Tritium-labeled Peptides Research Articles

Synacton and individual activity of synthetic and natural corticotropins.

Short endogenous peptides represent one of the most important constituents of the mammalian body's general regulatory system. Some synthesized analogs and modified natural peptides (eg, corticotropins) also show high biological activity. Nevertheless, the mechanism of action of regulatory peptides remains unclear. To explain the effects of peptides of intermolecular processes, the hypothesis that a synactonal mechanism underlies the action of regulatory peptides, exemplified by the heptapeptide Semax, has been proposed. Thus, in the total pool of Semax metabolites, which includes the cleavage products of the parental molecule, we can distinguish the functional core, represented by the major metabolic products-peptides HFPGP and PGP. These peptides have their own binding sites with similar although differing characteristics. Together with Semax, they constitute a single complex of bioregulators acting in a certain sequence and in interaction, ie, synacton. It can be assumed that the diverse clinically significant effects of the drug Semax are determined by its synacton. Specific interactions between some tritium-labeled peptides (basic constituents of the Semax synacton) and plasma membranes of neurons have been characterized. Only a few peptides of the Semax synacton showed competitive activity for the Semax binding sites. Fragments comprising 5 amino acid residues (EHFPG and HFPGP) showed the highest competitive activity. We also characterized the processes of specific ligand-receptor interactions of some tritium-labeled corticotropins ([3 H-Pro]MEHFPGP, [3 H-Pro]HFPGP, and [3 H-Pro]PGP) by applying mathematical discriminative models (Scatchard, Hill, Bjerrum, and Lineweaver-Burk plots). So the intermolecular interactions of these peptides with plasma membranes of neuronal brain targets are probably not limited by specific binding at orthosteric sites. The effect of peptides that act in the synacton considerably extends the regulatory potential of the initial molecule.

The Qualitative Analysis of the Amide Derivative of HLDF-6 Peptide and Its Metabolites with the Use of Tritium- and Deuterium-Labeled Derivatives

The goal of the study was to elaborate the pharmacokinetics methods of the amide derivative of peptide HLDF-6 (TGENHR-NH2) and its range of nootropic and neuroprotective activity is wide. The hexapeptide 41TGENHR46 is a fragment of the HDLF differentiation factor. It forms the basis for the development of preventive and therapeutic preparations for treating cerebrovascular and neurodegenerative conditions. Pharmacokinetic and molecular mechanisms of the action of the HLDF-6 peptide were studied using tritium- and deuterium-labeled derivatives of this peptide, produced with the use of the high-temperature solid-state catalytic isotope exchange reaction (HSCIE). This reaction was employed to produce the tritium-labeled peptide [3H]TGENHR-NH2 with a molar radioactivity of 230 Ci/mmol and the deuterium-labeled peptide [2H]TGENHR-NH2 with an average deuterium incorporation equal to 10.5 atoms. It was shown by the NMR spectroscopy that the isotope label distribution over the labeled peptide's molecule was uniform, which allowed qualitative analysis ofboth the peptide itself and its fragments in the organism's tissues to be conducted. The newly developed pharmacokinetics method makes it possible to avoid almost completely losses of the peptides under study due to biodegradation during the analysis of tissues. These labeled peptides were used in mice, rats and rabbits to study the pharmacokinetics of the peptide and to calculate the values of its principal pharmacokinetic parameters. Characteristics of its pharmacokinetic profile in the blood were obtained, the hypothesis of pharmacokinetics linearity tested, its metabolism analyzed and its bioavailability value, 34%, calculated. It has been shown that the studied TGENHR-NH2 peptide shows high resistance to hydrolysis in the blood plasma, with dipeptidyl aminopeptidases making the largest contribution to its hydrolysis.

Synthesis of Pro-Pro-Ile, a corticoliberin fragment, labeled with tritium in proline unit

Boc-Δ3Pro-Pro-Ile-OBzl and Boc-Pro-Δ3Pro-Ile-OBzl were synthesized. Tritium-labeled Pro-Pro-Ile was prepared by their hydrogenation in a tritium atmosphere in the presence of a catalyst. In the reduction of Boc-Δ3Pro-Pro-Ile-OBzl and Boc-Pro-Δ3Pro-Ile-OBzl, elimination of the Bzl protective group occurs faster than hydrogenation of the 3,4-dehydroproline residue (benzene and dioxane were used as solvents). The resulting Boc-Δ3Pro-Pro-Ile-OH and Boc-Pro-Δ3Pro-Ile-OH are poorly soluble in aprotic solvents, which accounts for the low yield of the desired tritium-labeled peptides.

