Proton magnetic resonance study of nucleosides,nucleotides, and dideoxynucleoside monophosphates containing a syn pyrimidine base

Proton magnetic resonance data have been obtained for 6-methyl-2′-deoxyuridine (dT*), its 3′- and 5′-monophosphates, and its 3′,5′-diphosphate, as well as for the corresponding thymine derivatives. The synthesis of the dideoxynucleoside monophosphates—d(TpT), d(T*pT), d(TpT*), and d(T*pT*)—was accomplished, and spectral data were obtained for these four dimers. The data show that the 6-methyluracil base prefers the syn conformation about the N-glycosyl bond at the monomer and dimer levels. The presence of the syn base leads to increases in the cis couplings of the sugar ring, J_1′2″ and J_2′3′, which indicate a trend towards eclipsing of the substituents on the C1′-C2′ and C2′-C3′ fragments. This trend is discussed in terms of changes in the pseudorotational parameters which describe the pucker of the ring. The syn base destabilizes the g⁺ conformer about the C4′-C5′ bond, leading to a preference for the t conformer in all dT* residues at the monomer and dimer levels. Preliminary work on the formation of cyclobutane-type photodimers in d(T*pT) and d(T*pT*) is discussed and presented as evidence for the capability of the syn 6-methyluracil base to form base-stacked complexes.     

Hydrogen bonding in nucleosides and nucleotides

An analysis of the hydrogen bonding in 76 nucleoside and 11 nucleotide crystal structures shows that the hydrogen bond lengths fall into well-defined categories according to the nature of the donor or acceptor groups. The shortest bonds are those involving P---OH or O=P groups. For donor groups, the sequence in bond lengths is

P—OH<C—OH< N−H<O_w(H)—H<N(H)—H<C−H

There are ten examples of two centre

H—H…O

bonds, which are comparable in length with P---OH …O bonds. The acceptor seqeunce is

O=P<OH₂<OH₂<O=CO(H)C<N N(H₂)C<Cl^…<O<S=C

The number of three-centre bonds, about 24%, is comparable to that observed in the carbohydrates and the amino acids. Most hydrogen bonds are involved in short finite chains. Only in the nucleotides are cyclic hydrogen bonding schemes observed.     

Novel structure of the N-acetylgalactosamine containing N-glycosidic carbohydrate chain of batroxobin, a thrombin-like snake venom enzyme.

The structure of the Asn-linked carbohydrate chain of batroxobin, a thrombin-like enzyme from Bothrops atrox moojeni snake venom, has been determined. The sugar chain was isolated from batroxobin by hydrazinolysis followed by pyridylamination (PA). The PA-oligosaccharide chain was purified by HPLC on an anion exchange or reverse phase columns, and its structure was examined by sequential exoglycosidase digestion, 600 MHZ 1H NMR spectroscopy and methylation analysis. The results indicate that the oligosaccharide chain has the following structure involving a novel linkage, NeuAc alpha 2----3GalNAc.     

Stereochemistry of hydrolysis by snake venom phosphodiesterase.

[18O]Adenosine 5'-O-phosphorothioate-O-p-nitrophenyl ester was prepared by saponification of the bis (-O,O-p-nitrophenyl ester) with K18OH. Only the diastereoisomer with the Rp configuration si a substrate for snake venom phosphodiesterase. The asymmetrically labeled [18O]adenosine 5'-O-phosphorothioate formed in this reaction was converted enzymatically to [18O]adenosine 5'-(1-thiodiphosphate) with the Sp configuration. The position of the 18O label, either bridging [1,2-mu-18O] or nonbridging [1-18O] was then determined. The results show that the reaction catalyzed by snake venom phosphodiesterase takes place with retention of configuration at phosphorus. This indicates that the hydrolysis proceeds via a covalent nucleotide enzyme intermediate.     

Purification of leaf nucleotides and nucleosides on insoluble polyvinylpyrrolidone

Of the 19 nucleotides and nucleosides tested, all were eluted by 1 mM HCl in less than 60 ml from 2 × 6-cm columns of Polyclar AT (an insoluble polyvinylpyrrolidone). Recoveries were good and, with the possible exceptions of ADPG and UDPG, the presence of cotton leaf extract did not decrease recovery of known nucleotides and nucleosides.Passing leaf extracts through Polyclar AT removed most, but not all, of the uv-absorbing impurities that interfere with quantitation of nucleotides and nucleosides. The optimum pH for purification of HClO₄ extracts from leaves of alfalfa, cotton, grape, and orange appeared to be between 2.0 and 3.0. In this pH range Polyclar AT removed from 59 to 91% of the substances in leaf extracts that absorbed at 230 nm and from 93 to 97% of the substances that absorbed at 320 nm.Extraction of leaf extract with isoamyl alcohol was relatively ineffective and extraction with ether was almost completely ineffective in removing uv-absorbing impurities.Because nucleotides and nucleosides quickly pass through a short column of Polyclar AT at pH 3.0 while plant phenols are retained, this procedure provides a simple and rapid method for bulk purification of leaf extracts prior to chromatography and assay of nucleotides and nucleosides.     

