Calculations of intrinsic isotope effects and the detection of tunneling in enzyme-catalyzed reactions

The equation of Northrop [1975, Biochemistry, 14, 2644] for calculating intrinsic isotope effects from observed deuterium and tritium isotope effects of V/K, in which hydrogen is the reference isotope, has been extended to experimental designs using either deuterium or tritium as a reference. Partial derivatives of the intrinsic equations allow calculation of the relative precision of the three referenced isotope effects and these favor the order deuterium > tritium > hydrogen. In comparisons of observed and calculated isotope effects when hydrogen tunneling is present, both the precision and the magnitude of the difference was greater for intrinsic calculations than for exponentiations based upon a breakdown in the Swain-Schaad relationship.     

稳定氢氧同位素定量植物水分来源的不确定性解析

稳定氢氧同位素技术被广泛运用于生态系统、特别是干旱区生态系统中植物水分来源的研究,其理论假设为"水分被植物根系吸收并向木质部运输过程中不发生氢氧同位素分馏"。生态系统中不同水源的氢氧同位素组成普遍存在显著差异,为从水源混合体中区分出各水源的贡献率提供了前提条件。但在实际应用过程中,诸多因素导致稳定氢氧同位素技术定量植物水分来源的结果具有不确定性。综合已有研究并加以分析,举证说明植物吸收水分相对于水源同位素变化的滞后性、水源同位素的季节性变化、蒸发作用和水源之间的混合作用对水源同位素的影响等导致植物水分来源定量结果不确定性的几个因素,以期为今后稳定氢氧同位素技术在植物水分来源领域的应用提供参考。     

Incorporation of tritium into the DNA of hematopoietic tissues during prolonged administration of tritium oxide

With long-term (90 days) administration of tritium oxide (0.37 MBq/g body weight) to ras the carbon-bound tritium accumulated in DNA of haemopoietic tissues during two-month administration of the isotope (the accumulation half-time of 15-25 days); during the next month, the isotope level remained nearly constant (about 20 X 10(6) decay/min/g DNA). Elimination of tritium from DNA started 3 days after termination of its administration and proceeded with two half-times (4-8 days and 12-18 days). The ratio of the tritium content per 1 M hydrogen of DNA to tritium content per 1 M hydrogen of tissue water increased up to 0.5-0.7 during the uptake of tritium oxide, and up to 4-7 after the administration of the isotope had ceased.     

Tritium partitioning and isotope effects in adenosylcobalamin-dependent glutamate mutase.

Tritiated adenosylcobalamin, labeled at the exchangeable position, has been used to investigate the partitioning of tritium between substrate and product in the reaction catalyzed by glutamate mutase. The isotope partitions between glutamate and methylaspartate in nearly 1:1 ratio, regardless of the direction in which the overall reaction is proceeding. This is consistent with a free-energy profile in which the interconversion of the intermediate glutamyl and methylaspartyl radicals is rapid relative to the transfer of tritium from 5'-deoxyadenosine to either substrate or product. Initial velocity measurements have been used to measure the tritium isotope effects for the transfer of tritium from adenosylcobalamin to product in each direction. The isotope effect is 21 for the formation of glutamate and 19 for the formation of methylasparate. The large magnitude of these isotope effects makes it likely that the rate-determining step may be altered by the substitution of tritium for hydrogen in the reaction. The results of these experiments are compared with previous isotope effect measurements made on other adenosylcobalamin-dependent enzymes.     

Kinetic mechanism and nucleotide specificity of NADH peroxidase

NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between [14C]NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols.     

Stereochemical probes of bovine plasma amine oxidase: evidence for mirror image processing and a syn abstraction of hydrogens from C-1 and C-2 of dopamine

