Comparison of rates and degrees of isoleucine epimerization in dipeptides and tripeptides

The degrees and rates of isoleucine epimerization in heating experiments with pure di- and tripeptides are dependent on: (1) the position of isoleucine in the peptide chain; (2) the formation from dipeptides of diketopiperazines, cyclic intermediates that are in equilbrium with, and hydrolyze to, both the parent and inverted dipeptides as follows: NH₂-terminal?diketopiperazine?COOH-terminal. The results of heating experiments show that isoleucine residues in diketopiperazine are the most highly epimerized form in both di- and tripeptide solutions. NH₂-terminal isoleucine also undergoes relatively rapid epimerization, while the rates of epimerization of COOH-terminal and free isoleucine are much slower. Through hydrolysis reactions, high degrees of isoleucine epimerization are transferred to slower epimerizing species. Consequently, the relative rate of isoleucine epimerization in the various positions differs from the relative degree of epimerization.The relative rates of isoleucine epimerization are: NH₂ ? diketopiperazine ? COOH ≈ interior ≈ free, while the relative degrees of isoleucine epimerization are: diketopiperazine > NH₂ > COOH ? interior. The high degree of epimerization exhibited by COOH-terminal isoleucine in fossil dipeptides is due to prior preferential epimerization in diketopiperazine. The high degree of epimerization of free isoleucine in carbonate fossils is due to the hydrolysis of highly epimerized terminal isoleucine. These results are applicable to peptides both in fossil shells and in aqueous solutions and form the basis for a revised model of isoleucine epimerization in fossil shells.     

Comparison of isoleucine epimerization in a model dipeptide and fossil protein

The dipeptide isoleucyl-glycine in aqueous solution has activation energies for isoleucine epimerization and hydrolysis of 23.1 and 20.9 kcal mol^?1, respectively. The activation energy for epimerization of NH₂-terminal isoleucine is 5–8 kcal mol^?1 less than activation energies reported for epimerization of isoleucine in the free state or in various protein systems. Furthermore, the rate of isoleucine epimerization in the NH₂-terminal position exceeds the rate of hydrolysis at temperatures between 0 and 152°C. Consequently, a high rate of epimerization in fossils should only occur when isoleucine is in the terminal position. Partially epimerized isoleucine will be converted through hydrolysis from the terminal form to the slower epimerizing free isoleucine. Over the course of the total isoleucine epimerization reaction, either in dipeptides or proteins, the activation energy will increase and the reaction rate will decrease as terminal isoleucine is converted to free isoleucine. This process may explain the non-linear kinetics observed for isoleucine epimerization in carbonate fossils.The degree of isoleucine epimerization is low in different molecular weight fractions of a fossil protein from Mercenaria in all fractions greater than 500 mol. wt, where most isoleucine is likely to be in the interior position. In the less than 500 mol. wt peptide fraction, where a considerable portion of the isoleucine is likely to be in the terminal position, the degree of epimerization is significantly greater. These analytical results from fossil protein support the interpretation of isoleucine kinetics obtained by study of dipeptides.     

Amino acids in fossil corals: racemization (epimerization) reactions and their implications for diagenetic models and geochronological studies

Thirty-eight fossil coral samples of known age from late Pleistocene uplifted reef terraces have been analyzed for amino acid composition and for the degree of racemization of selected amino acids. Particular attention has been given to the epimerization (racemization about the α-carbon) of isoleucine. The $\text{D}\text{L}$ ratios observed in many of these samples do not conform to the concept of increasing racemization being associated with increasing fossil age. It is shown that sixteen of the samples demonstrate concordance between their known age and the age estimated from racemization data. Racemization ages are based upon a kinetic model derived from the kinetics observed in foraminifera in marine sediments and published estimates of the temperature dependence of the isoleucine epimerization reaction. Lack of concordance for the remaining samples is explained by either of two separate diagenetic phenomena: extensive leaching of free amino acids from the fossil, or contamination by ‘young’ amino acids. Ambiguities observed in many of the results may be due to the fact that coralline organic matter can have a complex history because of variations in its source and in its relation to the mineral phase. Neither of these factors has been observed to exert such a significant effect on racemization kinetics observed in fossil foraminifera or molluscs. Fossil corals are of only modest value as specimens for amino acid geochronological studies.     

Amino acid geochronology of selected interglacial sites in the North Sea area.

Isoleucine epimerization (alle/Ile) ratios have been measured on foraminiferal tests from interglaical sites in the southern North Sea region. In order to evaluate the possibilities of this amino acid method on benthic foraminifera, sites were chosen from areas where the stratigraphy was already well known. The study demonstrates that the isoleucine epimerization reaction in benthic foraminifera can be used for correlation and as a means of relative age estimation within a limited geographic area. Previous suggestions of a Holsteinian age for the Rugrd, Tornskov, and Wacken sites are supported for the Kås Hoved site. The age of most presumed Eemian sites is supported by the amino acid results; the Reighton site also seems to belong in this interglacial.     

