Photochemically induced nuclear polarization study of the accessibility of tyrosines in insulin

The accessible tyrosines of bovine insulin were studied by the photochemically induced dynamic nuclear polarization (photo-CIDNP) method. Tyrosine 1H nuclear polarization is observed in acidic, neutral, and basic solutions at all concentrations studied, in the absence of added salts as well as in the presence of 0.05-0.1 M chloride or phosphate. At pH 2.1 in the presence of chloride, at concentrations of 640 microM and above, most of the nuclear polarization at delta 6.82 originates from one group of tyrosines. On the basis of the crystallographic model, these are assumed to be the A14 tyrosines. We explored the possibility of a genuine concentration dependence of the photo-CIDNP intensity of insulin due to aggregation. In order to discern between such effects and trivial kinetic effects traceable to the optical irradiation method, the effects of concentration changes on polarization were examined in three apparently nonassociating trypsin inhibitor proteins. In insulin, the intensity of Tyr-A 14 polarization changes slowly at concentrations above 1 mM, suggesting that these residues are similarly accessible in all association states. At insulin concentrations below 320 microM, additional tyrosine emission signals were observed. These signals are probably due to B16 and B26 tyrosines of monomers. Polarization transfer effects from Tyr-A14 are evident in the tetramer and hexamer. Enhanced absorption effects in the two histidines (B5 and B10) of the insulin monomer were observed at pH 10 in the presence of 0.1 M phosphate.     

Photochemically induced dynamic nuclear polarization study on microsomal NADPH-cytochrome P-450 reductase

Sedimentation equilibrium experiments with NADPH-cytochrome P-450 reductase showed that increasing 1-O-n-octyl-beta-D-glucopyranoside levels promoted disaggregation of the flavoprotein. The reductase was monomeric at a molar ratio of detergent to protein above 10(3). Addition of N3-carboxymethyllumiflavin to the flavoprotein in the presence of 1-O-n-octyl-beta-D-glucopyranoside results in photochemically induced dynamic nuclear polarization (CIDNP) signals in the aromatic region. The CIDNP spectrum of the holoprotein shows sharp resonances due to histidine residues. On removal of FMN from the protein, CIDNP signals originating from a tyrosine residue appeared, suggesting that the tyrosine residue is exposed to solvent after the depletion of FMN. However, this tyrosine residue appears to become inaccessible to the external dye after full incubation of FMN-depleted reductase with FMN. This suggests that the tyrosine residue could be located in the vicinity of the FMN-binding domain which constitutes the active center of the reductase.     

Photochemically induced dynamic nuclear polarization NMR study of yeast and horse muscle phosphoglycerate kinase

A photochemically induced dynamic nuclear polarization (photo-CIDNP) study of yeast and horse muscle phosphoglycerate kinase with flavin dyes was undertaken to identify the histidine, tryptophan, and tyrosine resonances in the aromatic region of the simplified 1H NMR spectra of these enzymes and to investigate the effect of substrates on the resonances observable by CIDNP. Identification of the CIDNP-enhanced resonances with respect to the type of amino acid residue has been achieved since only tyrosine yields emission peaks and the dye 8-aminoriboflavin enhances tryptophan but not histidine. By use of the known amino acid sequences and structures derived from X-ray crystallographic studies of the enzymes from the two species, assignment of the specific residues in the protein sequences giving rise to the CIDNP spectra was partially achieved. In addition, flavin dye accessibility was used to probe any changes in enzyme structure induced by substrate binding. The nine resonance peaks observed in the CIDNP spectrum of yeast phosphoglycerate kinase have been assigned tentatively to five residues: histidines-53 and -151, tryptophan-310, and tyrosines-48 and -195. The accessibility of a tyrosine to photoexcited flavin is reduced in the presence of MgATP. Since the tyrosine residues are located some distance from the MgATP binding site of the catalytic center, it is proposed either that this change is due to a distant conformational change or that a second metal-ATP site inferred from other studies lies close to one of the tyrosines. Horse muscle phosphoglycerate kinase exhibits seven resonances by CIDNP NMR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Classification of elapid snake neurotoxins and cytotoxins according to chain length: Evolutionary implications