Bruce Merrifield's contribution to antimicrobial peptide research

Although within the scientific community at large Bruce Merrifield is and will remain celebrated for the pivotal discovery and dedicated crafting of solid phase peptide synthesis methodology, there are also other areas where his unique scientific acumen was applied to with distinctive success. Among them, those of glucagon antagonists and antimicrobial peptides are arguably the better known. Over my 25 year-long relationship with Bruce, I had occasion to work with him in both areas, but it is to the latter I will devote these brief reminiscences, as our collaboration in this fast expanding research field reached from its very dawn till relatively recently. 2006, the year of Bruce’s passing, marked the silver jubilee of antimicrobial peptide research, officially initiated with the publication of the seminal paper on the cecropins by Boman (see Figure 1) and coworkers. 1 Hans had befriended Bruce during his postdoc (1958–60) in Fritz Lipmann’s group at Rockefeller, and his logical enthusiasm over the finding of a family of eukaryotic, geneencoded peptides with remarkably potent antimicrobial activities must have been contagious for Bruce, who started to collaborate with Hans on the cecropins in late 1981. On joining Bruce’s lab in January 1982, the very first project given to me was to synthesize the putative 37-residue sequence of cecropin A, to confirm some structural assignations tentatively proposed for the C-terminal region. A previous synthetic attempt 2 had turned out an active compound, albeit nonidentical with the natural peptide. Building on that result and benefiting from several improvements emanating at the time from Bruce and Jimmy Tam’s joint research—not least the very useful high-low HF cleavage method 3 —I completed my assignment in two-and-a-half months, which at the time was regarded a reasonable time for a relative novice charged with the fully manual synthesis of a 37-residue, tritiumlabeled peptide. Our synthetic product was completely identical with the native peptide by microbiological as well as biochemical (enzyme digest + HPLC) criteria, and we were able to confirm the hitherto uncertain structure of the C-terminal end by means of plasma desorption MS, a forerunner of modern MALDI-TOF MS. In addition, our report 4 discussed the relevance of the Trp-2 residue for antimicrobial activity and thus set the basis for further analogue work. 5 In retrospect, that initial 2-year stint in the cecropin project appears as a decisive period, where Bruce’s gracious support and encouragement became the beacon blazing away the often usual angst of a nascent scientific career. A thorough and constructive draft reviewer if ever I saw one, Bruce returned each one of my versions generously endowed with pencil scribbles which, readily deciphered, brought gradual improvement to my struggling manuscripts. To Bruce I owe a great deal of any abilities I may have achieved as a (non-native) writer of English scientific texts. Those early 1980s cecropin manuscripts, first examples of the substantial contri

Evenly tritium labeled peptides in study of peptide in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50–150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a μg scale of biological samples both in vitro and in vivo.

The solid-state catalytic isotope exchange of hydrogen in dalargin

A [3H]Dalargin preparation with a molar radioactivity of 52 Ci/mmol was obtained by the high temperature solid-state catalytic isotope exchange (HSCIE) of tritium for hydrogen at 150°C. This tritium-labeled peptide was shown to completely retain its biological activity in the test of binding to opioid receptors from rat brain. The dissociation constant of the Dalargin-opioid receptor complex was found to be 4.3 nM. The dependences of the chemical yield and the molar radioactivity on the reaction time and temperature of HSCIE were determined. The activation energy of the HSCIE reaction for the peptide was calculated to be 32 kcal/mol. The amino acid analysis showed that tritium is distributed between all the amino acid residues of [3H]Dalargin at the HSCIE reaction, with the temperature growth significantly increasing the total tritium incorporation and, especially, enhancing the radioactivity incorporation into aromatic residues.

Adsorption studies of tritium-labeled peptides on polystyrene surfaces

In this study, three presentation formats of an epitope peptide (hepta-peptide), derived from the human chorionic gonadotropin amino acid sequence, were compared for adsorption to the polystyrene wells of a microELISA plate. The peptides had either a free N-terminus, an Ata-group or a linear (Lys) 7-extension at the N-terminal. In order to measure the adsorption properties, all peptides were tritiated by synthesizing an additional 3 H− labeled glycyl residue to the N-terminus of their peptide sequence. Over a broad range of peptide concentrations used as coat solution, extension of the peptide by an Ata-group consistently increased adsorption by a factor of 1.5 to 3 compared to the free parent peptide. Of the three peptides studied, the Ata-peptide showed the highest surface coverage of 0.6 mg/m 2 when 1.0 mmol/l was offered as the concentration of peptide in the coating solution. The highest surface coverage observed for the parent peptide was 0.4 mg/m 2 (at 1.5 mmol/l). The lysyl (K 7) peptide showed a maximum plateau value of 0.2 mg/m 2, and therefore the lysyl (K 7) extension reduced the peptide surface coverage at relatively high coat concentrations (above 0.1 mmol/l) compared to the parent peptide. At lower input concentrations (below 0.1 μmol/l), however, the packing density of the lysyl (K 7) peptide was up to 25 times higher when compared to the other two peptide analogs. We conclude that better adsorption as well as improved antibody binding activity and (functional) affinity could explain the higher reactivity observed in ELISA procedures when peptides are N-terminally extended by an Ata-group or lysyl (K 7) extension.

