Nuclear spin effects in biological processes.

Traditionally, nuclear spin is not considered to affect biological processes. Recently, this has changed as isotopic fractionation that deviates from classical mass dependence was reported both in vitro and in vivo. In these cases, the isotopic effect correlates with the nuclear magnetic spin. Here, we show nuclear spin effects using stable oxygen isotopes (16O, 17O, and 18O) in two separate setups: an artificial dioxygen production system and biological aquaporin channels in cells. We observe that oxygen dynamics in chiral environments (in particular its transport) depend on nuclear spin, suggesting future applications for controlled isotope separation to be used, for instance, in NMR. To demonstrate the mechanism behind our findings, we formulate theoretical models based on a nuclear-spin-enhanced switch between electronic spin states. Accounting for the role of nuclear spin in biology can provide insights into the role of quantum effects in living systems and help inspire the development of future biotechnology solutions.

High-precision measurements of δ(17)O and (17) Oexcess of NBS19 and NBS18.

RATIONALE: Measurements of oxygen-17 excess ((17)Oexcess) in carbonates have become of great importance. However, to compare results obtained by different laboratories, it is necessary to normalize them to international standards. With this purpose in mind, we measured accurate and high precision δ(17)O and (17)Oexcess values for NBS19 and NBS18, two international standards, for which δ(18)O values are already widely used as the references for carbonates. METHODS: The measurements are based on isotopic exchange at steady state between O2 and CO2 over hot platinum sponge at 750°C. Dual-inlet isotope ratio mass spectrometry (IRMS) measurements of the δ(17)O and δ(18)O values of this O2 allow δ(17)O values of CO2 to be obtained with very high precision (0.01 to 0.03‰) and, correspondingly, accurate (17)Oexcess values with a precision of 5 per meg. RESULTS: We measured, for the first time, the δ(17)O values and (17)Oexcess of CO2 liberated from NBS19 at 25°C (39.196 ± 0.026; 20.276 ± 0.015‰ and -227 ± 4 per meg, respectively) and NBS18 (17.591 ± 0.041‰; 9.253 ± 0.018‰ and 3 ± 5 per meg, respectively). The values are given versus VSMOW. CONCLUSIONS: Accurate values of δ(17)O and (17)Oexcess of the international standards NBS19-CO2 and NBS18-CO2 are now available. The new values should be used for normalization of measured oxygen isotope ratios of carbonates to allow meaningful comparison of results among different laboratories.

High-precision measurements of 17O/16O and 18O/16O ratios in CO2.

RATIONALE: Measurements of δ(17)O and δ(18)O values of tropospheric CO(2) are of great importance. However, to be useful, such measurements must be an order of magnitude more precise than in current published literature. With this purpose we developed a new method for high-precision mass spectrometric measurements of (17)O/(16)O and (18)O/(16)O ratios in CO(2), which is presented in this study. METHODS: The method is based on isotopic exchange equilibration between H(2)O and CO(2) in sealed glass ampoules followed by water fluorination to produce O(2). Dual inlet isotope ratio mass spectrometric (IRMS) measurements of the δ(17)O and δ(18)O values of this O(2) allow δ(17)O values of CO(2) to be obtained with very high precision (0.01 to 0.03 ‰). This method requires about 70 µmol of CO(2). RESULTS: Measurements of standard CO(2) gas and atmospheric CO(2) yield reproducibility of 0.01 to 0.03 ‰ for both δ(17)O and δ(18)O values, and 5 per meg for (17)O(excess). Fractionation factors (17)α and (18)α were determined in the H(2)O-CO(2) equilibrium at 25 °C as 1.021254 ± 0.00004 and 1.041036 ± 0.00008, respectively, and the ratio ln(17)α/ln(18)α as 0.5229 ± 0.0001. CONCLUSIONS: The results demonstrate that the new method is the most accurate analytical procedure ever presented for measurements of (17)O(excess) of CO(2) gas. It is suitable for measurements of CO(2) extracted from relatively small samples of air (~5 L), and is thus useful for monitoring the (17)O(excess) of CO(2) on a broad global scale.

