Nuclear spin catalysis in biochemical physics

Magnetic isotope effects have been recently discovered in living nature. They were observed for the first time in experiments on cells enriched with various magnesium isotopes, magnetic ²⁵Mg or non-magnetic ones. A catalytic effect of the magnetic isotope of magnesium was discovered in experiments with myosin, the most important biomolecular motor utilizing the energy of ATP to perform mechanical work. The rate of ATP hydrolysis with the magnetic ²⁵Mg isotope is 2.0–2.5 times higher than that obtained with nonmagnetic ²⁴Mg or ²⁶Mg. A similar effect of the nuclear spin catalysis was experimentally observed for zinc isotopes. The rate of ATP hydrolysis in the case of magnetic ⁶⁷Zn increased by 40–50% as compared to that observed experimentally for nonmagnetic isotopes (⁶⁴Zn or ⁶⁸Zn). Catalytic effects of the magnetic isotope of magnesium were also experimentally found for H⁺-ATPase isolated from yeast mitochondria and ATPase of the plasma membrane of the myometrium. The magnetic isotope effect indicates unambiguously that the chemomechanical processes involve a limiting step catalyzed by biomolecular motors, which depends on the electronic spin state, and that this step is accelerated in the presence of nuclear spin of the magnetic isotope.

     

Firefly Luciferase Bioluminescence as a Tool for Searching Magnetic Isotope Effects in ATP-Dependent Enzyme Reactions

Cells and tissues are composed from atoms of chemical elements, some of which have two kinds of stable isotopes, magnetic and nonmagnetic ones. Not long ago, magnetic isotope effects (MIEs) have been discovered in experiments with cells enriched with magnetic or nonmagnetic isotopes of magnesium. These MIEs can stem from higher efficiency of the enzymes of bioenergetics in the cells enriched with magnetic magnesium isotope. In the studies of MIEs in biological systems, it is needed to monitor the ATP concentrations as the major energy source in cells. The most sensitive and rapid method of the ATP measurements is based on the use of the firefly luciferase–luciferin system. Since luciferase is the ATP-dependent enzyme and activated by Mg-ions, it is necessary to elucidate whether this enzyme is sensitive to magnetic field of the magnesium isotope’s nuclear spin. Herein we present the results of studying the effects of different isotopes of magnesium, magnetic ²⁵Mg and nonmagnetic ²⁴Mg and ²⁶Mg, on bioluminescence spectra and enzymatic activity of firefly luciferase. It was shown, that neither kinetics of the bioluminescence signal nor the bioluminescence spectra manifest any statistically significant dependence on the type of magnesium isotope. So, no MIEs have been revealed in the luciferase-catalyzed oxidation of luciferin. It means that firefly luciferase bioluminescence can serve as the tool for search and studies of magnetic isotope effects in ATP-dependent enzyme reactions in biological systems, including the enzymatic synthesis and hydrolysis of ATP.     

Effect of the 67Zn isotope on leukemic cells and normal lymphocytes

Porphyrinofullerene nanoparticles (NP) containing magnetic isotopes ²⁵Mg and ⁶⁷Zn (²⁵Mg-NP and ⁶⁷Zn-NP) and the natural isotopic composition of zinc (Zn) were tested on human leukemic cells of patients with acute leukemia and on lymphocytes of healthy donors. The fundamental differences in the cytotoxic effect of magnetic and nonmagnetic zinc isotopes on tumor cells were observed, as well as the complete absence of the influence of the magnetic magnesium isotope and pristine nanoparticles. The ⁶⁷Zn-NP manifested high cytotoxicity towards cells of acute B-lymphoblast leukemia with LD₅₀ almost three times lower than that of healthy donors and four times lower than that of the Zn-NP. Apoptosis was evaluated by cytofluorimetry for the drugs used.     