Testing the aminostratigraphy of fluvial archives: the evidence from intra-crystalline proteins within freshwater shells

Until recently few studies of amino acid racemization of fossil bivalves and gastropods collected from river terrace deposits in Europe were based on the analysis of the intra-crystalline fraction. Instead they were based on the epimerization (racemization) of a single amino acid, isoleucine, and its inter-conversion to alloisoleucine. This paper presents data from the analysis of the intra-crystalline fraction of the shells, using a preparation technique of sample bleaching to remove the leachable matrix, thus leaving a component that exhibits closed-system behaviour. Reverse-phase HPLC separation with fluorescence detection allows the interpretation of four amino acids in detail: aspartic acid, glutamic acid, alanine and valine. The intra-crystalline fraction offers greater potential for improved resolution, especially when combined with the analysis of multiple amino acid d/l values, which racemize at different rates. This is explored using three species of freshwater gastropods (Bithynia tentaculata and troschelii, Valvata piscinalis) and the bivalve Corbicula. Sites of different ages within the Lower Thames river terrace sequence are used as a stratigraphical framework, with samples from other southern UK sites providing supplementary evidence. The results indicate better resolution using the intra-crystalline fraction over that obtained using unbleached shells, with differentiation possible at sites of up to MIS 7 age. However, for older sites, although values are always higher, the separation is less successful. A species effect has been identified between the gastropod shells. Despite the analysis of intra-crystalline protein, amino acid data from Corbicula remain problematical. Preliminary data on the opercula from Bithynia indicate that better resolution is possible, particularly at older sites.     

Kinetics and mechanism of the epimerization and decomposition of threonine in fossil foraminifera

Determination of alloThr/Thr ratios in foraminiferal tests ranging in age from Recent to ～ 700,000 yr B.P. has shown that threonine epimerization ratios approach a steady-state value of ～0.2. This is well below the equilibrium ratio of ～0.9 as measured in buffered aqueous solutions. It has also been found that the initial rate of decomposition of threonine in fossil foraminfera is about an order of magnitude faster than would be predicted on the basis of extrapolation from high temperature kinetic studies on free threonine in buffered aqueous solution. These results are apparently a consequence of metal cation catalysis of both the decomposition and the epimerization of free threonine, which is being continually released by the slow hydrolysis of peptide and proteinaceous material present in the fossil shells.     

Amino acid racemization dating of quaternary deposits of central and Southern Italy

Results are discussed of amino acid racemization dating of numerous samples of bones, teeth, tooth enamel and fossil shells. Racemization dating proved to be a very useful tool to date non-marine and marine deposits which are a few hundred thousand years old, as well as to correlate and chronologically classify even more ancient marine deposits. Aspartic acid racemization was used to date samples aged less than 80,000–100,000 yr; for more ancient samples, isoleucine epimerization, instead, was resorted to.In this work, dates were established for some important fragments of human bones and of large-mammalian fauna weighing a few grams, which would have been impossible to date with the ¹⁴C technique, requiring a large amount of bone material.The extent of isoleucine epimerization was measured in Glycymeris genera to estimate the age of numerous marine deposits of the Tyrrhenian coast of Centra-Southern Italy and of Sardinia. In the Tarquinia area (Latium) three marine terraces were identified, which were estimated to be approx. 120,000, 200,000 and 350,000 yr old. In Sardinia, five marine horizons were identified; the youngest deposit (Neotyrrhenian) proved to be present at Punta tramontana and Santa Reparata, while, at Riola and Stagno di Sassu, the most ancient marine deposits of Sardinia were found. In the Rome area, the Monte Mario Formation was estimated to be coeval with Monte delle Piche Formation. Finally, from the terraced coastal deposits of Capo Milaazzo (Sicily), Panarea (Eolian archipelago), Archi (Calabria) and Gallipoli (Apulia), a Tyrrhenian age range of 100,000–120,000 yr was obtained.     

Amino acid diagenesis in the marine bivalve Arctica islandica Linné from northwest European sites: Only time and temperature?