Summary The amino acid sequences of the 139 homologous short neurotoxins, long neurotoxins and cytotoxins so far characterised from elapid snake venoms were compared on the basis of the amino acid deletion/insertion events that have occurred during evolution. Systematic grouping of the toxins according to similarity suggests that the short neurotoxins resemble the cytotoxins more closely than they do the long neurotoxins. The significance of this finding is discussed in relation to the methodology, the conformations of the toxins (as represented by circular dichroism spectra) and the outcome of the study that would have been obtained had more traditional methods been used. It appears probable that the cytotoxins evolved relatively recently from neurotoxic ancestors.     

Tryptophan modification of phospholipase A2 enzymes and presynaptic neurotoxins from snake venoms

Three phospholipases A₂ purified from cobra venoms and two presynaptically acting neurotoxins that exhibit phospholipase A₂ activity were subjected to tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Associated with the modification of an increasing number of Trp residues were marked decreases in enzymatic activity and lethality, whereas antigenicity remained unchanged. The degree of exposure of tryptophanyl groups as determined by acrylamide quenching was consistent with the relative reactivity toward 2-hydroxy-5-nitrobenzyl bromide, except for Hemachatushaemachatus phospholipase A₂, which showed unusually high reactivity due to its characteristic dimeric conformation. Difference spectra of Trp-modified derivatives differed from those of their native enzymes by the presence of a new positive perturbation between 350 and 500 nm, with a maximum at 415 nm. Scatchard plots revealed only one type of binding site for Ca²⁺, and the binding abilities of the modified enzymes were not impaired. At pH 8.0, all native enzymes enhanced the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, and the emission intensity of the ANS-enzyme complex increased or decreased in parallel with increasing concentration of Ca²⁺ for the respective enzyme. The Trp-modified derivatives did not enhance the emission intensity of ANS at all either in the presence or absence of Ca²⁺. By means of tryptophan modification, we were able to infer that the tryptophan residues are in the vicinity of the Ca²⁺ binding site and are directly involved in the binding with ANS. This, together with the suggestion that the hydrophobic pocket that interacts with ANS might be the site of binding of the phospholipase A₂ enzyme with the substrate, suggests that the Trp residues in phospholipase A₂ enzymes and presynaptic toxins are involved in substrate binding.     

Post-translational modification accounts for the presence of varied forms of nerve growth factor in Australian elapid snake venoms

The Australian elapid snakes are amongst the most venomous snakes in the world, but much less is known about the overall venom composition in comparison to Asian and American snakes. We have used a combined approach of cDNA cloning and 2-DE with MS to identify nerve growth factor (NGF) in venoms of the Australian elapid snakes and demonstrate its neurite outgrowth activity. While a single 730 nucleotide ORF, coding for a 243 amino acid precursor protein was detected in all snakes, use of 2-DE identified NGF proteins with considerable variation in molecular size within and between the different snakes. The variation in size can be explained at least in part by N-linked glycosylation. It is possible that these modifications alter the stability, activity and other characteristics of the snake NGFs. Further characterisation is necessary to delineate the function of the individual NGF isoforms.     

Role of C-terminal tail of long neurotoxins from snake venoms in molecular conformation and acetylcholine receptor binding: proton nuclear magnetic resonance and competition binding studies

The role of the "C-terminal tail" segment of long neurotoxins has been investigated. The C-terminal four to five residues of alpha-bungarotoxin and Laticauda colubrina b have been cleaved off by carboxypeptidase P. The effect of such deletion on the toxin conformation has been monitored in proton nuclear magnetic resonance spectra and circular dichroism spectra. The removal of the C-terminal residues primarily affects the chemical shifts of proton resonances of the residues close to the cleavage site and does not induce a major conformational change. Therefore, the C-terminal tail of long neurotoxins does not appear to be important in maintaining the specific polypeptide chain folding. On the other hand, competition binding with tritium-labeled toxin alpha to Narke japonica acetylcholine receptor has revealed that cleavage of the C-terminal residues reduces the binding activity of alpha-bungarotoxin or Laticauda colubrina b to acetylcholine receptor. Thus it is likely that (the basic amino acid residues in) the C-terminal tail is directly involved in the binding of long neurotoxins to electric organ (and muscle) acetylcholine receptor.     

