Yu. A. Zolotarev

New development in the tritium labelling of peptides and proteins using solid catalytic isotopic exchange with spillover-tritium.

Summary. The mechanism of the reaction of high temperature solid state catalytic isotope exchange (HSCIE) of hydrogen in peptides with spillover-tritium at 140–180°C was analyzed. This reaction was used for preparing [3H]enkephalins such as [3H]DALG with specific activity of 138 Ci/mmol and [3H]LENK with specific activity of 120 Ci/mmol at 180°C. The analogues of [3H]ACTG4–10 with specific activity of 80 Ci/mmol, [3H]zervamicin IIB with specific activity of 70 Ci/mmol and [3H]conotoxin G1 with specific activity 35 Ci/mmol were produced. The obtained preparations completely retained their biological activity. [3H]Peptide analysis using 3H NMR spectroscopy on a Varian UNITY-600 spectrometer at 640 MHz was carried out. The reaction ability of amino fragments in HSCIE was shown to depend both of their structures and on the availability and the mobility of the peptide chain. The reaction of HSCIE with the β-galactosidase from Termoanaerobacter ethanolicus was studied. The selected HSCIE conditions allow to prepare [3H] β-galactosidase with specific activity of 1440 Ci/mmol and completely retained its the enzymatic activity.

The solid-state catalytic synthesis of tritium labeled amino acids, peptides and proteins.

SummaryNew catalytic reaction between a solid bioorganic compound and activated spillover tritium (ST), based on High-temperature Solid-state Catalytic Isotopic Exchange (HSCIE) was examined. The HSCIE mechanism and determination of the reactivity of hydrogen atoms in amino acids, peptides and proteins was investigated. Quantum mechanical calculations of the reactivity of hydrogen atoms in amino acids in the HSCIE reaction were done. The carbon atom with a greater proton affinity undergoes a greater exchange of hydrogen for tritium in HSCIE. The electrofilic nature of spillover hydrogen in the reaction of HSCIE was revealed. The isotope exchange between ST and the hydrogen of the solid organic compound proceeds with a high degree of configuration retention at the carbon atoms. The HSCIE reaction enables to synthesize tritium labeled proteins with a specific activity of 20–30 mCi/mg and kept biological activity.

Characteristics of non-opioid β-endorphin receptor

Tritium-labeled selective agonist of non-opioid β-endorphin receptor, the decapeptide immunorphine ([2H]SLTCLVKGFY) with specific activity of 24 Ci/mmol has been prepared. By its use, non-opioid β-endorphin receptors were revealed and characterized on mouse peritoneal macrophages and rat myocardium, spleen, adrenal, and brain membranes. The non-opioid β-endorphin receptor of macrophages has in addition to immunorphine (Kd of the [2H]immunorphinereceptor complex was 2.4 ± 0.1 nM) and β-endorphin (Ki of the [2H]immunorphine specific binding was 2.9 ± 0.2 nM) a high affinity for Fc-fragment of human IgG1, pentarphine (VKGFY), cyclopentarphine [cyclo(VKGFY)], and [Pro2]pentarphine (VKPFY) (Ki values were 0.0060 ± 0.0004, 2.7 ± 0.2, 2.6 ± 0.2, and 2.8 ± 0.2 nM, respectively) and is insensitive to naloxone and [Met5]enkephalin (Ki > 100 μM). Treatment of macrophages with trypsin resulted in the loss of their ability for the specific binding of [2H]immunorphine. Values of the specific binding of 8.4 nM [2H]immunorphine to rat adrenal, spleen, myocardium, and brain membranes were determined to be 1146.0 ± 44.7, 698.6 ± 28.1, 279.1 ± 15.4, and 172.2 ± 1.8 fmol/mg protein, respectively. Unlabeled β-endorphin, pentarphine, [Pro2]pentarphine, cyclopentarphine, cyclodipentarphine [cyclo(VKGFYVKGFY)], and Fc-fragment of IgG1 inhibited the binding of [2H]immunorphine to membranes from these organs. No specific binding of [2H]immunorphine to rat liver, lung, kidney, and intestine membranes was found.

