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PubMed2004

Superimposed dual-isotope SPECT using 99mTc-hydroxymethylene diphosphonate and 201Tl-chloride to assess cartilage invasion in laryngohypopharyngeal cancer.

OBJECTIVE: Cartilage invasion in laryngohypopharyngeal cancer has a significant impact on the choice of treatment modality and outcome of the disease. We examined invasion of cartilage in laryngohypopharyngeal cancer by simultaneous bone and tumor dual-isotope SPECT using 99mTc-hydroxymethylene diphosphonate and 201Tl-chloride. METHODS: Early and delayed simultaneous bone and tumor dual-isotope SPECT were performed on 19 patients with laryngohypopharyngeal cancer. Dual-isotope SPECT images were superimposed to project tumor location from tumor SPECT onto the osseous structures shown by bone SPECT. The presence or absence of cartilage invasion was evaluated histopathologically or by radiological studies such as CT and/or MRI. RESULTS: Histopathological or radiological examination of the cartilage revealed invasion in 5 patients and no invasion in 14 patients. The results of both early and delayed dual-isotope SPECT were exactly the same. Using dual-isotope SPECT, the sensitivity, specificity, and accuracy in detecting cartilage invasion by laryngohypopharyngeal cancer were: 80% (4/5), 92.9% (13/14), and 89.5% (17/19), respectively. CONCLUSIONS: Results of the present study suggest that superimposed early bone and tumor dual-isotope SPECT images may be sufficient for the diagnostic evaluation of cartilage invasion by laryngohypopharyngeal cancer. Superimposed dual-isotope SPECT imaging is a useful technique in the evaluation of cartilage invasion in laryngohypopharyngeal cancer.

Aged↗
PubMed2003

Evaluation of the combined measurement uncertainty in isotope dilution by MC-ICP-MS.

The combination of metrological weighing, the measurement of isotope amount ratios by a multicollector inductively coupled plasma mass spectrometer (MC-ICP-MS) and the use of high-purity reference materials are the cornerstones to achieve improved results for the amount content of lead in wine by the reversed isotope dilution technique. Isotope dilution mass spectrometry (IDMS) and reversed IDMS have the potential to be a so-called primary method, with which close comparability and well-stated combined measurement uncertainties can be obtained. This work describes the detailed uncertainty budget determination using the ISO-GUM approach. The traces of lead in wine were separated from the matrix by ion exchange chromatography after HNO(3)/H(2)O(2) microwave digestion. The thallium isotope amount ratio ( n((205)Tl)/ n((203)Tl)) was used to correct for mass discrimination using an exponential model approach. The corrected lead isotope amount ratio n((206)Pb)/ n((208)Pb) for the isotopic standard SRM 981 measured in our laboratory was compared with ratio values considered to be the least uncertain. The result has been compared in a so-called pilot study "lead in wine" organised by the CCQM (Comité Consultatif pour la Quantité de Matière, BIPM, Paris; the highest measurement authority for analytical chemical measurements). The result for the lead amount content k(Pb) and the corresponding expanded uncertainty U given by our laboratory was:k(Pb)=1.329 x 10-10mol g-1 (amount content of lead in wine)U[k(Pb)]=1.0 x 10-12mol g-1 (expanded uncertainty U=kxuc, k=2)The uncertainty of the main influence parameter of the combined measurement uncertainty was determined to be the isotope amount ratio R(206,B) of the blend between the enriched spike and the sample.

Lead↗
PubMed2003

Isotope-dilution ICP-MS for trace element determination and speciation: from a reference method to a routine method?

