated and characterized, a quantum mechanical study of various structures of the dieter is carried out using the INDO method. The results indicate that the lowest energy isomer is the iminoacetonitrile, with the aminocyanocarbene being next lowest. The triplet and singlet energy surfaces of the aminocyanocarbene intersect, so that for some geometries the singlet is lower in energy than the triplet, for others the reverse is true. The minima of both surfaces correspond to the linear configuration, with slightly different bond lengths. The triplet state minimum is 8·8 kcal/mol lower in energy than the singlet minimum. The calculated spin density distribution for the ground state of the carbene can be qualitatively described as a 1,3 biradical, in agreement with the early proposal of Kliss &; Matthews. The charge distribution of the singlet at its minimum energy geometry was also calculated. We found the charge separation to be less than that proposed for the dipolar structure of Moser et al. These calculations indicate that while the lowest energy isomer is the iminoacetonitrile, the aminocyanocarbene, the lowest-energy triplet, does have the appropriate spin distribution in its ground state for the biradical polymerization proposed in the theory of chemical evolution of Kliss &; Matthews. Experiments are suggested to determine the nature of the HCN polymerization mechanism, especially in the gas phase. By application of standard techniques used in polymer science, the nature of the gas phase polymerization of HCN can be determined, and the role of such reactions in chemical evolution can be better understood.     

Quantitative histochemical determination of succinic dehydrogenase activity in skeletal muscle fibres

Summary A histochemical technique was developed for the quantitative determination of succinic dehydrogenase (SDH) activity in muscle cross-sections using 1-methoxyphenazine methosulphate (mPMS) as the exogenous electron carrier, and azide as an inhibitor of cytochrome oxidase. The optimal composition of the incubation medium for the SDH reaction was determined. This histochemical procedure was compared to one using phenazine methosulphate (PMS) instead of mPMS and cyanide instead of azide. The substitution of mPMS and azide resulted in a substantial decrease in the non-specific reduction of nitroblue tetrazolium (NBT; the reaction indicator), i.e., nothing dehydrogenase activity. With mPMS and azide in the reaction medium, the production of NBT formazan was linear for at least 9 min during the enzymic reaction. This compared to a non-linear reduction of NBT during the initial stages of the reactions (SDH and nothing dehydrogenase) when using PMS and cyanide. The use of both mPMS and azide also eliminated the production of NBT monoformazan which occurred with PMS and cyanide. This procedure was shown to meet various criteria established for the quantification of histochemical reactions.     

Studies on the mutagenic action of formaldehyde in Drosophila

Summary Drosophila males were exposed to a sublethal concentration of cyanide gas prior to the injection of formaldehyde solutions. Compared to the controls which only received formaldehyde the frequency of sex-linked lethals was increased after the cyanide pretreatment in altogether six independent experiments. These results are taken as further proof that formaldehyde exerts at least part of its mutagenic effects via the formation of peroxides. It is suggested that an excessive amount of hydrogen peroxide, due to inhibition of the cytochrome and catalase enzyme systems, favours the formation of a mutagenic, organic peroxide, presumably dihydroxydimethyl peroxide. The fact that formaldehyde exerts an inhibiting effect on catalase in its own right might be of importance for the interpretation of its mutagenic action.It was also observed that after cyanide pretreatment, the mutagenic effectiveness of a mixture of formaldehyde and hydrogen peroxide was lower than that of formaldehyde alone. These findings can be interpreted by assuming that high concentrations of dihydroxydimethyl peroxide or of a combination of cyanide and this peroxide, eliminate selectively germ cells with induced mutations. It is possible that the same explanation applies to the low mutagenic effectiveness of a mixture of formaldehyde and hydrogen peroxide compared with that of formaldehyde alone when both are preceded by cyanide.With 3 figures in the text     

Chemical evolution of iron containing enzymes: Mixed ligand complexes of iron as intermediary steps