Solid State Isotope Exchange with Spillover Hydrogen in Organic Compounds

The term spillover, as used in heterogeneous catalysis, refers to the transport of active particles which are either absorbed or formed in one phase and transported to another phase where, under these reaction conditions, such particles are not absorbed or formed. An example is that of hydrogen, which after dissociative adsorption on platinum particles can migrate onto a nonorganic support such as aluminum oxide, barium sulfate, or others. Such active hydrogen atoms were given the name spillover hydrogen (SH).1 The first direct evidence of spillover was obtained during the reduction of wolfram trioxide to tungsten bronze at room temperature:2 the reaction proceeds in a mechanical mixture of 0.5% Pt/ Al2O3 + WO3. It was assumed that the hydrogen dissociated on the platinum and migrated through the alumina onto WO3 in the form of atoms or H+ ions. The solid state hydrogenation of asymmetric crystals of 2-isopropyl-5-methylphenol (also known as thymol) occurs with the participation of SH and leads to the formation of a series of asymmetric menthols and menthones.3 Despite the fact that processes involving SH have been known for over 40 years, the nature of this phenomenon has not been fully investigated or clarified. According to various hypotheses, hydrogen can migrate in the form of a solvated proton,4 as a proton-electron pair,5 or as atomic hydrogen.6 The main difficulty in explaining the SH phenomenon is that the SH concentration is too small to detect using direct instrumental analysis such as modern spectroscopic methods. Because the importance of SH in heterogeneous catalysis cannot be underestimated, the debate regarding the nature of the activated hydrogen particles and their diffusion is still ongoing.1

The Heptapeptide SEMAX stimulates BDNF Expression in Different Areas of the Rat Brain in vivo

N-terminal fragments of adrenocorticotropic hormone (ACTH) and different types ( α , β and γ ) of melanocyte-stimulating hormone (MSH) form the family of melanocortin peptides exerting a marked action on the functions of the central nervous system (CNS). Peptides from this family possess neurotrophic, nootropic and neuroprotective properties [1]. The heptapeptide SEMAX (Met–Glu–His–Phe–Pro–Gly–Pro) is an analog of the ACTH(4–10) fragment completely devoid of any hormonal activity present in the full-length ACTH molecule. It shown to stimulate the learning and memory formation processes in laboratory animals [2, 3]. As a regulator of CNS functions, this peptide, if administered at very small doses (15–50 μ g/kg), produces a marked nootropic effect [4, 5]. It also stimulates forebrain functions by increasing selective attention at the moment of information reception, improving memory consolidation and raising the learning ability [4]. At the cellular level, SEMAX has a neuroprotective effect, preventing the death of cholinergic neurons in culture, and stimulates an activity of choline acetyl transferase [6, 7].

Immunostimulating effect of the synthetic peptide octarphin corresponding to β-endorphin fragment 12–19

We have synthesized the peptide TPLVTLFK corresponding to β-endorphin fragment 12–19 (dubbed octarphin) and its analogs (LPLVTLFK, TLLVTLFK, TPLVLLFK, TPLVTLLK, TPLVTLFL). The octarphin peptide was labeled with tritium (specific activity 28 Ci/mol), and its binding to murine peritoneal macrophages was studied. [3H]Octarphin was found to bind to macrophages with high affinity (Kd = 2.3 ± 0.2 nM) and specificity. The specific binding of [3H]octarphin was inhibited by unlabeled β-endorphin and the selective agonist of nonopioid β-endorphin receptor synthetic peptide immunorphin (SLTCLVKGFY) (Ki = 2.7 ± 0.2 and 2.4 ± 0.2 nM, respectively) and was not inhibited by unlabeled nalox-one, α-endorphin, γ-endorphin, or [Met5]enkephalin (Ki > 10 μM). Inhibitory activity of unlabeled octarphin analogs was more than 100 times lower than that of unlabeled octarphin. Octarphin was shown to stimulate activity of murine immuno-competent cells in vitro and in vivo: at concentration of 1–10 nM it enhanced the adhesion and spreading of peritoneal macrophages as well as their ability to digest bacteria of Salmonella typhimurium virulent strain 415 in vitro; the peptide administered intraperitoneally at a dose of 20 μg/animal on day 7, 3, and 1 prior to isolation of cells increased activity of peritoneal macrophages as well as spleen T- and B-lymphocytes.