This critical review discusses the conditions under which inductively coupled plasma-isotope dilution mass spectrometry (ICP-IDMS) is suitable as a routine method for trace element and element-speciation analysis. It can, in general, be concluded that ICP-IDMS has high potential for routine analysis of trace elements if the accuracy of results is of predominant analytical importance. Hyphenated techniques with ICP-IDMS suffer both from lack of commercially available isotope-labeled spike compounds for species-specific isotope dilution and from the more complicated system set-up required for species-unspecific ICP-IDMS analysis. Coupling of gas or liquid chromatography with species-specific ICP-IDMS, however, enables validation of analytical methods involving species transformations which cannot easily be performed by other methods. The potential and limitations of ICP-IDMS are demonstrated by recently published results and by some unpublished investigations by our group. It has been shown that possible loss of silicon as volatile SiF4 during decomposition of a sample by use of hydrofluoric acid has no effect on trace silicon determination if the isotope-dilution step occurs during digestion in a closed system. For powder samples, laser ablation ICP-IDMS can be applied with an accuracy comparable with that only available from matrix-matched standardization, whereas the accuracy of electrothermal vaporization ICP-IDMS was strongly dependent on the element determined. The significance of easy synthesis of isotope-labeled spike compounds for species-specific ICP-IDMS is demonstrated for monomethylmercury and Cr(VI). Isotope-exchange reactions between different element species can prevent the successful application of ICP-IDMS, as is shown for iodinated hydrocarbons. It is also shown for monomethylmercury that species transformations during sample-pretreatment steps can be followed by species-specific ICP-IDMS without loss of accuracy. A relatively simple and time-efficient procedure for determination of monomethylmercury in environmental and biological samples is discussed. The method, which entails a rapid microwave-assisted isotope dilution step and in-situ extraction of the derivatized species, has good potential for routine application in the future.

Journal Article↗
PubMed2005

Isotope dilution ICP-MS with laser-assisted sample introduction for direct determination of sulfur in petroleum products.

Inductively coupled plasma isotope dilution mass spectrometry (ICP-IDMS) with direct laser-assisted introduction of isotope-diluted samples into the plasma, using a laser ablation system with high ablation rates, was developed for accurate sulfur determinations in different petroleum products such as 'sulfur-free' premium gasoline, diesel fuel, and heating oil. Two certified gas oil reference materials were analyzed for method validation. Two different 34S-enriched spike compounds, namely, elementary sulfur dissolved in xylene and dibenzothiophene in hexane, were synthesized and tested for their usefulness in this isotope dilution technique. The isotope-diluted sample was adsorbed on a filter-paper-like material, which was fixed in a special holder for irradiation by the laser beam. Under these conditions no time-dependent spike/analyte fractionation was only observed for the dibenzothiophene spike during the laser ablation process, which means that the measured 34S/32S isotope ratio of the isotope-diluted sample remained constant-a necessary precondition for accurate results with the isotope dilution technique. A comparison of LA-ICP-IDMS results with the certified values of the gas oil reference materials and with results obtained from ICP-IDMS analyses with wet sample digestion demonstrated the accuracy of the new LA-ICP-IDMS method in the concentration range of 9.2 microg g(-1) ('sulfur-free' premium gasoline) to 10.4 mg g(-1) (gas oil reference material BCR 107). The detection limit for sulfur by LA-ICP-IDMS is 0.04 microg g(-1) and the analysis time is only about 10 min, which therefore also qualifies this method for accurate determinations of low sulfur contents in petroleum products on a routine level.

Lasers↗
PubMed2001

Certification of the chlorine content of the isotopic reference materials IRMM-641 and IRMM-642.

The Slovak Institute of Metrology and the Institute for Reference Materials and Measurements have collaborated in the certification of the two chlorine reference materials IRMM-641 and IRMM-642. Until now no isotopically enriched chlorine isotopic reference material certified for isotopic composition and content has been available commercially. The isotopic reference materials IRMM-641 and IRMM-642 described herein are certified for isotopic composition and for chlorine content. The chlorine content of the reference material IRMM-641 was certified by use of high-precision argentometric coulometric titration at the Slovak Institute of Metrology. The base material used for IRMM-641 is NIST Standard Reference Material 975. The chlorine content of the reference material IRMM-642 was measured by isotope dilution, using negative thermal ionization mass spectrometry at the Institute for Reference Materials and Measurements. Both standard reference materials were prepared by dissolving NaCl in water. The reference material IRMM-641 contains 0.025022 +/- 0.00011 mol kg(-1) chlorine of natural isotopic composition with an n(37Cl)/n(35Cl) ratio of 0.31977 +/- 0.00082. The reference material IRMM-642 contains 0.004458 +/- 0.000028 mol kg(-1) chlorine with an n(37Cl)/n(35Cl) ratio of 0.01914 +/- 0.00088.