Activities of the iron complexes of evolutionary importance like K₄[Fe(CN)₆], K₄[Fe(CN)₅(gly)], and K₄[Fe(CN)₅(trigly)] have been tested towards some redox reactions of biological significance, namely, decomposition of hydrogen peroxide, dehydrogenation of NADH and ascorbic acid both coupled with reduction of methylene blue. It has been observed that the catalytic activities of iron (II) complexes towards the redox reactions studied at pH 9.18 followed the order, K₄[Fe(CN)₆]4[Fe(CN)₅(gly)]4[Fe(CN)₅(trigly)]. Decomposition of H₂O₂ catalysed by cyanocomplexes of iron (II) has been discussed through the formation of an innersphere complex in which loosly bound ligands like, glycine and triglycine are replaced by hydroperoxide ion. A tentative mechanism for the catalysed decomposition of H₂O₂ has been discussed.Based upon the experimental observations a hypothesis on the evolution of iron containing enzymes has been envisaged as: iron(II) ion iron(II) cyanide complexes mixed ligand iron(II) cyanide and amino acid complexes iron(II) complexes of macromolecules proenzyme or early enzyme containing iron(II).     

Isolation and characterization of a cyanide-utilizing Burkholderia cepacia strain

A bacterium that utilizes cyanide as a nitrogen source was isolated from soil after enrichment in a liquid medium containing potassium cyanide (10mM) and glucose (1.0%, w/v). The strain could tolerate and grow in potassium cyanide at concentrations of up to 25mM. It could also utilize potassium cyanate, potassium thiocyanate, linamarin and a range of aliphatic and aromatic nitriles. The isolate was tentatively identified as Burkholderia cepacia strain C-3. Ammonia and formic acid were found in the culture supernatant of the strain grown on fructose and potassium cyanide, no formamide was detected, suggesting a hydrolytic pathway for the degradation of cyanide. The cyanide-degrading activity was higher in early and the stationary phase cells. Crude cell extracts of strain C-3 grown on nutrient broth exhibited cyanide-degrading activity. The characteristics of strain C-3 suggest that it would be useful in the bioremediation of cyanide-containing waste.     

The origin of proteins: Heteropolypeptides from hydrogen cyanide and water

Evidence from laboratory and extraterrestrial chemistry is presented consistent with the hypothesis that the original heteropolypeptides on Earth were synthesized spontaneously from hydrogen cyanide and water without the intervening formation of α-amino acids, a key step being the direct polymerization of atmospheric hydrogen cyanide to polyaminomalononitrile (IV) via dimeric HCN. Molecular orbital calculations (INDO) show that the most probable structure for (HCN)₂ is azacyclopropenylidenimine. Successive reactions of hydrogen cyanide with the reactive nitrile side chains of IV then yield heteropolyamidines which are converted by water to heteropolypeptides. To study this postulated modification of a homopolymer to a heteropolymer, poly-α-cyanoglycine (IX) was prepared from the N-carboxyanhydride of α-cyanoglycine. Hydrolysis of IX, a polyamide analog of the polyamidine IV, yielded glycine. However, when IX was hydrolysed after being treated with hydrogen cyanide, other α-amino acids were also obtained including alanine, serine, aspartic acid and glutamic acid, suggesting that the nitrile groups of IX (and therefore of IV) are indeed readily attacked by hydrogen cyanide as predicted. Further theoretical and experimental studies support the view that hydrogen cyanide polymerization along these lines is a universal process that accounts not only for the past formation of primitive proteins on Earth, but also for the yellow-brown-orange colors of Jupiter today and for the presence of water-soluble compounds hydrolyzable to α-amino acids in materials obtained from environments as diverse as the moon, carbonaceous chondrites and the reaction chambers used to simulate organic synthesis in planetary atmospheres.     