Bovine plasma amine oxidase (PAO) has previously been shown to catalyze a nonstereospecific loss of tritium from [2(R)-3H]- and [2(S)-3H]dopamines, attributed to multiple, catalytically active binding sites for substrate [Summers, M. C., Markovic, R., & Klinman, J. P. (1979) Biochemistry 18, 1969-1979]. Analysis of products formed from incubation of dopamine with PAO in tritiated water indicates a stereospecific, pro-R, incorporation of label at C-2. Thus, tritium washout (random) and washin (pro-R) are not the microscopic reverse of one another. We conclude that the (enamine) intermediates leading to tritium washin are nonequivalently bound. The observation of pro-R incorporation has provided a straightforward synthetic route to [1(R)-2H,2(R)-3H]- and [1(S)-2H,2(R)-3H]dopamines, which upon oxidation with PAO are expected to be processed preferentially by 1S and 1R cleavage, respectively. From previously measured isotope effects, we predict the loss of tritium from the 1(R)-2H and 1(S)-2H samples to be 74:8 for a syn relationship between cleavage at C-1 and C-2 vs. 21:90 for an anti relationship. The observation of a 68:18 ratio at 100% conversion provides strong evidence for a syn cleavage. The data support a mechanism in which a single base catalyzes a 1,3-prototrophic shift of hydrogen from C-1 of the substrate to cofactor, followed by exchange from C-2. Additionally, the results confirm the presence of alternate binding modes for dopamine at the active site of bovine plasma amine oxidase. This interaction of dopamine with plasma amine oxidase is a rare example of mirror-image catalysis in which a single substrate has two functional binding orientations on an enzyme surface.     

Tritium secondary kinetic isotope effect on phenylalanine ammonia-lyase-catalyzed reaction.

The mechanism by which phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) catalyzes the reversible elimination of ammonia from phenylalanine yielding (E)-cinnamic acid has gained much attention in the recent years. Dehydroalanine is essential for the catalysis. It was assumed that this prostetic group acts as the electrophile, leading to a covalently bonded enzyme-intermediate complex with quarternary nitrogen of phenylalanine. Recently, an alternative mechanism has been suggested in which the enzyme-intermediate complex is formed in a Friedel-Crafts reaction between dehydroalanine and orthocarbon of the aromatic ring. Using semiempirical calculations we have shown that these two alternative mechanisms can be distinguished on the basis of the hydrogen secondary kinetic isotope effect when tritium label is placed in the orthopositions. Our calculations indicated also that the kinetic isotope effect measured using ring-labeled d(5)-phenylalanine could not be used to differentiate these alternative mechanisms. Measured secondary tritium kinetic isotope effect shows strong dependence on the reaction progress, starting at the inverse value of k(H)/k(T) = 0.85 for 5% conversion and reaching the normal value of about 1.15 as the conversion increases to 20%. This dependence has been interpreted in terms of a complex mechanism with initial formation of the Friedel-Crafts type intermediate.     

氚水在模拟水稻－水－土壤生态系统中的行为

采用模拟污染物的同位素示踪技术研究HTO(流水)在水稻-水-土壤模拟生态系统中的迁移、消长行为,并应用三库室开系统模型和非线性回归方法确定了水稻、水和土壤分室的拟合方程．结果表明,田表水中的HTO不仅在系统各分室间转移和分配,而且迅速向系统外散逸;HTO中的流以自由水氚和结合态流形式存在于水稻中,以吸湿性水流和结晶水氚存在于土壤,其中自由水氚(或吸湿性水氚)的比活度大于结合态流(或结晶水氚);水稻植株和土壤中HTO比活度随时间增加至最大值后又趋于下降,而结合态氚则呈缓慢增加;水稻茎秆中的总氚比活度高于其它各部位,而后逐渐趋于动态平衡．对实验数据进行回归分析得：田表水、土壤和水稻植株中的总氚比活度分别为Cw(t)=32.19c^-0.0353t 99.94c^-0.330t、Cs(t)=20.42(e^-0.0353t-e^-0.330t)和Cr(t)=38.49c^-0.0353t-10.13e^-0.330t-28.36c^-2.5744t。方差分析结果表明,各回归方程较好地反映了HTO在水稻-水-土壤生态系统中的行为．     

Bile acid production in human subjects: rate of oxidation of [24,25-3H]cholesterol compared to fecal bile acid excretion