Alpha and beta-fibrinogenases from Trimeresurus gramineus snake venom.

By means of DEAE-Sephadex A-50 Column chromatography, Trimeresurus gramineus venom was separated into twelve fractions. The fibrinogenolytic activities were distributed in Fractions 1 and 10. These enzymes were further purified by gel filtration and were homogeneous as judged by cellulose acetate membrane, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ultracentrifugal analysis. Both of them were single peptide chains. The sedimentation constants of alpha- (Fraction 1) and beta-fibrinogenases (Fraction 10) were 2.20 and 3.60, respectively. The molecular weights of alpha- and beta-fibrinogenases were 23 500 and 25 000 respectively. The contents of proline and glycine were higher in beta-fibrinogenase than in alpha-fibrinogenase. The isoelectric points of alpha-fibrinogenase and beta-fibrinogenase were pH greater than 10 and 4.5, respectively. The optimal pH of alpha-fibrinogenase was approx. 7.4 and that of beta-fibrinogenase was approx. 9.0. The activity of alpha-fibrinogenase was completely destroyed after 30 min at 60 degrees C, pH 5.4, 7.4 and 9.0, while that of beta-fibrinogenase was much less affected by the same treatment. The specific fibrinogenolytic activity alpha-fibrinogenase was 31 mg fibrinogen/min per mg protein, while that of beta-fibrinogenase was 9 mg fibrinogen/min per mg protein. alpha-Fibrinogenase cleaved specifically the alpha(A) chain of monomeric fibrinogen without cleaving the beta(B) chain and gamma-chain. beta-fibrinogenase preferentially cleaved the beta(B) chain, and the alpha(A) chain was also partially cleaved by beta-fibrinogenase, if the incubation time was prolonged. Both enzymes showed proteolytic activities toward fibrinogen, fibrin and casein, but were devoid of phospholipase A, alkaline phosphomonoesterase and phosphodiesterase activities found in the crude venom. The tosyl-L-arginine methylester esterase activity of beta-fibrinogenase was about 14 times that of crude venom, while alpha-fibrinogenase was completely devoid of this activity. The fibrinogenolytic activity of alpha-fibrinogenase was markedly inhibited by EDTA and cysteine, while that of beta-fibrinogenase was inhibited markedly by phenylmethanesulfonylfluoride. alpha- and beta-fibrinogenases exert their fibrinogenolytic activity by a direct action on fibrinogen or fibrin without activation of plasminogen.     

The venom and venom apparatus of the sea snake Lapemis hardwicki Gray

The venom apparatus of Lapemis hardwicki , consisting of two functional fangs, their venom glands, and associated musculature, are described. The yield of venom per snake ranged from 2.4-5.2 mg. The LD₅₀ of the crude venom varied from 0.7-1.4 mg/kg intravenously in mice. The toxicological, chemical and immunological properties of the venom are discussed.     

Structural features of carbohydrate moieties in snake venom glycoproteins.

The structures of the carbohydrate moieties of glycoproteins in snake venoms are largely unknown. In the present study, we have analyzed venoms of several species of snakes as well as plasma and tissue glycoproteins from one species of cobra (Naja naja kaouthia) by lectin affinity staining of Western blots. The data demonstrate that glycoproteins in cobra venom invariably contain terminal alpha-galactosyl residues with negligible proportions of sialic acids. Interestingly, however, terminal alpha-galactosyl residues are present in significantly lower proportions in cobra tissues such as brain, liver, lung, kidney, spleen, muscle, and totally absent in cobra plasma glycoproteins. In sharp contrast to cobras, venom glycoproteins of other snakes do not contain terminal alpha-galactosyl residues but do contain terminal 2,3- and/or 2,6-linked sialic acids as well as beta-galactosyl residues. Cobra venom also contains high molecular weight heavily glycosylated proteins bearing poly-N-acetyllactosaminyl oligosaccharides, the majority of which appear to be linked to the protein core via O-glycosidic bonds.