Elucidation and Characteristics of Non-opioid β-Endorphin Receptors in Rat Adrenal Cortex

Abstractβ-Endorphin-like decapeptide immunorphin (SLTCLVKGFY), a selective agonist of non-opioid β-endorphin receptor, was labeled with tritium to specific activity of 24 Ci/mmol. It was used for the detection and characterization of nonopioid β-endorphin receptors on rat adrenal cortex membranes (Kd1 = 39.6 ± 2.0 nM, Bmax1 = 40.7 ± 2.3 pmol/mg protein; Kd2 = 0.25 ± 0.01 μM, Bmax2 = 187.8 ± 9.4 pmol/mg protein). β-Endorphin was found to inhibit the [3H]immunorphin specific binding to membranes (Ki = 70.0 ± 9.2 nM); naloxone, [Met5]enkephalin, and α- and γ-endorphins tested in parallel were inactive. Immunorphin at concentrations of 10–9-10–6 M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, while intramuscular injection of immunorphin at doses of 10-100 μg/kg was found to reduce the secretion of 11-oxycorticosteroids from the adrenals to the bloodstream.

Effect of Semax peptide on survival of cultured rat pheochromocytoma cells during oxidative stress.

We studied the effects of Semax (antiinsulin peptide with neuroprotective effect) on the survival of cultured rat pheochromocytoma cell after oxidative stress induced by short-term incubation with hydrogen peroxide. Studies with fluorescent dyes propidium iodide and Hoechst 33258 showed that cell incubation with hydrogen peroxide led to the formation of damaged cells with characteristic signs of necrosis. Semax dose-dependently reduced the number of cells damaged by oxidative stress. The efficiency of Semax depended on the time of its addition to the culture medium. The results suggest that the neuroprotective effect of Semax in ischemic stroke can be due to its capacity to protect neurons from damage caused by oxidative stress.

Application of tritium high resolution NMR spectroscopy to analysis of tritium-labelled amino acids and peptides

SummaryA method has been developed for the qualitative and quantitative analysis of complex isotopic mixtures of tritium-labelled amino acids and peptides by using high resolution3H NMR spectroscopy at 266.8 MHz. Determined were tritium distribution in alanine, glycine, tryptophan and 4-hydroxyproline amino acids, as well as in glycine and valine residues of peptides. Approaches have been worked out for the determination of spin coupling constants and isotope chemical shifts for the strongly coupled nonequivalent atoms of the methylene groups.

Degradation of the ACTH(4-10) analog Semax in the presence of rat basal forebrain cell cultures and plasma membranes.

Summary.Here a new approach of the elucidation of paths of proteolytic biodegradation of physiologically active peptides, based on the use of a peptide with isotopic label at all amino acid residues and the enrichment of HPLC samples with unlabeled peptide fragments in UV-detectable concentration, has been proposed. The method has been applied for the investigation of degradation dynamics of the neuroactive heptapeptide MEHFPGP (Semax) in the presence of plasma membranes, and cultures of glial and neuronal cells obtained from the rat basal forebrain. The splitting away of ME and GP, and formation of pentapeptides are the predominant processes in the presence of all tested objects, whereas the difference in patterns of resulting peptide products for glial and neuronal cells has been detected. In conclusion, the approach applied allows analyzing physiologically active peptide concentrations in biological tissues and degradation pathways of peptides in the presence of targets of their action.