Journal Article↗
PubMed1991

Corticosteroid side-chain oxidations--I. Structural effects on the excreted isotope ratios of 4-14C- and 21-3H-labelled corticosteroid metabolites in rabbit urine.

The metabolic fates of 4-14C- and 21-3H-labelled corticosteroids have been investigated in the rabbit by analysis of the normalized isotope ratios of neutral and acidic metabolites excreted in the urine. Isotope ratios of excreted radioactivity declined in the order cortisol (F) greater than corticosterone (B) greater than 11-desoxycortisol (S) greater than deoxycorticosterone (DOC). Steroid acids, isolated in alumina fraction C, represented 19.0, 15.0, 9.7 and 2.7% of the doses of DOC, B, S and F, respectively, and the isotope ratios declined in the order F greater than B greater than S greater than DOC. HPLC of steroid acid methyl ester derivatives indicated generally low isotope ratios for DOC and S steroid acids, consistent with complete side-chain oxidation to 20-oxo-21-oic acids and/or 17-carboxylic acids. Several B metabolite methyl esters peaks also exhibited low isotope ratios, but both B and F metabolites gave methyl esters that retained significant tritium consistent with the presence of 20-hydroxysteroid acids. The 21-hydroxy-steroid metabolite fractions had isotope ratios of F = S greater than B greater than DOC. HPLC showed that 20-oxo (tetrahydro) metabolites of B and F had reduced isotope ratios unlike the C-20 reduced (hexahydro) metabolites of DOC and S. It may be concluded that the metabolic fate of the corticoid side-chain in the rabbit is dependent on the steroid structure and may result in the excretion of both 20-oxo and 20-hydroxysteroid acids.

Adrenal Cortex Hormones↗
PubMed2001

A trend analysis of the relative value of blue dye and isotope localization in 2,000 consecutive cases of sentinel node biopsy for breast cancer.

BACKGROUND: Among the advocates of blue dye, isotope, or combined dye-isotope mapping of the sentinel lymph node (SLN) for breast cancer, there is no universal consensus as to which technique is optimal and whether the relative value of each method changes with increasing experience. The objective of this study was to examine the relative contributions of blue dye and radioisotope to successful identification of the SLN as the SLN-mapping technique evolved over our first 2,000 consecutive cases. STUDY DESIGN: Using the first 2,000 consecutive SLN biopsy procedures for breast cancer, performed by eight surgeons (none previously experienced in SLN techniques) at one institution, using a combined technique of blue dye and isotope mapping, we report the institutional learning curve and the relative contributions of dye and isotope to identifying both the SLN and the positive SLN, by increments of 500 cases. RESULTS: Comparing the first 500 with the most recent 500 cases, success in identifying the SLN by blue dye did not improve with experience, although success in isotope localization steadily increased, from 86% to 94% (p < 0.00005). With the increasing success of isotope mapping, the marginal benefit of blue dye (the proportion of cases in which the SLN was identified by blue dye alone) steadily declined, from 9% to 3% (p = 0.0001). Parallel to this trend, the proportion of positive SLNs identified by blue dye did not change with experience (89% to 90%), but isotope success steadily increased, from 88% to 98% (p = 0.0015). The proportion of positive SLNs identified by blue dye alone declined from 12% to 2% (p = 0.0015). CONCLUSIONS: Using a combined technique of blue dye and radioisotope mapping, and with refinement of the radioisotope technique, we report 97% success identifying the SLN. Although we continue to recommend the use of both methods in SLN mapping for breast cancer, we observe with experience a declining marginal benefit for blue dye.