Separation of magnesium isotopes by a 2'-aminomethyl-18-crown-6 bonded Merrifield peptide resin

Separation of magnesium isotopes was investigated by chemical ion exchangewith synthesized 2'-aminomethyl-18-crown-6 (AM18C6) bonded Merrifieldpeptide resin using an elution chromatographic technique. The capacity ofthe novel crown ion exchanger was found to be 2.3 meq/g dry resin. The heavierisotopes of magnesium were enriched in the solution phase, while the lighterisotopes were enriched in the resin phase. The single stage separation factorwas determined according to the method of GLUECKAUF from the elution curveand isotopic assays. The separation factors of ²⁴Mg–²⁵ Mg, ²⁵ Mg–²⁶ Mg, and ²⁴ Mg–²⁶ Mg isotope pair fractionations were 1.012, 1.005, and 1.022, respectively.     

Syntheses and separating properties of triazacrown and AM18C6 bonded Merrifield peptide resins

Separation of lithium and magnesium isotopes by cation exchange elution chromatography was carried out with a synthesized 1,13,16-trioxa-4,7,10-triazacyclooctadecane (N₃O₃)-4,7,10-trimerrifield peptide resin and with a 2^′-aminomethyl-18-crown-6 (AM18C6) bonded Merrifield peptide resin. The resins have a capacity of 0.1 and 2.3 meq/g dry resin. A single stage separation factor of lithium isotopes, 1.018 was obtained by the Glueckauf theory from the elution curve and isotopic assays. The heavier isotope, ⁷Li was concentrated in the resin phase, while the lighter isotope, ⁶Li concentrated in the solution phase. On the other hand, the heavier isotopes of magnesium were concentrated in the solution phase, while the lighter isotopes were concentrated in the resin phase. The separation factors of ²⁴Mg-²⁵Mg, ²⁴Mg-²⁶Mg, and ²⁵Mg-²⁶Mg isotope pair fractionations were 1.012, 1.022, and 1.012, respectively.     

Magnesium isotope separation by ion exchange using hydrous manganese(IV) oxide

The magnesium isotope effects were investigated by chemical ion exchange with a hydrous manganese(IV) oxide. The capacity of manganese(IV) oxide was 0.5 meq g(-1). The distribution coefficient of magnesium ions on the MnO(2) was determined by a batch method. The heavier isotopes of magnesium were enriched in the solution phase, while the lighter isotopes were enriched in the hydrous MnO(2) phase. The separation factor was determined according to the method of Glueckauf from the elution curve and isotopic assays. The separation factors of (24)Mg(2+)-(25)Mg(2+), (24)Mg(2+)-(26)Mg(2+), and (25)Mg(2+)-(26)Mg(2+) isotope pair fractionations were 1.011, 1.021, and 1.011, respectively.     

Adsorption and isotope effects by ion exchange with 1-aza-12-crown-4 bonded Merrifield peptide resin

Magnesium isotope effects were investigated by chemical ion exchange with synthesized 1-aza-12-crown-4 bonded Merrifield peptide resin using elution chromatography. The capacity of azacrown ion exchanger was 0.89 meq/g dry resin. The heavier isotopes of magnesium were enriched in the resin phase, while the lighter isotopes were enriched in the solution phase. The hydration effect is less than the complexation and isotope mass effects. The single stage separation factor was determined according to the method of Glueckauf from the elution curve and isotopic assays. The separation factors of 24Mg(2+)-25Mg(2+), 24Mg(2+)-26Mg(2+), and 25Mg(2+)-26Mg(2+) were 1.012, 1.023, and 1.011, respectively.     

Enrichment of Magnesium Isotopes by N3O2 Azacrown Ion Exchange Resin

The elution chromatographic separation of magnesium isotopes was investigated by chemical ion exchange with the synthesized 1,7-dioxa-4,10,13-triazacyclopentadecane-4,10,13-trimerrifield peptide resin [N₃O₂·3M]. The capacity of novel N₃O₂ azacrown ion exchanger was 0.21 meq/g dry resin. The heavier isotopes of magnesium concentrated in the resin phase, while the lighter isotopes are enriched in the solution phase. The glass ion exchange column used in our experiment was 30 cm long with inner diameter of 0.2 cm, and the 2.0M NH₄Cl solution was used as an eluent. The separation factors of ²⁴Mg-²⁵Mg, ²⁵Mg-²⁶Mg, and ²⁴Mg-²⁶Mg were 1.030, 1.009, and 1.027, respectively.     