Bile acid production has been quantitated in seven subjects by methods that compare the results of two independent approaches, namely, quantitation of cholesterol side-chain oxidation and fecal bile acid excretion. Six hypertriglyceridemic (HT) subjects and one normolipidemic control were studied by both techniques. A further control subject was studied by the cholesterol side-chain oxidation method alone. Cholesterol side-chain oxidation was quantitated by measuring the appearance of 3H2O after intravenous administration of [24,25-3H]cholesterol, using multicompartmental analysis of plasma cholesterol and [3H]water specific activity. Body water kinetics were independently defined by use of oral D2O. Two HT subjects were restudied while they were taking cholestyramine, 16 g/day. In all ten studies, multicompartmental analysis closely simulated the observed appearance of 3H2O. Values obtained for bile acid production suggest that cholesterol oxidation, or bile acid input, was significantly greater than fecal bile acid output in the HT subjects (P less than 0.05). Cholesterol side-chain oxidation rates in the two normal subjects were lower than those encountered in HT subjects, being similar to published values for normal subjects both for bile acid synthesis as determined by isotope dilution kinetics and fecal bile acid excretion. Studies conducted with two, synthetically different, preparations of [24,25-3H]cholesterol indicated that, in one of the two preparations, approximately 20% of the tritium label was at positions proximal to C24. In the other preparation examined, all of the tritium was located at, or distal to, C24. Further studies revealed that 0.055-0.24% of the dose was present as labile tritium by virtue of its appearance as 3H2O following in vitro incubation with human plasma. Provided these isotope effects are taken into account, multicompartmental analysis of plasma [24,25-3H]cholesterol and body water appears to be a useful technique for quantitating cholesterol oxidation in human subjects.     

Stereochemistry and kinetic isotope effects in the bovine plasma amine oxidase catalyzed oxidation of dopamine.

The stereochemistry of the bovine plasma amine oxidase catalyzed oxidation of 2-(3,4-dihydroxyphenyl)-ethylamine (domapine) has been investigated by comparing 3H/14C ratios of 3,4-dibenzyloxyphenethyl alcohols, derived from 3,4-dihydroxyphenylacetaldehydes, to starting dopamines chirally labeled at C-1 and C-2. The oxidation of [2RS-3H]-, [2R-3H]-, and [2S-3H]dopamine leads to products which have retained 53, 59, and 47% of their tritium. Similarly, oxidation of [1RS-3H]-, [1R-3H]-, and [1S-3H]dopamine leads to an 80, 80, and 92% retention of tritium. The configurational purity of tritium at C-2 of dopamine and C-1 of the dopamine precursor 3-methoxy-4-hydroxyphenethylamine has been confirmed employing dopamine-beta-hydroxylase (specific for the pro-R hydrogen at C-2) and pea seedling amine oxidase (specific for the pro-S hydrogen at C-1). In addition, chromatographically resolved isozymes of bovine plasma amine oxidase have been demonstrated to lead to the same stereochemical result as pooled enzyme fractions. We have been able to rule out carbon interchange and tritium transfer in the ethylamine side chain of dopamine as the source of the apparent nonstereospecificity. Estimated primary tritium isotope effects are 1 for [2-3H]dopamines and 5--6 and 26--34 for [1R-3H]- and [1S-3H]dopamine, respectively. We propose the presence of alternate dopamine binding modes, characterized by absolute but opposing stereochemistries and differential primary tritium isotope effects at C-1.     

Intermolecular hydrogen transfer catalyzed by a flavodehydrogenase, bakers' yeast flavocytochrome b2

Bakers' yeast flavocytochrome b2 is a flavin-dependent L-2-hydroxy acid dehydrogenase which also exhibits transhydrogenase activity. When a reaction takes place between [2-3H]lactate and a halogenopyruvate, tritium is found in water and at the halogenolactate C2 position. When the halogenopyruvate undergoes halide ion elimination, tritium is also found at the C3 position of the resulting pyruvate. The amount tau of this intermolecular tritium transfer depends on the initial keto acid-acceptor concentration. At infinite acceptor concentration, extrapolation yields a maximal transfer of 97 +/- 11%. This indicates that the hydroxy acid-derived hydrogen resides transiently on enzyme monoprotic heteroatoms and that exchange with bulk solvent occurs only at the level of free reduced enzyme. Using a minimal kinetic scheme, the rate constant for hydrogen exchange between Ered and solvent is calculated to be on the order of 10(2) M-1 S-1, which leads to an estimated pK approximately equal to 15 for the ionization of the substrate-derived proton while on the enzyme. It is suggested that this hydrogen could be shared between the active site base and Flred N5 anion. It is furthermore shown that some tritium is incorporated into the products when the transhydrogenation is carried out in tritiated water. Finally, with [2-2H]lactate-reduced enzyme, a deuterium isotope effect is observed on the rate of bromopyruvate disappearance. Extrapolation to infinite bromopyruvate concentration yields DV = 4.4. An apparent inverse isotope effect is determined for bromide ion elimination. These results strengthen the idea that oxidoreduction and elimination pathways involve a common carbanionic intermediate.     