Breast Neoplasms↗
PubMed2003

Reduction of chemical formulas from the isotopic peak distributions of high-resolution mass spectra.

A method has been developed for the reduction of the chemical formulas of compounds in complex mixtures from the isotopic peak distributions of high-resolution mass spectra. The method is based on the principle that the observed isotopic peak distribution of a mixture of compounds is a linear combination of the isotopic peak distributions of the individual compounds in the mixture. All possible chemical formulas that meet specific criteria (e.g., type and number of atoms in structure, limits of unsaturation, etc.) are enumerated, and theoretical isotopic peak distributions are generated for each formula. The relative amount of each formula is obtained from the accurately measured isotopic peak distribution and the calculated isotopic peak distributions of all candidate formulas. The formulas of compounds in simple spectra, where peak components are fully resolved, are rapidly determined by direct comparison of the calculated and experimental isotopic peak distributions. The singular value decomposition linear algebra method is used to determine the contributions of compounds in complex spectra containing unresolved peak components. The principles of the approach and typical application examples are presented. The method is most useful for the characterization of complex spectra containing partially resolved peaks and structures with multiisotopic elements.

Journal Article↗
PubMed1993

Product dependence of deuterium isotope effects in enzyme-catalyzed reactions.

Theory for enzyme-catalyzed reactions is developed for the dependence on product concentration of deuterium isotope effects on V and V/K. Generally, a product that decreases the off-rate for a second product to zero causes the isotope effect on V/K to decrease to DKeq and that on V to decrease to a value between 1 and DKeq. If the second product off-rate is decreased to a finite value, DV and D(V/K) will decrease to a value greater than DKeq, while if there is no effect on the off-rate for the second product, DV and D(V/K) will not change. Interestingly, for a ping-pong mechanism, the presence of the product that provides a reversible connection between the isotope-sensitive step and the isotope-insensitive half-reaction will give an isotope effect on V/K for the latter. (In the absence of the product, the isotope effect on V/K for the isotope-insensitive half-reaction will be unity.) Theory is supported with data for alcohol and lactate dehydrogenases. For lactate dehydrogenase, D(V/Kpyruvate) decreases from 1.93 +/- 0.02 at zero to 1.16 +/- 0.02 at infinite lactate concentration, while DV decreases from a value of 1.75 +/- 0.03 at zero to a value of 0.93 +/- 0.05 at infinite lactate concentration. Thus, it appears that the pathway in which lactate is released first is greatly preferred, but the pathway in which NAD+ is released before lactate is observed at high lactate concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1994

Intrinsic primary, secondary, and solvent kinetic isotope effects on the reductive half-reaction of D-amino acid oxidase: evidence against a concerted mechanism.

D-Amino acid oxidase catalyzes the oxidation of D-amino acids to imino acids with subsequent transfer of the electrons to molecular oxygen. Proposed mechanisms for the mode of cleavage of the substrate CH bond include stepwise formation of a carbanion, followed by attack of the carbanion on the enzyme-bound FAD, direct hydride transfer of the substrate alpha-hydrogen to the FAD, and transfer of a hydride from the substrate amino group to the FAD. Conditions have previously been established under which large, limiting, primary deuterium kinetic isotope effects can be measured with D-alanine, D-serine, and glycine as substrates for D-amino acid oxidase [Denu, J. M., & Fitzpatrick, P. F. (1992) Biochemistry 31, 8207-8215]. To determine whether these values are the intrinsic isotope effects, primary tritium kinetic isotope effects have been determined with these three substrates. The values are 12.6, 8.6, and 6.4, respectively. These values are consistent with expression of the intrinsic isotope effects under these conditions, allowing for determination of the values of the intrinsic deuterium effects as 5.7, 4.5, and 3.6 for D-alanine, D-serine, and glycine, respectively. Under these conditions, the alpha-secondary tritium kinetic isotope effect with glycine, the beta-secondary deuterium kinetic isotope effect with D-alanine, and the solvent kinetic isotope effect with D-serine are all indistinguishable from unity. These results are not consistent with concerted mechanisms for CH bond cleavage with this enzyme, but are fully consistent with the involvement of a carbanion intermediate.