Enrichment of oxygen heavy isotopes during photosynthesis in phytoplankton.

Some of the oxygen produced during oxygenic photosynthesis is consumed but little is known about the extent of the processes involved. We measured the (17)O/(16)O and (18)O/(16)O ratios in O(2) produced by certain marine and freshwater phytoplankton representing important groups of primary producers. When the cells were performing photosynthesis under very low dissolved oxygen concentrations (< 3 µM), we observed significant enrichment in both (18)O and (17)O with respect to the substrate water. The difference in δ(18)O between O(2) and water was about 4.5, 3, 5.5, and 7‱ in the diatom Phaeodactylum tricornutum, Nannochloropsis sp. (Eustigmatophyceae), the coccolithophore Emiliania huxleyi and the green alga Chlamydomonas reinhardtii, respectively. The difference in δ(17)O was about 0.52 that of δ(18)O. As explained, the observed enrichments most probably stem from considerable oxygen consumption during photosynthesis even when major O(2)-consuming reactions such as photorespiration were minimized. These enrichments increased linearly with rising O(2) levels but with different δ(17)O/δ(18)O slopes for the various organisms, suggesting engagements of different O(2)-consuming reactions with rising O(2) levels. Consumption of O(2) may be important for energy dissipation during photosynthesis. The isotope enrichment observed here, not accounted for in earlier assessments, closes an important gap in our understanding of the difference between the isotopic compositions of atmospheric oxygen and that of seawater, i.e., the Dole effect.

Mass-dependent isotopic fractionation in ozone produced by electrolysis.

During the electrolysis of water in an acidified medium, ozone is produced, in association with oxygen, at the anode. This ozone is found to be depleted in heavy isotopes ((18)O and (17)O), with respect to the source water, following a strict mass-dependent rule. Our experiments also suggest that the isotopes are distributed at the apex and base positions of the bent ozone molecule in a random fashion, without obeying the zero-point energy constraint. Endowed with these characteristics, the electrolytic ozone provides a source of reference that has a known internal heavy isotope distribution for spectroscopic studies. In addition, this ozone, when subjected to photolytic decomposition, can be used as a source of atomic oxygen with mass-dependent isotope ratios that can be varied by simply changing the water composition. Such an oxygen source is important for studying isotope effects in gas-phase recombination/exchange reactions such as COO + O* --> [COOO*] --> COO* + O.

Diffusivity fractionations of H2(16)O/H2(17)O and H2(16)O/H2(18)O in air and their implications for isotope hydrology.

We have determined the isotope effects of (17)O and (18)O substitution of (16)O in H(2)O on molecular diffusivities of water vapor in air by the use of evaporation experiments. The derived diffusion fractionation coefficients (17)alpha(diff) and (18)alpha(diff) are 1.0146 +/- 0.0002 and 1.0283 +/- 0.0003, respectively. We also determined, for the first time, the ratio ln((17)alpha(diff))/ln((18)alpha(diff)) as 0.5185 +/- 0.0002. This ratio, which is in excellent agreement with the theoretical value of 0.5184, is significantly smaller than the ratio in vapor-liquid equilibrium (0.529). We show how this new experimental information gives rise to (17)O excess in meteoric water, and how it can be applied in isotope hydrology.

High precision measurements of 17O/16O and 18O/16O ratios in H2O.

We have optimized the method of water fluorination using the solid reagent CoF3 to produce O2. This allows isotope ratio measurements by dual-inlet mass spectrometry with very high precision of 0.01 to 0.03/1000 for both delta17O and delta18O. Using this method, delta17O and delta18O of atmospheric O2 were determined as 12.08 and 23.88/1000 vs. VSMOW, respectively. Likewise, delta17O and delta18O of GISP were -13.12 and -24.73/1000, and for SLAP they were -29.48 and -55.11/1000 vs. VSMOW, respectively. Analysis of these data in a ln(delta17O + 1) vs. ln(delta18O + 1) plot yields a line with a regression coefficient (lambda) of 0.5279 +/- 0.0001 (R2 = 0.999999). We also determined the fractionation factors 17alpha and 18alpha in liquid-vapor equilibrium, and found that the ratio ln 17alpha/ln 18alpha is constant (0.529 +/- 0.001) over the temperature range 11.4 to 41.5 degrees C.