Metabolic processes account for the majority of the intracellular water in log-phase Escherichia coli cells as revealed by hydrogen isotopes

It is generally believed that water transport across biological membranes is essentially a near-instantaneous process, with water molecules diffusing directly across the membrane as well as through pores such as aquaporins. As a result of these processes by which water can equilibrate across a membrane, a common assumption is that intracellular water is isotopically indistinguishable from extracellular water. To test this assumption directly, we measured the hydrogen isotope ratio of intracellular water in Escherichia coli cells. Our results demonstrate that more than 50% of the intracellular water hydrogen atoms in log-phase E. coli cells are isotopically distinct from the growth medium water and that these isotopically distinct hydrogen atoms are derived from metabolic processes. As expected, the (2)H/(1)H isotope ratio of intracellular water from log-phase cells showed an appreciably larger contribution from metabolic water than did intracellular water from stationary-phase cells (53 +/- 12 and 23 +/- 5%, respectively). The (2)H/(1)H isotope ratio of intracellular water was also monitored indirectly by measuring the isotope ratio of fatty acids, metabolites that are known to incorporate hydrogen atoms from water during biosynthesis. Significantly, the difference in the isotopic composition of intracellular water from log- to stationary-phase E. coli cells was reflected in the hydrogen isotope ratio of individual fatty acids harvested at the two different times, indicating that the isotope ratio of metabolites can be used as an indirect probe of metabolic activity. Together, these results demonstrate that contrary to the common assumption that intracellular water is isotopically identical to extracellular water, these two pools of water can actually be quite distinct.     

A metabolic derivation of tritium transfer coefficients in animal products

Tritium is a potentially important environmental contaminant originating from the nuclear industry, and its behaviour in the environment is controlled by that of hydrogen. Animal food products represent a potentially important source of tritium in the human diet and a number of transfer coefficient values for tritium transfer to a limited number of animal products are available. In this paper we present an approach for the derivation of tritium transfer coefficients which is based on the metabolism of hydrogen in animals. The derived transfer coefficients separately account for transfer to and from free (i.e. water) and organically bound tritium. A novel aspect of the approach is that tritium transfer can be predicted for any animal product for which the required metabolic input parameters are available. The predicted transfer coefficients are compared to available independent data. Agreement is good (R ²=0.97) with the exception of the transfer coefficient for transfer from tritiated water to organically bound tritium in ruminants. This may be attributable to the particular characteristics of ruminant digestion. We show that tritium transfer coefficients will vary in response to the metabolic status of an animal (e.g. stage of lactation, diet digestibility etc.) and that the use of a single transfer coefficient from diet to animal product is inappropriate. It is possible to derive concentration ratio values from the estimated transfer coefficients which relate the concentration of tritiated water and organically bound tritium in an animal product to their respective concentrations in the animals diet. These concentration ratios are shown to be less subject to metabolic variation and may be more useful radioecological parameters than transfer coefficients. For tritiated water the concentration ratio shows little variation between animal products ranging from 0.59 to 0.82. In the case of organically bound tritium the concentration ratios vary between animal products from 0.15 (goat milk) to 0.67 (eggs). Received: 28 May 2001 / Accepted: 20 August 2001     

Climatic significance of isotope ratios

The hydrogen and oxygen isotope ratios of water, which can be measured by Isotope Ratio Mass Spectrometry (IRMS), exhibit climatic dependencies and are commonly exploited in hydrogeology. More generally, the overall carbon or hydrogen isotope ratios of plant organic matter, and in particular of tree-ring cellulose, have been frequently used for climatic reconstruction. However, since many physicochemical and biochemical fractionation phenomena are likely to contribute to the isotopic values, the interpretation of the climatic significance of isotopic parameters is not always straightforward. In the case of hydrogen and oxygen for instance, the climatic profile of the source meteoric water is not simply transferred to leaf water and many steps of the biosyntheses are accompanied by kinetic and thermodynamic isotope effects that depend on the individual mechanistic pathways. The information brought about by overall isotope ratios determined by IRMS is averaged over all fractionation effects undergone at the different molecular positions. In contrast, the NMR investigation of Site-specific Natural Isotope Fractionation (SNIF-NMR) gives simultaneous access to isotope ratios specific to individual positions in the molecule. Since the different atoms do not necessarily exhibit the same climatic dependency, the method provides complementary responses to the environmental conditions. In particular, the isotopic parameters of ethanol and water obtained by fermenting sugars in standardized conditions reflect climatic influences which took place at different periods of plant growth. As a consequence, statistical analyses based on multi-site isotopic variables provide powerful criteria for distinguishing geographical regions of cultivation characterized by different climatic features. Although the sensitivity to climatic variations is the most pronounced for plant water and for sugars formed at the first step of photosynthesis, other components such as lipids or minor metabolites also exhibit climatic dependencies. The combination of isotopic values pertaining to different atomic species and either averaged over the whole molecule (IRMS) or associated with different molecular sites (SNIF-NMR), provides complementary criteria, which can be exploited in terms of both climatic significance and mechanistic pathways of the individual atoms.     

