Position-specific isotope analysis, also called site-specific isotope analysis, is a branch of isotope analysis aimed at determining the isotopic composition of a particular atom position in a molecule. Isotopes are elemental variants with different numbers of neutrons in their nuclei, thereby having different atomic masses. Isotopes are found in varying natural abundances depending on the element; their abundances in specific compounds can vary from random distributions (i.e., stochastic distribution) due to environmental conditions that act on the mass variations differently. These differences in abundances are called "fractionations," which are characterized via stable isotope analysis.
Isotope abundances can vary across an entire substrate (i.e., “bulk” isotope variation), specific compounds within a substrate (i.e., compound-specific isotope variation), or across positions within specific molecules (i.e., position specific isotope variation). Isotope abundances can be measured in a variety of ways (e.g., isotope ratio mass spectrometry, laser spectrometry, NMR, ESI-MS). Early analyses varied in technique, but were commonly limited by their ability to only measure average isotope compositions over molecules or samples. While this allows isotope analysis of the bulk substrate, it eliminates the ability to distinguish variation between different sites of the same element within the molecule. The field of position-specific isotope biogeochemistry studies these intramolecular variations, known as “position-specific isotope” and “site-specific isotope” enrichments. It focuses on position-specific isotope fractionations in many contexts, development of technologies to measure these fractionations and the application of position-specific isotope enrichments to questions surrounding biogeochemistry, microbiology, enzymology, medicinal chemistry, and Earth history.
Position-specific isotope enrichments can retain critical information about synthesis and source of the atoms in the molecule. Indeed, bulk isotope analysis averages site-specific isotope effects across the molecule, and so while all those values have an influence on the bulk value, signatures of specific processes may be diluted or indistinguishable. While the theory of position-specific isotope analysis has existed for decades, new technologies exist now to allow these methods to be much more common. The potential applications of this approach are widespread, such as understanding metabolism in biomolecules, environmental pollutants in air, inorganic reaction mechanisms, etc. Clumped isotope analysis, a subset of position-specific isotope analysis, has already proven useful in characterizing sources of methane, paleoenvironment, paleoaltimetry, among many other applications. More specific case studies of position-specific isotope fractionation are detailed below.
Theory Stable isotopes do not decay, and the heavy and light isotope masses affect how they partition within the environment. Any deviation from a random distribution of the light and heavy isotopes within the environment is called fractionation, and consistent fractionations as a result of a particular process or reaction are called "isotope effects."
Isotope Effects Isotope effects are recurring patterns in the partitioning of heavy and light isotopes across different chemical species or compounds, or between atomic sites within a molecule. These isotope effects can come about from a near infinite number of processes, but most of them can be narrowed down into two main categories, based on the nature of the chemical reaction creating or destroying the compound of interest:
(1) Kinetic isotope effects manifest in irreversible reactions, when one isotopologue is preferred in the transition state due to the lowest energy state. The preferred isotopologue will depend on whether the transition state of the molecule during a chemical reaction is more like the reactant or the product. Normal isotope effects are defined as those which partition the lighter isotope into the products of the reaction. Inverse isotope effects are less common as they preferentially partition the heavier isotope into the products.
(2) Equilibrium isotope effects manifest in reversible reactions, when molecules can exchange freely to reach the lowest possible energy state.
These variations can occur on a compound-specific level, but also on a position-specific level within a molecule. For instance, the carboxyl site of amino acids is exchangeable and therefore its carbon isotope signature can change over time and may not represent the original carbon source of the molecule.
Biological fractionation Chemical reactions in biological processes are controlled by enzymes that catalyze the conversion of substrate to product. Since enzymes can alter the transition state structure for reactions, they also change kinetic and equilibrium isotope effects. Placed in the context of a metabolism, the expression of isotope effects on biomolecules is further controlled by branch points. Different pathways of biosynthesis will use different enzymes, yielding a range of position specific isotope enrichments. This variability allows position-specific isotope measurements to discern multiple biosynthetic pathways from the same metabolic product. Biogeochemists use position specific isotope enrichments from amino acids, lipids, and sugars in nature to interpret the relative importance of different metabolisms.
Mechanism The position-specific isotope effect of an enzymatic reaction is expressed as the ratio of rate constants for a monoisotopic substrate and a substrate substituted with one rare isotope. For example, enzyme formate dehydrogenase catalyzes the reaction of formate and NAD+ to carbon dioxide and NADH. The hydrogen of formate is directly transferred to NAD+. This step has an isotope effect, because the rate of protium transfer from formate to NAD+ is nearly three times faster than the rate of the same reaction with a deuterium transfer. This is also an example of a primary isotope effect. A primary isotope effect is one in which the rare isotope is substituted where a bond is broken or formed. Secondary isotope effects occur on other positions in the molecule and are controlled by the molecular geometry of the transition state. These are generally considered to be negligible but do arise in certain cases, especially for hydrogen isotopes.