Histochemical observations on endogenous lactoperoxidase activity of the bovine submandibular salivary gland

Synopsis Seromucous demilunar cells of glutaraldehyde-fixed bovine submandibular salivary glands are intensely stained when sections are incubated in a benzidine-or a 3,3-diaminobenzidine-hydrogen peroxide medium in the pH range 6.0–9.0 whereas mucous acinar cells are completely unreactive. The histochemical reaction is completely inhibited by 3-amino-1,2,4-triazole. In contrast 2,4-dichlorophenol or potassium cyanide has little or no effect on the staining of demilunar cells. Striated duct cells also display a positive reaction with the diaminobenzidine method; this staining reaction, however, is most intense at pH 6.0. Furthermore, this reaction is markedly affected by potassium cyanide. The positive histochemical benzidine and diaminobenzidine reactions of demilunar cells probably corresponds to endogenous lactoperoxidase activity. On the other hand, the positive reaction shown by striated ducts, with optimal staining at pH 6.0 and which is completely inhibited by potassium cyanide, seems to be due to cytochromal oxidation of diaminobenzidine.     

Effective formaldehyde fixation of the reversibly interacting ribosomal system

The possiblity of using formaldehyde fixation for the quantitative studies of the reversibly interacting ribosomal system 50S–30S 50S+30S was investigated. Conditions have been found under which formaldehyde fixation of a reaction mixture of purified ribosomal particles proved to be possible without any change in the initial ratio of its components. This fixation technique was used in studying the dependence of the association level on Mg⁺⁺ ion concentration. An estimation has been made of the number of Mg⁺⁺ ions which bind during the association of 50S and 30S subparticles.     

Catalysis of an isotopic exchange between CO2 and the carboxyl group of acetate by Methanosarcina barkeri grown on acetate

Cell suspensions of Methanosarcina barkeri (strain Fusaro) grown on acetate were found to catalyze the formation of methane and CO₂ from acetate (30–40 nmol/min·mg protein) and an isotopic exchange between the carboxyl group of acetate and ¹⁴CO₂ (30–40 nmol/min·mg protein). An isotopic exchange between [¹⁴C]-formate and acetate was not observed. Cells grown on methanol mediated neither methane formation from acetate nor the exchange reactions. The data indicate that the isotopic exchange between CO₂ and the carboxyl group of acetate is a partial reaction of methanogenesis from acetate. Both reactions were completely inhibited by low concentrations of cyanide (20 M) or of hydrogen (0.5% in the gas phase). Methane formation from acetate was also completely inhibited by low concentrations of carbon monoxide (0.2% in the gas phase) whereas only significantly higher concentrations of CO had an effect on the exchange reaction. In the concentration range tested KCN, H₂ and CO had no effect on methane formation from methanol or from H₂ and CO₂; however, cyanide (20 M) also affected methane formation from CO. The results are discussed with respect to proposed mechanisms of methane and CO₂ formation from acetate.     

Was ferrocyanide a prebiotic reagent?

Hydrogen cyanide is the starting material for a diverse array of prebiotic syntheses, including those of amino acids and purines. Hydrogen cyanide also reacts with ferrous ions to give ferrocyanide, and so it is possible that ferrocyanide was common in the early ocean. This can only be true if the hydrogen cyanide concentration was high enough and the rate of reaction of cyanide with ferrous ions was fast enough. We show experimentally that the rate of formation of ferrocyanide is rapid even at low concentrations of hydrogen cyanide in the pH range 6–8, and therefore an equilibrium calculation is valid. The equilibrium concentrations of ferrocyanide are calculated as a function of hydrogen cyanide concentration, pH and temperature. The steady state concentration of hydrogen cyanide depends on the rate of synthesis by electric discharges and ultraviolet light and the rate of hydrolysis, which depends on pH and temperature. Our conclusions show that ferrocyanide was a major species in the prebiotic ocean only at the highest production rates of hydrogen cyanide in a strongly reducing atmosphere and at temperatures of 0°C or less, although small amounts would have been present at lower hydrogen cyanide production rates. The prebiotic application of ferrocyanide as a source of hydrated electrons, as a photochemical replication process, and in semi-permeable membranes is discussed.     