Fractionation of the three stable oxygen isotopes by oxygen-producing and oxygen-consuming reactions in photosynthetic organisms.

The triple isotope composition (delta17O and delta18O) of dissolved O2 in the ocean and in ice cores was recently used to assess the primary productivity over broad spatial and temporal scales. However, assessment of the productivity with the aid of this method must rely on accurate measurements of the 17O/16O versus 18O/16O relationship in each of the main oxygen-producing and -consuming reactions. Data obtained here showed that cleavage of water in photosystem II did not fractionate oxygen isotopes; the delta18O and delta17O of the O2 evolved were essentially identical to those of the substrate water. The fractionation slopes for the oxygenase reaction of Rubisco and respiration were identical (0.518 +/- 0.001) and that of glycolate oxidation was 0.503 +/- 0.002. There was a considerable difference in the slopes of O2 photoreduction (the Mehler reaction) in the cyanobacterium Synechocystis sp. strain PCC 6803 (0.497 +/- 0.004) and that of pea (Pisum sativum) thylakoids (0.526 +/- 0.001). These values provided clear and independent evidence that the mechanism of O2 photoreduction differs between higher plants and cyanobacteria. We used our method to assess the magnitude of O2 photoreduction in cyanobacterial cells maintained under conditions where photorespiration was negligible. It was found that electron flow to O2 can be as high as 40% that leaving photosystem II, whereas respiratory activity in the light is only 6%. The implications of our findings to the evaluation of specific O2-producing or -consuming reactions, in vivo, are discussed.

High-precision measurements of 17O/16O and 18O/16O of O2 and O2/Ar ratio in air.

A method for high-precision and high-accuracy mass spectrometric measurements of the ratios among the three oxygen isotopes, and of the O(2)/Ar ratio, is presented. It involves separation of the O(2)-Ar mixture from air and includes a fully automated system that ensures highly reliable sample processing. Repeated measurements of atmospheric oxygen yield the repeatability (+/-SE x t, standard error of the mean (n = 12) multiplied by Student's t-factor for a 95% confidence limit) of 0.004, 0.003 and 0.2 per thousand for delta(18)O, delta(17)O and delta O(2)/Ar, respectively.

Massive light-dependent cycling of inorganic carbon between oxygenic photosynthetic microorganisms and their surroundings.

Membrane inlet mass spectrometry indicated massive light-dependent cycling of inorganic carbon between the medium and the cells of various phytoplankton species representing the main groups of aquatic primary producers. These included diatoms, symbiotic and free living dinoflagellates, a coccolithophorid, a green alga and filamentous and single cell cyanobacteria. These organisms could maintain an ambient CO(2) concentration substantially above or below that expected at chemical equilibrium with HCO(3) (-). The coccolithophorid Emiliania huxleyi shifted from net CO(2) uptake to net CO(2) efflux with rising light intensity. Differing responses of CO(2) uptake and CO(2) fixation to changing light intensity supported the notion that these two processes are not compulsorily linked. Simultaneous measurements of CO(2) and O(2) exchange and of the fluorescence parameters in Synechococcus sp. strain PCC 7942, showed that CO(2) uptake can serve as a sensitive probe of the energy status of the photosynthetic reaction centers. However, during transitions in light intensity, changes in CO(2) uptake did not accord with those expected from fluorescence change. Quantification of the net fluxes of CO(2), HCO(3) (-) and of photosynthesis at steady-state revealed that substantial HCO(3) (-) efflux accompanied CO(2) uptake and fixation in the case of 'CO(2) users'. On the other hand, 'HCO(3) (-) users' were characterized by a rate of net CO(2) uptake below that of CO(2) fixation. The results support the notion that entities associated with the CCM function not only in raising the CO(2) concentration at the site of Rubisco; they may also serve as a means of diminishing photodynamic damage by dissipating excess light energy.