Separation of magnesium isotopes by N4S2 azacrown ion exchanger

The chromatographic separation of magnesium isotopes was investigated by chemical ion exchange with 1,16-dithia-4,7,10,13-tetraazacyclooctadecane-4,7,10,13-tetramerrifield peptide resin[N₄S₂·4M] synthesized recently. The capacity of novel N₄S₂ azacrown ion exchanger was 0.34 meq/g dry resin. The heavier isotopes of magnesium concentrated in the resin phase, while the lighter isotopes are enriched in the solution phase. The glass ion exchange column used was 30 cm long with inner diameter of 0.2 cm, and the 1.0M NH₄Cl solution was used as an eluent. The separation factors of²⁴Mg−²⁵Mg,²⁵Mg−²⁶Mg, and²⁴Mg−²⁶Mg were 1.047, 1007, and 1.008, respectively.     

Adsorption and isotope effects by ion exchange with aqueous-2-aminomethyl-18-crown-6 bonded merrifield resin

Magnesium isotopes effects were investigated by chemical ion exchange using synthesized 2-aminomethyl-18-crown-6 (AM18C6) bonded Merrifield peptide resin. It was found that separation factors larger those reported previously were obtained, and the hydration and isotope mass effects are more significant than that of the complexation. The capacity of the crown ion exchanger was 2.3 meq/g dry resin. The adsorption capacity of the resin for magnesium ion was 26.8 mg/g dry resin at pH 7. The heavier isotopes of magnesium were enriched in the solution phase, while the lighter isotopes were enriched in the resin phase. The separation factors of (24)Mg-(25)Mg, (24)Mg-(26)Mg, and (25)Mg-(26)Mg were 1.0085, 1.0162, and 1.0081, respectively.     

Magnesium isotope effects in enzymatic phosphorylation

Recent discovery of magnesium isotope effect in the rate of enzymatic synthesis of adenosine triphosphate (ATP) offers a new insight into the mechanochemistry of enzymes as the molecular machines. The activity of phosphorylating enzymes (ATP-synthase, phosphocreatine, and phosphoglycerate kinases) in which Mg(2+) ion has a magnetic isotopic nucleus 25Mg was found to be 2-3 times higher than that of enzymes in which Mg(2+) ion has spinless, nonmagnetic isotopic nuclei 24Mg or 26Mg. This isotope effect demonstrates unambiguously that the ATP synthesis is a spin-dependent ion-radical process. The reaction schemes, suggested to explain the effect, imply a reversible electron transfer from the terminal phosphate anion of ADP to Mg(2+) ion as a first step, generating ion-radical pair with singlet and triplet spin states. The yields of ATP along the singlet and triplet channels are controlled by hyperfine coupling of unpaired electron in 25Mg+ ion with magnetic nucleus 25Mg. There is no difference in the ATP yield for enzymes with 24Mg and 26Mg; it gives evidence that in this reaction magnetic isotope effect (MIE) operates rather than classical, mass-dependent one. Similar effects have been also found for the pyruvate kinase. Magnetic field dependence of enzymatic phosphorylation is in agreement with suggested ion-radical mechanism.     

Enrichment of Magnesium Isotopes by 2′-Aminomethyl-15-Crown-5 Bonded Merrifield Peptide Resin

Magnesium isotope enrichment was investigated by chemical ion exchange with a synthesized 2-aminomethyl-15-crown-5 bonded Merrifield peptide resin using elution chromatography. The capacity of the novel crown ion exchanger was found to be 2.25 meq/g dry resin. The heavier isotopes of magnesium were enriched in the solution phase, while the lighter isotopes were enriched in the resin phase. The separation factor was determined according to the method of GLUECKAUF from the elution curve and isotopic assays. The separation factors of ²⁴Mg²⁺–²⁵Mg²⁺, ²⁴Mg²⁺–²⁶Mg²⁺, and ²⁵Mg²⁺–²⁶Mg²⁺ isotope pair fractionations were 1.00095, 1.00857, and 1.00014, respectively.     