The Sugar Model: Catalysis by Amines and Amino Acid Products

Ammonia and amines (including amino acids) were shown tocatalyze the formation of sugars from formaldehyde andglycolaldehyde, and the subsequent conversion of sugars tocarbonyl-containing products under the conditions studied (pH5.5 and 50°C). Sterically unhindered primary amineswere better catalysts than ammonia, secondary amines, andsterically hindered primary amines (i.e.-aminoisobutyric acid). Reactions catalyzed by primaryamines initially consumed formaldehyde and glycolaldehyde about15–20 times faster than an uncatalyzed control reaction. Theamine-catalyzed reactions yielded aldotriose (glyceraldehyde),ketotriose (dihydroxyacetone), aldotetroses (erythrose andthreose), ketotetrose (erythrulose), pyruvaldehyde, acetaldehyde,glyoxal, pyruvate, glyoxylate, and several unindentifiedcarbonyl products. The concentrations of the carbonyl products,except pyruvate and ketotetrose, initially increased and thendeclined during the reaction, indicating their ultimateconversion to other products (like larger sugars or pyruvate).The uncatalyzed control reaction yielded no pyruvate orglyoxylate, and only trace amounts of pyruvaldehyde, acetaldehyde and glyoxal. In the presence of 15 mM catalyticprimary amine, such as alanine, the rates of triose andpyruvaldehyde of synthesis were about 15-times and 1200-timesfaster, respectively, than the uncatalyzed reaction. Sinceprevious studies established that alanine is synthesized fromglycolaldehyde and formaldehyde via pyruvaldehyde as its directprecursor, the demonstration that the alanine catalyzes theconversion of glycolaldehyde and formaldehyde to pyruvaldehydeindicates that this synthetic pathway is capable ofautocatalysis. The relevance of this synthetic process, namedthe Sugar Model, to the origin of life is discussed.     

Prebiotic Amino Acid Thioester Synthesis: Thiol-Dependent Amino Acid Synthesis from Formose Substrates (Formaldehyde and Glycolaldehyde) and Ammonia

Formaldehyde and glycolaldehyde (substrates of the formose autocatalytic cycle) were shown to react with ammonia yielding alanine and homoserine under mild aqueous conditions in the presence of thiol catalysts. Since similar reactions carried out without ammonia yielded -hydroxy acid thioesters (Weber, 1984a, b), the thiol-dependent synthesis of alanine and homoserine is presumed to occur via amino acid thioesters – intermediates capable of forming peptides (Weber and Orgel 1979). A pH 5.2 solution of 20 mM formaldehyde, 20 mM glycolaldehyde, 20 mM ammonium chloride, 23 mM 3-mercaptopropionic acid, and 23 mM acetic acid that reacted for 35 days at 40°C yielded (based on initial formaldehyde) 1.8% alanine and 0.08% homoserine. In the absence of thiol catalyst, the synthesis of alanine and homoserine was negligible. Alanine synthesis required both formaldehyde and glycolaldehyde, but homoserine synthesis required only glycolaldehyde. At 25 days the efficiency of alanine synthesis calculated from the ratio of alanine synthesized to formaldehyde reacted was 2.1%, and the yield (based on initial formaldehyde) of triose and tetrose intermediates involved in alanine and homoserine synthesis was 0.3 and 2.1%, respectively. Alanine synthesis was also seen in similar reactions containing only 10 mM each of aldehyde substrates, ammonia, and thiol. The prebiotic significance of these reactions that use the formose reaction to generate sugar intermediates that are converted to reactive amino acid thioesters is discussed.     

Studies on the Degradation Mechanisms of Proteins and Their Derivatives in the Foods

Cysteine mercaptals and mercaptoles were prepared by the reactions of l-cystine with formaldehyde, acetaldehyde, n-butyraldehyde, benzaldehyde, furfural, pyruvic acid and levulinic acid in 6 n hydrochloric or sulfuric acid. Hydrogen sulfide released from cysteine mercaptals and mercaptoles in heated aqueous solutions (oil bath: 120°C) was determined. Although a small amount of hydrogen sulfide was liberated from l-cystine on one hour heating, its amount increased suddenly after three hours. Among these compounds l-cystine mercaptal of furfural was most unstable and a large amount of hydrogen sulfide was produced.