Alanine↗
PubMed1983

Isotopic probes yield microscopic constants: separation of binding energy from catalytic efficiency in the bovine plasma amine oxidase reaction.

Bovine plasma amine oxidase catalyzes the oxidative deamination of primary amines. The reaction can be viewed as two half-reactions: enzyme reduction by substrate followed by enzyme reoxidation by dioxygen. Anaerobic stopped-flow kinetic measurements of the first half-reaction indicate very large deuterium isotope effects for benzylamine, m-tyramine, and dopamine, Dk = 13.5 +/- 1.3, which are ascribed to an intrinsic isotope effect. From the insensitivity of these isotope effects to amine concentration, stopped-flow data provide substrate dissociation constants, K1, and rate constants for the C-H bond cleavage step, k3, directly. Steady-state isotope effects have also been measured for benzylamine and six ring-substituted phenethylamines. Whereas a small range of values for kcat, 0.38-1.2 s-1, and Dkcat, 5.4-8.8, is observed, kcat/Km = 1.3 X 10(2) to 3.8 X 10(4) M-1 S-1 and D(kcat/Km) = 5.6-16.1 indicate a marked effect of ring substituent. As described earlier [Miller, S., & Klinman, J.P. (1982) Methods Enzymol. 87, 711], the availability of an intrinsic isotope effect for an enzymatic reaction permits calculation of microscopic constants from steady-state data. By employment of a minimal mechanism for bovine plasma amine oxidase involving a single precatalytic and multiple postcatalytic enzyme-substrate complexes, equations have been derived that allow calculation of k3 and K1 when DKeq congruent to 1 less than Dk. Unexpectedly, in the case of K1, we have shown that this parameter can be calculated from steady-state parameters without the requirement for an intrinsic isotope effect. This result should have general application to both ping-pong and sequential ternary-complex enzyme mechanisms. Of significance for future applications of steady-state isotope effects to the calculation of microscopic constants, values for K1 and k3 derived from steady-state parameters and single turnover measurements indicate excellent agreement. Compilation of parameters among six ring-substituted phenethylamines reveals alteration in delta G for enzyme-substrate complex formation by 2.8 kcal/mol, together with an essentially invariant rate constant for C-H bond activation. A detailed discussion of the relevance of these findings to the interrelationship of binding energy and catalytic efficiency in enzyme reactions is presented.

Amine Oxidase (Copper-Containing)↗
PubMed1985

Peroxidase-catalyzed N-demethylation reactions: deuterium solvent isotope effects.

The effect of D2O on the kinetic parameters for the hydroperoxide-supported N-demethylation of N,N-dimethylaniline catalyzed by chloroperoxidase and horseradish peroxidase was investigated in order to assess the roles of exchangeable hydrogens in the demethylation reaction. The initial rate of the chloroperoxidase-catalyzed N-demethylation of N,N-dimethylaniline supported by ethyl hydroperoxide exhibited a pL optimum (where L denotes H or D) of 4.5 in both H2O and D2O. The solvent isotope effect on the initial rate of the chloroperoxidase-catalyzed demethylation reaction was independent of pL, suggesting that the solvent isotope effect is not due to a change in the pK of a rate-controlling ionization in D2O. The solvent isotope effect on the Vmax for the chloroperoxidase-catalyzed demethylation reaction was 3.66 +/- 0.62. In contrast, the solvent isotope effect on the Vmax for the horseradish peroxidase catalyzed demethylation reaction was approximately 1.5 with either ethyl hydroperoxide or hydrogen peroxide as the oxidant, indicating that the exchange of hydrogens in the enzyme and hydroperoxide for deuterium in D2O has little effect on the rate of the demethylation reaction. The solvent isotope effect on the Vmax/KM for ethyl hydroperoxide in the chloroperoxidase-catalyzed demethylation reaction was 8.82 +/- 1.57, indicating that the rate of chloroperoxidase compound I formation is substantially decreased in D2O. This isotope effect is suggested to arise from deuterium exchange of the hydroperoxide hydrogen and of active-site residues involved in compound I formation. A solvent isotope effect of 2.96 +/- 0.57 was observed on the Vmax/KM for N,N-dimethylaniline in the chloroperoxidase-catalyzed reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloride Peroxidase↗
PubMed1999