Separation of magnesium isotopes by chemical exchange with a polymer-bound azacrown compound

The chromatographic separation of magnesium isotopes was investigated by chemical exchange with the recently synthesized 1-oxa-4,7,10,13-tetraazacyclopentadecane-4,7,10,13-tetramerrifield peptide resin [N₄O·4M]. The capacity of the novel N₄O-4 Merrifield ion exchanger was 1.0 meq/g dry resin. The heavier isotope²⁶Mg concentrated in the resin phase, while the lighter isotopes²⁴Mg, and²⁵Mg are enriched in the fluid phase. The maximum separation factors , for²⁵Mg–²⁶Mg and²⁴Mg–²⁶Mg were found to be 1.048 and 1.022, respectively, at 20.0±0.02 °C with 2.0 M ammonium chloride solution as an eluent.     

Chemically induced magnetic isotope effect on the tin nuclei during the photolysis of (1-fluorenyl)trimethyltin

A magnetic isotope effect on the ¹¹⁷Sn and ¹¹⁹Sn nuclei, accompanied by the fractionation of magnetic and nonmagnetic tin isotopes, was observed during the photolysis of (9-fluorenyl)trimethyltin. The magnetic and nonmagnetic isotopes were accumulated, respectively, in the initial compound and a photolysis product (hexamethyldistannane).     

Measuring magnesium,calcium and potassium isotope ratios using ICP-QMS with an octopole collision cell in tracer studies of nutrient uptake and translocation in plants

The ability of a quadrupole-based ICP-MS with an octopole collision cell to obtain precise and accurate measurements of isotope ratios of magnesium, calcium and potassium was evaluated. Hydrogen and helium were used as collision/reaction gases for ICP-MS isotope ratio measurements of calcium and potassium in order to avoid isobaric interference with the analyte ions from (mainly) argon ions ⁴⁰Ar⁺ and argon hydride ions ⁴⁰Ar¹H⁺. Mass discrimination factors determined for the isotope ratios ²⁵Mg/²⁴Mg, ⁴⁰Ca/⁴⁴Ca and ³⁹K/⁴¹K under optimized experimental conditions varied between 0.044 and 0.075. The measurement precisions for ²⁵Mg/²⁴Mg, ⁴⁰Ca/⁴⁴Ca and ³⁹K/⁴¹K were found to be 0.09%, 0.43% and 1.4%, respectively. This analytical method that uses ICP-QMS with a collision cell to obtain isotope ratio measurements of magnesium, calcium and potassium was used in routine mode to characterize biological samples (nutrient solution and small amounts of digested plant samples). The mass spectrometric technique was employed to study the dynamics of nutrient uptake and translocation in barley plants at different root temperatures (10 °C and 20 °C) using enriched stable isotopes (²⁵Mg, ⁴⁴Ca and ⁴¹K) as tracers. For instance, the mass spectrometric results of tracer experiments demonstrated enhanced ²⁵Mg and ⁴⁴Ca uptake and translocation into shoots at a root temperature of 20 °C 24 h after isotope spiking. In contrast, results obtained from ⁴¹K tracer experiments showed the highest ⁴¹K contents in plants spiked at a root temperature of 10 °C.     

Magnetic field affects enzymatic ATP synthesis

The rate of ATP synthesis by creatine kinase extracted from V. xanthia venom was shown to depend on the magnetic field. The yield of ATP produced by enzymes with 24Mg2+ and 26Mg2+ ions in catalytic sites increases by 7-8% at 55 mT and then decreases at 80 mT. For enzyme with 25Mg2+ ion in a catalytic site, the ATP yield increases by 50% and 70% in the fields 55 and 80 mT, respectively. In the Earth field the rate of ATP synthesis by enzyme, in which Mg2+ ion has magnetic nucleus 25Mg, is 2.5 times higher than that by enzymes, in which Mg2+ ion has nonmagnetic, spinless nuclei 24Mg or 26Mg. Both magnetic field effect and magnetic isotope effect demonstrate that the ATP synthesis is an ion-radical process, affected by Zeeman interaction and hyperfine coupling in the intermediate ion-radical pair.     