State of functionally essential Trp-29 in snake venom neurotoxins: A proton nuclear magnetic resonance study

Proton nuclear magnetic resonance (NMR) spectra have been recorded of various neurotoxins from snake venoms.pH dependence of the chemical shifts and resonance intensity has been followed for the functionally essential Trp-29. The indole N-1 proton of Trp-29 in -bungarotoxin, toxin B, and cobrotoxin exhibits appreciably large upfield shifts as thepH is lowered and the suppressed exchange with the solvent hydrogen atpH 3–4, but not inNaja haje annulifera 10 where Asp-31 is replaced with Gly-31. This observation strongly suggests the presence of a hydrogen bond between Trp-29 and Asp-31 that is probably important in stabilizing the arrangement of the functionally essential residues to form a distinct binding region for the receptor.     

p-Diazobenzoyl biocytin--a new biotinylating reagent for the labeling of tyrosines and histidines in proteins

A new biotin-containing reagent, p-diazobenzoyl biocytin (DBB), has been developed for labeling tyrosines and histidines in proteins. The reagent has used to label these residues in both model proteins and erythrocyte membrane proteins on dot blots and blot transfers. In some cases, sub-nanogram levels of individual proteins could be detected. The utility of DBB as a versatile alternative to biotin-containing N-hydroxysuccinimide esters for the general labeling of proteins is discussed.     

Hydrogen-1 nuclear magnetic resonance studies of staphylococcal nuclease variant H124L: pH dependence of histidines and tyrosines

The pH dependence of the 1H NMR spectrum of staphylococcal nuclease H124L was investigated as a function of the binding of Ca2+, the ion required for enzymatic activity, and deoxythymidine-3',5'-diphosphate (pdTp), a competitive inhibitor. The protein studied was the product of a cloned gene expressed in Escherichia coli which yields a protein having a sequence identical to that of the nuclease isolated from the V8 strain of Staphylococcus aureus. Of the observable ring protons of the three histidine residues, only the C delta 1H of His46 shows a large chemical shift perturbation on formation of the ternary complex, (nuclease H124L).pdTp.Ca2+. The pKa of His46 is lowered by 0.2 pH unit in the binary complex. All seven tyrosines titrate with normal pKa values between 9 and 11 in the unligated nuclease. In the ternary complex, however, the pKa values of Tyr85 and Tyr93 increase above pH 11.0. The chemical shift perturbations of the ring protons of the Tyr27, Tyr85, Tyr113, and Tyr115 were observed between pH 4 and 6; these spectral perturbations are attributed to interactions with carboxylate groups. Binding Ca2+ alone acted opposite to the perturbation in Tyr113 and Tyr115. Ca2+ binding leads to deshielding the ring protons of Tyr113, but this effect is removed in the ternary complex. Binding of pdTp and Ca2+ stabilizes the protein against high pH denaturation up to pH 11.5.     

蛇伤药酒体外抗5种蛇毒的实验研究

目的探讨蛇伤药酒抗五步蛇、竹叶青蛇、眼镜蛇、蝰蛇、蝮蛇蛇毒的效果。方法采用鲎试剂试管凝集反应法。结果蛇伤药酒浓度为1.0 ml/ml时,对5种蛇毒均有破坏产生凝胶反应的作用;浓度为0.5ml/ml时,对五步蛇、竹叶青蛇、眼镜蛇蛇毒有破坏产生凝胶反应的作用;浓度为0.3 ml/ml时,仅对五步蛇毒有破坏产生凝胶反应的作用;浓度降至0.1 ml/ml时,对5种蛇毒均无破坏产生凝胶反应的作用。结论蛇伤药酒有较好的抗蛇毒作用,且对五步蛇毒作用最强。     

Toxicities, LD50 prediction and in vivo neutralisation of some elapid and viperid venoms.