Identification of the Domains of Photoincorporation of the 3‘- and 7-Benzophenone Analogues of Taxol in the Carboxyl-Terminal Half of Murine mdr1b P-glycoprotein

P-glycoprotein is an ATP-dependent drug-efflux pump that can transport a diverse range of structurally and functionally unrelated hydrophobic compounds across the plasma membrane. The transporter is composed of two homologous halves, each containing a nucleotide binding fold and six putative transmembrane spanning segments. The contact domains between the murine mdr1b P-glycoprotein and two photoreactive Taxol analogues have been mapped by a combination of CNBr digestion and immunoprecipitation studies. We had demonstrated previously that the 3'-p-benzoyldihydrocinnamoyl (BzDC) analogue of Taxol specifically photolabeled mdr1b P-glycoprotein and now show that the corresponding C-7 analogue likewise specifically photoincorporates into the transporter. CNBr digestion of both photolabeled P-glycoproteins gave rise to an approximate 10 kDa tritium-labeled peptide, each of which was a distinct polypeptide. The CNBr fragment generated from the 3'-BzDC-Taxol-photolabeled P-glycoprotein was immunoprecipitated by a polyclonal antibody (Ab7) raised against amino acid residues 1008-1019 of the mdr1b isoform. In contrast, the CNBr fragment generated from the 7-BzDC-Taxol-photolabeled P-glycoprotein was immunoprecipitated by a polyclonal antibody (Ab4) raised against amino acid residues 740-750. The specificity of these reactions was demonstrated by showing that the presence of the appropriate synthetic peptide blocked the immunoprecipitation. Moreover when the antibodies were reversed, no immunoprecipitation occurred. Based on the deduced amino acid sequence of mdr1b P-glycoprotein, and its hydropathy plot analysis, our data indicated that the 3'-BzDC group photoincorporates into amino acid residues 985-1088, a region of the transporter that includes half of TM 12 and terminates just after the Walker A motif in the second nucleotide binding fold. The 7-BzDC group photoincorporates into amino acid residues 683-760, a region of the transporter that includes all of TM 7 and half of TM 8 plus the intervening extracellular loop.

Fast association rates suggest a conformational change in the MHC class I molecule H-2Db upon peptide binding.

Major histocompatibility complex (MHC) class I molecules bind peptides in the endoplasmic reticulum (ER). For this binding reaction, when performed in vitro, widely differing association rates have been reported. We have expressed empty soluble H-2Db class I molecules in Chinese hamster ovary (CHO) cells and generated complete sets of association, dissociation, and equilibrium constants of unmodified peptides using tritium-labeled peptides and stopped-flow fluorescence spectroscopy. We find that (i) the transition midpoint of temperature denaturation (Tm) of the protein is shifted from 30.5 to 56 degrees C upon the binding of a high-affinity peptide. (ii) With the peptide SV-324-332 (sequence FAPGNYPAL) at 4 degrees C, the dissociation rate constant of 1.02 x 10(-5) s-1 and an equilibrium constant of 8.5 x 10(7) M-1 predict an association rate constant of 870 M-1 s-1 for a simple one-step model of binding. (iii) In contrast, binding of this peptide proceeds much faster, with 1.4 x 10(6) M-1 s-1. These "mismatch kinetics" suggest that peptide binding occurs in several steps, most likely via a conformational rearrangement of the peptide binding groove. The structure of the peptide-class I complex at the time-point of peptide recognition may therefore be different from the equilibrium crystal structures. (iv) Association of modified peptides, in the presence of detergent, or above the Tm of the empty molecule is considerably slower. This might explain why fast on-rates have not been observed in previous studies.

Protein Kinase Assay Using Tritiated Peptide Substrates and Ferric Adsorbent Paper for Phosphopeptide Binding

The recently described synthesis of ferric adsorbent paper has made possible the modification of protein kinase assays. The adsorbent contains ferric chelate groups, which are responsible for the binding of phosphopeptide via phosphate group. The selective adsorption of phosphopeptide contra nonphosphorylated peptide allows the use of tritium-labeled peptides and unlabeled ATP as substrates. The binding of the reaction product to the adsorbent was complete and was not affected by the amino acid sequence of the peptide. The conditions required for the separation of the produced phosphopeptide from the initial peptide have been worked out as well. The firmly bound phosphopeptide should be released from the ferric adsorbent paper prior to liquid scintillation counting. Using 0.1 M NH4HCO3 solution (pH 8.0), the elution of phosphopeptides was almost complete. The modified protein kinase assay proposed herein is rapid and allows handling of multiple samples simultaneously. In addition, the ferric paper method avoids the use of 32P-isotope, replacing it with 3H which has lower radiation energy and a much longer half-life.