Evidence for coupled motion and hydrogen tunneling of the reaction catalyzed by glutamate mutase

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze unusual isomerizations that proceed through organic radical intermediates generated by homolytic fission of the coenzyme's unique cobalt-carbon bond. These enzymes are part of a larger family of enzymes that catalyze radical chemistry in which a key step is the abstraction of a hydrogen atom from an otherwise inert substrate. To gain insight into the mechanism of hydrogen transfer, we previously used pre-steady-state, rapid-quench techniques to measure the alpha-secondary tritium kinetic and equilibrium isotope effects associated with the formation of 5'-deoxyadenosine when glutamate mutase was reacted with [5'-(3)H]adenosylcobalamin and L-glutamate. We showed that both the kinetic and equilibrium isotope effects are large and inverse, 0.76 and 0.72, respectively. We have now repeated these measurements using glutamate deuterated in the position of hydrogen abstraction. The effect of introducing a primary deuterium kinetic isotope effect on the hydrogen transfer step is to reduce the magnitude of the secondary kinetic isotope effect to a value close to unity, 1.05 +/- 0.08, whereas the equilibrium isotope effect is unchanged. The significant reduction in the secondary kinetic isotope effect is consistent with motions of the 5'-hydrogen atoms being coupled in the transition state to the motion of the hydrogen undergoing transfer, in a reaction that involves a large degree of quantum tunneling.     

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].     

Tritiated water as a measure of body water in immature rats growing at different rates

Rats were reared from birth in litters of 4, 10, and 16 to achieve different growth rates. Pups in the litters of 16 had no access to rat chow until days 21-28, when chow was made available to one of the litters to induce catch-up growth. Total body water was estimated by tritiated water (TBWHTO) on days 7, 14, 21, and 28 and then calculated from desiccation (TBWdes). TBWHTO was consistently larger than TBWdes for all groups. Differences were 10.9-16.9% on day 7 and 3.7-6.4% on day 28. On day 28, percent difference was higher in the slower-growing than the faster-growing groups. Nonaqueous hydrogen exchange was determined from tritium activity in the dried carcass. Less than 1% of the injected tritium exchanged with nonaqueous hydrogen during the equilibration period. Thus differences between TBWHTO and TBWdes in the younger animals could not be accounted for by nonaqueous hydrogen exchange but may have resulted from a larger loss of injected tritium, possibly in insensible water.     

Three applications of path integrals: equilibrium and kinetic isotope effects, and the temperature dependence of the rate constant of the [1,5] sigmatropic hydrogen shift in (Z)-1,3-pentadiene

Recent experiments have confirmed the importance of nuclear quantum effects even in large biomolecules at physiological temperature. Here we describe how the path integral formalism can be used to describe rigorously the nuclear quantum effects on equilibrium and kinetic properties of molecules. Specifically, we explain how path integrals can be employed to evaluate the equilibrium (EIE) and kinetic (KIE) isotope effects, and the temperature dependence of the rate constant. The methodology is applied to the [1,5] sigmatropic hydrogen shift in pentadiene. Both the KIE and the temperature dependence of the rate constant confirm the importance of tunneling and other nuclear quantum effects as well as of the anharmonicity of the potential energy surface. Moreover, previous results on the KIE were improved by using a combination of a high level electronic structure calculation within the harmonic approximation with a path integral anharmonicity correction using a lower level method.     

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

Summary New 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.Presented at the 3rd International Congress on Amino Acids, Peptides and Analogues. Vienna, August, 23–27, 1993