Unlike abiotic reactions, enzymatic reactions occur through a series of steps, including substrate-enzyme binding, conversion of substrate to product, and dissociation of enzyme-product complex. The observed isotope effect of an enzyme will be controlled by the rate limiting step in this mechanism. If the step that converts substrate to product is rate limiting, the enzyme will express its intrinsic isotope effect, that of the bond forming or breaking reaction.
Abiological fractionation Like biotic molecules, position specific isotope enrichments in abiotic molecules can reflect the source of chemical precursors and synthesis pathways. The energy for abiotic reactions can come from many different sources, which will affect fractionation. For instance, metal catalysts can speed up abiotic reactions. Reactions can be slowed down or sped up by different temperature and pressure conditions, which will affect the equilibrium constant or activation energy of reversible and irreversible reactions, respectively.
For example, carbon in the interstellar medium and solar nebula partition into distinct states based on thermodynamic favorability. Measuring site-specific isotope enrichments of carbon from organic molecules extracted from carbonaceous chondrites can elucidate where each carbon atom comes from, and how organic molecules can be synthesized abiotically. More broadly, these isotope enrichments can provide information about physical processes in the region where the molecular precursors were formed, and where the molecule formed in the solar system (i.e., nucleosynthetic heterogeneity, mass independent fractionation, self-shielding, etc.).
Another example of distinct site-specific fractionations in abiotic molecules is Fischer-Tropsch-type synthesis, which is thought to produce abiogenic hydrocarbon chains. Through this reaction mechanism, site enrichments of carbon would deplete as carbon chain length increases, and be distinct from site-specific enrichments of hydrocarbons of biological origins.
Analysis Substrates need to be prepared and analyzed in a specific way to elucidate site specific isotope enrichments. This requires clean separation of the compound of interest from the original sample, which can require a variety of different preparatory chemistries. Once isolated, position-specific isotope enrichments can be analyzed with a variety of instruments, which all have different advantages and provide varying degrees of precision.
Enzymatic Reaction To measure the kinetic isotope effects of enzymatic reactions, biochemists perform in vitro experiments with enzymes and substrates. The goal of these experiments is to measure the difference in the enzymatic reaction rates for the monoisotopic substrate and the substrate with one rare isotope. There are two popularly used techniques in these experiments: Internal competition studies and direct comparison experiments. Both measure position-specific isotope effects.
Direct Comparison Direct comparison experiments are primarily used for measuring hydrogen/deuterium isotope effects in enzymatic reactions. The monoisotopic substrate and a deuterated form of the substrate are separately exposed to the enzyme of interest over a range of concentrations. The Michaelis-Menten kinetic parameters for both substrates are determined and the position-specific isotope effect at the site of deuteration is expressed as the ratio of the monoisotopic rate constant over the rare isotope rate constant.
Internal Competition For isotopes of elements like carbon and sulfur, the difference in kinetic parameters is too small, and the measurement precision too low, to measure an isotope effect by directly comparing the rates of the monoisotopic and rare isotope substrates. Instead, the two are mixed together using the natural abundance of stable isotopes in molecules. The enzyme is exposed to both isotopes simultaneously and its preference for the light isotope is analyzed by collecting the product of the reaction and measuring its isotope composition. For example, if an enzyme removes a carbon from a molecule by turning it into carbon dioxide, that carbon dioxide product can be collected and measured on an Isotope Ratio Mass Spectrometer for its carbon isotope composition. If the carbon dioxide has less 13C than the substrate mixture, the enzyme has preferentially reacted with the substrate that has a 12C at the site that is decarboxylated. In this way, internal competition experiments are also position-specific. If only the CO2 is measured, then only the isotope effect on the site of decarboxylation is recorded.
Chemical degradation Before the advent of technologies that analyze whole molecules for their intramolecular isotopic structure, molecules were sequentially degraded and converted to CO2 and measured on an Isotope Ratio Mass Spectrometer, revealing position-specific 13C enrichments.
Ninhydrin Reaction In 1961, Abelson and Hoering developed a technique for removing the carboxylic acid of amino acids using the ninhydrin reaction. This reaction converts the carboxylic acid to a molecule of CO2 which is measured via an Isotope Ratio Mass Spectrometer.
Ozonolysis Reaction