Alpha-secondary tritium kinetic isotope effects indicate hydrogen tunneling and coupled motion occur in the oxidation of L-malate by NAD-malic enzyme.

The NAD-malic enzyme from Ascaris suum catalyzes the divalent metal ion-dependent oxidative decarboxylation of L-malate to give pyruvate and CO2, with NAD+ as the oxidant. Alpha-secondary tritium kinetic isotope effects were measured with NAD+ or APAD+ and L-malate-2-H(D) and several different divalent metal ions. The alpha-secondary tritium kinetic isotope effects are slightly higher than 1 with NAD+ and L-malate as substrates, much larger than the expected inverse isotope effect for a hybridization change from sp2 to sp3. The alpha-secondary tritium kinetic isotope effects are reduced to values near 1 with L-malate-2-D as the substrate, regardless of the metal ion that is used. Data suggest the presence of quantum mechanical tunneling and coupled motion in the malic enzyme reaction when NAD+ and malate are used as substrates. Isotope effects were also measured using the D/T method with NAD+ and Mn2+ as the substrate pair. A Swain-Schaad exponent of 2.2 (less than the value of 3.26 expected for strictly semiclassical behavior) is estimated, suggesting the presence of other slow steps along the reaction pathway. With APAD+ and Mn2+ as the substrate pair, inverse alpha-secondary tritium kinetic isotope effects are observed, and a Swain-Schaad exponent of 3.3 is estimated, consistent with rate-limiting hydride transfer and no quantum mechanical tunneling or coupled motion. Data are discussed in terms of the malic enzyme mechanism and the theory developed by Huskey for D/T isotope effects as an indicator of tunneling [Huskey, W. P. (1991) J. Phys. Org. Chem. 4, 361-366].

Animals↗
PubMed2000

Effect of pressure on deuterium isotope effects of yeast alcohol dehydrogenase: evidence for mechanical models of catalysis.

Moderate pressure accelerates hydride transfer catalyzed by yeast alcohol dehydrogenase, indicative of a large negative volume of activation [Cho and Northrop (1999) Biochemistry 38, 7470-7475]. A comparison of the effects of pressure on the oxidation of normal versus dideuteriobenzyl alcohol generates a monophasic decrease in the intrinsic isotope effect; therefore, the volume of activation for the transition-state of deuteride transfer must be even more negative, by 10.4 mL/mol. This finding appears consistent with hydrogen tunneling previously proposed for this dehydrogenase [Cha, Y., Murray, C. J., and Klinman, J. P. (1989) Science 243, 1325-1330]. However, a global fit of the primary data shows that the entire isotope effect arises from a transition-state phenomenon, unlike normal isotope effects, which arise from different vibrational frequencies in reactant states, and tunneling isotope effects, which arise from a mixture of both states. Assuming the phenomenon is tunneling, the isotopic data are consistent with a Bell tunneling correction factor of Q(H) = 12 and an imaginary frequency of nu(H) = 1220 cm(-1), the first so calculated from experimental enzymatic data. This excessively large correction factor and the large difference in the isotopic activation volumes, plus the low isotope effects at extrapolated pressures, challenge traditional applications of physical organic chemistry and transition-state theory to enzymatic catalysis. They suggest instead that something other than transition-state stabilization or tunneling is responsible for the rate acceleration, something unique to the enzymatic transition state that does not occur in nonenzymatic reactions. Arguments for the vibrational model of coupled atomic motions and the fluctuating enzyme model of protein domain motion are put forward as possible interpretations.