The magnetic isotope effect as a tool for apoptosis modulation in leukemic cells

Lymphocytes from healthy donors and leukemic cells of patients with acute B-lymphoblastic leukemia (BALL-1) and acute myeloid leukemia were exposed to nanoparticles bearing magnetic (Zn-67) and nonmagnetic (total isotope pool) nuclei of zinc. The values of the corresponding magnetic isotope effects determined as the ratio of LD₅₀ magnitudes of preparations with magnetic and nonmagnetic zinc isotopes were 0, 3.5, and 1.5. Morphological studies using confocal and fluorescence microscopy showed apoptotic death of cells with the preparations; as well, there was an increase in the cell aggregation and better aggregation of nanoparticles in the case of ⁶⁷Zn-NP, which resulted in a decrease of cytotoxicity. However the magnetic isotope effect was observed even in the case of aggregation.     

A new nuclear spin selective reaction of molecular oxygen

During high-temperature (623–673 K) oxidation of polyarylenes (polypyromellitimide and polyphenylquinoxaline), the molecular oxygen is enriched in the¹⁸O nonmagnetic isotope and impoverished in the¹⁷O magnetic isotope. The isotope selection increases with the increase in the degree of conversion of oxygen. The spin-selective reaction responsible for the selection of the¹⁷O isotope is the addition of molecular oxygen to triplet exited aromatic fragments of macromolecules to give endoperoxides. This reaction, which is selective in terms of the electron spin, is also nuclear-spin selective resulting in a magnetic isotope effect. The selection of nonmagnetic isotopes,¹⁶O and¹⁸O, is caused by competition between the reversible and irreversible decomposition of endoperoxide and by the classical isotope effect in these reactions.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1402–1405, August, 1994.The authors are grateful to E. M. Galimov and I. V. Nikulina for high-quality isotope analyses and to the Russian Foundation for Basic Research for financial support (grant 93-03-5227).     

Separation of magnesium isotopes by NTOE bonded merrifield peptide resin

Separation of magnesium isotopes was investigated by chemical ion exchange with synthesyzed 1,12-diaza-3,4:9,10-dibenzo-5,8-dioxacyclo pentadecane(NTOE) bonded merrifield peptide resin using elution chromatographic technique. The capacity of novel diazacrown ion exchanger was 0.29 meq/g dry resin. The heavier isotopes of magnesium were concentrated in the solution phase, while the lighter isotopes were enriched in the resin phase. The glass ion exchange column used in our experiment was 32 cm long with inner diameter of 0.2 cm, and 0.5M NH₄Cl solution was used as an eluent. The single stage separation factor was determined according to the method of GLUECKAUF from the elution curve and isotopic assays. The separation factors of ²⁴Mg²⁺–²⁵Mg²⁺, ²⁴Mg²⁺–²⁶Mg²⁺, and ²⁵Mg²⁺–₂₆Mg²⁺ were 1.063, 1.080, and 1.021, respectively.     

Crystal Chemistry Study of Two Magnesium Complexes with Trifluoroacetylacetone

Single crystal X-ray diffraction at a temperature of 150(2) K is used to determine the structures of two magnesium complexes with trifluoroacetylacetone: [Mg(tfac)₂]₃I and [Mg(H₂O)₂(tfac)₂]·H₂O II. Crystallographic data for I: space group P2₁/n, a = 12.5226(10) Å, b = 13.0591(7) Å, c = 12.6034(13) Å, β = 95.243(2)°, V = 2052.5(3) ų, Z = 2; for II: space group P2₁/c, a = 10.826(2) Å, b = 7.0742(13) Å, c = 21.858(4) Å, β = 102.712(5)°, V = 1632.9(5) ų, Z = 4. The isle structure of I is formed by linear trimeric molecules; in the structure of II the molecules of the complex and crystallization water form a layered framework using hydrogen bondings; the coordinated water molecules are in a trans position. The magnesium atoms have a distorted octahedral coordination environment, the Mg–O distances are 1.991(4)- 2.146(4) Å and 2.040(5)-2.073(5) Å in molecules of I and II respectively.