Purification and characterization from human parotid secretion of a peptide which inhibits hemagglutination ofBacteroides gingivalis381

A peptide from human parotid secretion which inhibited hemagglutination of Bacteroides gingivalis 381 was purified by ultrafiltration followed by DEAE-Sephadex A-25 column chromatography and by gel filtration on Sephadex G-25, and then by reversed-phase HPLC. The complete amino acid sequence of the peptide, determined by automated Edman degradation was as follows; Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr. The peptide contained 12 residues and the charged amino acids predominated with 4 histidine, 2 lysine, 1 arginine and 1 glutamic acid residues, thus being a histidine-rich peptide. The peptide was an active inhibitor of the hemagglutinating activity of B. gingivalis. Specific binding of tritium-labeled peptide to B. gingivalis cells was demonstrated. These results suggest that the histidine-rich peptide may function as a binding domain for the hemagglutinins of B. gingivalis during agglutination.

Purification and characterization from human parotid secretion of a peptide which inhibits hemagglutination of Bacteroides gingivalis 381

A peptide from human parotid secretion which inhibited hemagglutination of Bacteroides gingivalis 381 was purified by ultrafiltration followed by DEAE-Sephadex A-25 column chromatography and by gel filtration on Sephadex G-25, and then by reversed-phase HPLC. The complete amino acid sequence of the peptide, determined by automated Edman degradation was as follows; Lys- Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr. The peptide contained 12 residues and the charged amino acids predominated with 4 histidine, 2 lysine, 1 arginine and 1 glutamic acid residues, thus being a histidine-rich peptide. The peptide was an active inhibitor of the hemagglutinating activity of B. gingivalis. Specific binding of tritium-labeled peptide to B. gingivalis cells was demonstrated. These results suggest that the histidine-rich peptide may function as a binding domain for the hemagglutinins of B. gingivalis during agglutination.

Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: essential role in biological activity of VirG

Agrobacterium tumefaciens virulence genes are induced by plant signals through the VirA-VirG two-component regulatory system. The VirA protein is a membrane-spanning sensor molecule that possesses an autophosphorylating activity, and the VirG protein is a sequence-specific DNA-binding protein. In this report, we demonstrate that the VirG protein is phosphorylated by the VirA protein and that the phosphate is directly transferred from the phosphorylated VirA molecule (phosphohistidine) to the VirG protein. The chemical stability of the phospho-VirG bond suggested that the VirG protein was phosphorylated at the aspartate and/or glutamate residue. The phosphorylated VirG protein was reduced with tritiated sodium borohydride and subjected to proteolytic digestion with the Achromobacter protease I enzyme. The resulting peptide fragments were separated by C8 reversed-phase high-pressure liquid chromatography, and the tritium-labeled peptide was sequenced. Amino acid sequence data showed that the aspartate residue at position 52 was the only site phosphorylated. Changing this aspartate into asparagine resulted in a nonphosphorylatable and biologically nonfunctional gene product. As a control, a randomly chosen aspartate was changed into an asparagine (position 72), and no effect on its phosphorylation or biological activity was observed. Unlike its homologs, including CheA-CheY, EnvZ-OmpR, and NtrB-NtrC, the phospho-VirG molecule was very stable in vitro. The possible implications of these observations and the function of VirG phosphorylation in vir gene activation are discussed.

A General Method of Tritium Labeling Utilizing Microwave Discharge Activation of Tritium Gas: METHODOLOGY AND APPLICATION TO BIOLOGICAL COMPOUNDS

A method of general tritium labeling is described which is applicable to a broad range of organic compounds of biological interest, including amino acids, peptides, and proteins. The method is rapid, inexpensive, and nondestructive, thereby making the purification of labeled material relatively uncomplicated. The reaction apparatus consists of a closed system in which tritium gas at a pressure of 4 mm Hg or less can be cycled by an internal pump. The labeling reaction(s) is initiated by microwave discharge activation of the tritium gas and the sample, cooled by liquid nitrogen, remains outside the region of the high ultraviolet and electron flux from the tritium plasma. The evidence suggests that tritium-hydrogen exchange occurs as a result of bombardment of the labeled substrate with tritium atoms. Studies of the chemistry of the labeling reaction with aromatic hydrocarbons, of the analytical requirements for radiochemical purity, of the characteristic specific activities noted for several classes of compounds, and of the application of the method to the preparation of tritium-labeled peptides are presented. Tritium-labeled adrenocorticotropin (ACTH) with a specific activity of 15 Ci per mmole and full biological activity has been prepared by this method. This represents a 1000-fold increase in the specific activity previously obtained by Wilzbach labeling of ACTH.