Alcohol Dehydrogenase↗
PubMed2002

Theoretical investigation of large kinetic isotope effects for proton-coupled electron transfer in ruthenium polypyridyl complexes.

A theoretical investigation of proton-coupled electron transfer in ruthenium polypyridyl complexes is presented. The three reactions studied are as follows: (1) the comproportionation reaction of [(bpy)(2)(py)Ru(IV)O](2+) and [(bpy)(2)(py)Ru(II)OH(2)](2+) to produce [(bpy)(2)(py)Ru(III)OH](2+); (2) the comproportionation reaction of [(tpy)(bpy)Ru(IV)O](2+) and [(tpy)(bpy)Ru(II)OH(2)](2+) to produce [(tpy)(bpy)Ru(III)OH](2+); and (3) the cross reaction of [(tpy)(bpy)Ru(III)OH](2+) and [(bpy)(2)(py)Ru(II)OH(2)](2+) to produce [(tpy)(bpy)Ru(II)OH(2)](2+) and [(bpy)(2)(py)Ru(III)OH](2+). This investigation is motivated by experimental measurements of rates and kinetic isotope effects for these systems (Binstead, R. A.; Meyer, T. J. J. Am. Chem. Soc. 1987, 109, 3287. Farrer, B. T.; Thorp, H. H. Inorg. Chem. 1999, 38, 2497.). These experiments indicate that the second reaction is nearly one order of magnitude faster than the first reaction, and the third reaction is in the intermediate regime. The experimentally measured kinetic isotope effects for these three reactions are 16.1, 11.4, and 5.8, respectively. The theoretical calculations elucidate the physical basis for the experimentally observed trends in rates and kinetic isotope effects, as well as for the unusually high magnitude of the kinetic isotope effects. In this empirical model, the proton donor-acceptor distance is predicted to be largest for the first reaction and smallest for the third reaction. This prediction is consistent with the degree of steric crowding near the oxygen proton acceptor for the three reactions. The second reaction is faster than the first reaction since a smaller proton donor-acceptor distance leads to a larger overlap between the reactant and product proton vibrational wave functions. The intermediate rate of the third reaction is determined by a balance among several competing factors. The observed trend in the kinetic isotope effects arises from the higher ratio of the hydrogen to deuterium vibrational wave function overlap for larger proton donor-acceptor distances. Thus, the kinetic isotope effect increases for larger proton donor-acceptor distances. The unusually high magnitude of the kinetic isotope effects is due in part to the close proximity of the proton transfer interface to the electron donor and acceptor. This proximity results in strong electrostatic interactions that lead to a relatively small overlap between the reactant and product vibrational wave functions.

Deuterium↗
PubMed2003

Binding equilibrium isotope effects for glucose at the catalytic domain of human brain hexokinase.

We have utilized tritium isotope effects to probe the in vitro binding equilibrium between glucose and human brain hexokinase (E.C.2.7.1.1). Replacing a backbone hydrogen atom in glucose with tritium can significantly increase or decrease the equilibrium association constant. Specifically, the equilibrium tritium isotope effects are 1.027 +/- 0.002, 0.927 +/- 0.0003, 1.027 +/- 0.004, 1.051 +/- 0.001, 0.988 +/- 0.001, and 1.065 +/- 0.003 for [1-t]-, [2-t]-, [3-t]-, [4-t]-, [5-t]-, and [6,6-t(2)]glucose, respectively. We have shown that the existence of prebinding equilibrium isotope effects can contribute to binding isotope effect studies but that this effect is insignificant for glucose binding to hexokinase. The binding isotope effects are interpreted in the context of structural studies of hexokinase-glucose complexes. Ab initio calculations on 2-propanol with or without a hydrogen bonding partner, in steric collision with formaldehyde or methane, and on ethanol, cyclohexanol and 1-hydroxymethyl-tetrahydropyran are presented to clarify the magnitude of isotope effects possible in such interactions and the accompanying changes in free energy. Position-specific binding isotope effects provide direct evidence of the partial deprotonation and activation of O6 by Asp657, of other hydrogen bonding interactions with ionic residues, and of the steric compression of CH2 by the backbone carbonyl of Ser603.