Toxicity levels of elapid (Naja naja and Naja oxiana) viperid (Vipera lebetina and Vipera russelli) venoms for mice and rat for intraperitoneal intravenous and intramuscular routes have been determined. The data have been analysed using a mathematical expression to calculate lethal venom concentrations in human snake bite cases. Further, in vivo neutralisation of snake venom potency (after experimental injection) using high voltage-low current electric shock treatment has been attempted. This treatment postponed the death further by 60-90 min in mice in case of elapid envenomation. In case of viperid envenomation such a postponement of death time was not noticed. The death postponement induced by the shock treatment probably refers to structural impairments that occur at molecular level in venom components and their consequent altered interactions with the target tissue or system.     

Photochemically induced dynamic nuclear polarization in the reaction center of the green sulphur bacterium Chlorobium tepidum observed by 13C MAS NMR

Photochemically induced dynamic nuclear polarization has been observed in reaction centres of the green sulphur bacterium Chlorobium tepidum by (13)C magic-angle spinning solid-state NMR under continuous illumination with white light. An almost complete set of chemical shifts of the aromatic ring carbons of a BChl a molecule has been obtained. All light-induced (13)C NMR signals appear to be emissive, which is similar to the pattern observed in the reaction centers of plant photosystem I and purple bacterial reaction centres of Rhodobacter sphaeroides wild type. The donor in RCs of green sulfur bacteria clearly differs from the substantially asymmetric special pair of purple bacteria and appears to be similar to the more symmetric donor of photosystem I.     

Photochemically induced dynamic nuclear polarisation NMR study of the aromatic residues of sea-anemone polypeptide cardiac stimulants

One-dimensional and two-dimensional photochemically induced dynamic nuclear polarisation (photo-CIDNP) nuclear magnetic resonance spectra have been recorded for the sea-anemone polypeptide cardiac stimulants anthopleurin-A and Anemonia sulcata toxins I and II. In anthopleurin-A and toxin II, all three Trp residues are accessible to the flavin dye, although Trp-23 in anthopleurin-A shows a weaker photo-CIDNP response than Trp-33 and Trp-45. Tyr-25 in anthopleurin-A also shows a strong response. In toxin I, Trp-23, Trp-33 and Tyr-45 (which replaces Trp in this molecule) are accessible to the dye. The pH dependences of the photo-CIDNP spectra of all three polypeptides have been examined. The response of Trp-33 increases significantly with pH. The two His residues of anthopleurin-A and toxin II display a response in their imidazole forms, but not their imidazolium forms. The surface accessibilities of Trp-23 and Trp-33 are discussed in relation to the interaction of these polypeptides with the Na+ channel.     

Photochemically induced dynamic nuclear polarization in the reaction center of the green sulphur bacterium Chlorobium tepidum observed by C MAS NMR

Photochemically induced dynamic nuclear polarization has been observed in reaction centres of the green sulphur bacterium Chlorobium tepidum by ¹³C magic-angle spinning solid-state NMR under continuous illumination with white light. An almost complete set of chemical shifts of the aromatic ring carbons of a BChl a molecule has been obtained. All light-induced ¹³C NMR signals appear to be emissive, which is similar to the pattern observed in the reaction centers of plant photosystem I and purple bacterial reaction centres of Rhodobacter sphaeroides wild type. The donor in RCs of green sulfur bacteria clearly differs from the substantially asymmetric special pair of purple bacteria and appears to be similar to the more symmetric donor of photosystem I.