2-Propanol↗
PubMed2006

Effect of pressure on a heavy-atom isotope effect of yeast alcohol dehydrogenase.

Hydrostatic pressure causes a monophasic decrease in the (13)C primary isotope effect expressed on the oxidation of benzyl alcohol by yeast alcohol dehydrogenase. The primary isotope effect was measured by the competitive method, using whole-molecule mass spectrometry. The effect is, therefore, an expression of isotopic discrimination on the kinetic parameter V/K, which measures substrate capture. Moderate pressure increases capture by activating hydride transfer, the transition state of which must therefore have a smaller volume than the free alcohol plus the capturing form of enzyme [Cho, Y.-K.; Northrop, D. B. Biochemistry 1999, 38, 7470-7475]. The decrease in the (13)C isotope effect with increasing pressure means that the transition state for hydride transfer from the heavy atom must have an even smaller volume, measured here to be 13 mL.mol(-1). The pressure data factor the kinetic isotope effect into a semiclassical reactant-state component, with a null value of k(12)/k(13) = 1, and a transition-state component of Q(12)/Q(13) = 1.028 (borrowing Bell's nomenclature for hydrogen tunneling corrections). A similar experiment involving a deuterium isotope effect previously returned the same volume and null value, plus a pressure-sensitive isotope effect [Northrop, D. B.; Cho, Y.-K. Biochemistry 2000, 39, 2406-2412]. Consistent with precedence in the chemical literature, the latter suggested a possibility of hydrogen tunneling; however, it is unlikely that carbon can engage in significant tunneling at ambient temperature. The fact that the decrease in activation volumes for hydride transfer is equivalent when one mass unit is added to the carbon end of a scissile C-H bond and when one mass unit is added to the hydrogen end is significant and suggests a common origin.

Alcohol Dehydrogenase↗
PubMed1990

Intrinsic isotope effects suggest that the reaction coordinate symmetry for the cytochrome P-450 catalyzed hydroxylation of octane is isozyme independent.

The mechanism of the omega-hydroxylation of octane by three catalytically distinct, purified forms of cytochrome P-450, namely, P-450b, P-450c, and P-450LM2, was investigated by using deuterium isotope effects. The deuterium isotope effects associated with the omega-hydroxylation of octane-1,1,1-2H3, octane-1,8-2H2, and octane-1,1,8,8-2H4 by all three isozymes were determined. From these data the intrinsic isotope effects were calculated and separated into their primary and secondary components. The primary intrinsic isotope effect for the reaction ranged from 7.69 to 9.18 while the secondary intrinsic isotope effect ranged from 1.13 to 1.25. Neither the primary nor secondary isotope effect values were statistically different for any of the isozymes investigated. These data are consistent with a symmetrical transition state for a mechanism involving initial hydrogen atom abstraction followed by hydroxyl radical recombination which is essentially independent of the specific isozyme catalyzing the reaction. It is concluded that (1) in general the porphyrin-[FeO]3+ complex behaves as a source of a triplet-like oxygen atom, (2) the regioselectivity for the site of oxidation is dictated by the apoprotein of the specific isozyme of cytochrome P-450 catalyzing the reaction, and (3) the maximum primary intrinsic isotope effect for any cytochrome P-450 catalyzed oxidation of a carbon center is about 9, assuming no tunneling effects.

Cytochrome P-450 Enzyme System↗