Collections of the molluscan species Arctica islandica from seven sites in Norway, Scotland and the North Sea, ranging in age from recent to Early Pleistocene, have been sampled for amino acid analyses. The shells were sampled in profiles through the valves at different distances from the umbo. In the fossil material a general trend of increasing degree of isoleucine (lle) epimerisation and decreasing amino acid concentrations were found going from the inner part of the valve to the outer. Although less pronounced, there is a similar trend from the central part of the valve to the margin. As the concentration of alloisoleucine (alle) remains nearly constant, the observed changes in alle/lle ratios are a result of variations in the amount of isoleucine. The amino acid composition is fairly uniform in recent shells and the observed gradients are established in mid-Holocene samples. Therefore, it is suggested that the gradients are established during rapid early degradation of the protein, possibly due to microbiological activity. Differences in alle/lle ratios observed in molluscs from sites of the same age and same thermal history could be explained by differences in the exposure to amino-acid-consuming microorganisms during the very early stage of diagenesis.     

我国“北京人”、“兰田人”和“元谋人”产地骨化石中氨基酸的地球化学

氨基酸是生命物质中普遍存在的一类重要的有机化合物,它是构成蛋白质的“砖块”。一个最简单的氨基酸分子至少必须含有一个羧基和一个氨基的官能团。在生物的蛋白质中通常只包括有二十种氨基酸,叫做蛋白氨基酸。但是自然界还存在其它种类的氨基酸,因为它们一般不存在于蛋白质中,所以叫做非蛋白氨基酸。除了最简单的甘氨酸外,所有的氨基酸都含有一个以上的不对称的碳原子。含有一个不对称碳原子的氨基酸可以存在L和D型两种结构的对映体。所有的蛋白质氨基酸都属L构型。     

Chronostratigraphic studies of sediments in the Negev desert, using amino acid epimerization analysis of land snail shells

The chronostratigraphy of mostly Holocene sediments in the Northern Negev desert of Israel was studied through amino acid epimerization analysis (alloisoleucine/isoleucine ratio, A/I) of individual land snail shells and ¹⁴C analysis of bulk samples of land snail shells contained within the sediments. Analysis of 31 deposits shows a very strong correlation between A/I and ¹⁴C age. A/I analyses indicate that age mixtures occur within all colluvial and in some fluvial deposits. Consequently, radiocarbon dates of bulk samples, which give an average age, overestimate the time of deposition. Sedimentation rates were generally rapid in both fluvial and colluvial deposits. Colluviation shows a maximum in the early Holocene and decreases progressively thereafter, in accordance with the change in average rainfall. Accumulation of fluvial sediments shows a distinct mid-Holocene maximum and apparently relates to the interaction between variation in rainfall, extent of bedrock exposure, and vegetation density. Late Holocene fluvial deposition may relate to agricultural activity. The understanding of the chronology of sedimentation is dependent on an integrated approach employing amino acid epimerization analysis to study age variation within a layer and ¹⁴C analyses for establishment of an absolute chronology.     

Geothermal and other effects on amino acid racemization in selected Deep-Sea Drilling Project cores

Kinetic parameters for the epimerization of isoleucine in multispecific foraminiferal asemblages were used to establish the effects of burial depth and the geothermal gradient on the extent of reaction. It was observed that with a little as thirty meters of burial in a normal thermal regime there were differences between the extent of epimerization measured and that which would have been predicted for thermal equilibrium with bottom water temperatures. As would be expected, these differences are greatest when the heat flow (the geothermal gradient) and/or the sedimentation rates are highest. These effects were observed in most of the DSDP samples studied, and have been used to estimate the average heat flux since the time of sample deposition. Occasional anomalous effects were observed which could not be related to past or present heat flux. These were determined to be due to such geologic occurrences as slumping and reworking or to recent sample contamination. Other problems emerged related to bottom water temperatures including changes over geologic time which are unknown and could not be deduced. Thus, the presence of epimerization anomalies in DSDP cores as noted above limits the effectiveness of amino acid geochronology in such cores, unless these anomalies can be recognized as ab initio.     

Interlaboratory comparison of amino acid enantiomeric ratios in fossil Pleistocene mollusks

Enantiomeric (d/l) ratios of eight amino acids in three homogeneous powdered fossil Pleistocene mollusk samples have been compared. Eleven laboratories have contributed results to this effort. Three gas chromatographic methods and one ion-exchange chromatographic method were used. In general, the coefficients of variation (c.v.) are best for alanine, glutamic acid, and aspartic acid (3–8%). For leucine and phenylalanine, the c.v.'s are between 5 and 10%. For isoleucine, proline, and valine the c.v.'s range from 10 to 18%. Individual laboratories usually report analytical precision of between 2 and 5%, but it is clear that significant differences between laboratories' results are often encountered, and that these analytical differences could, in some cases, lead to very significant differences (25% or more) in age estimates based upon enantiomeric ratios. Multiple analyses of desalted hydrolyzates of these powder samples suggest that interlaboratory differences are often caused by instrumental and/or derivatization procedures rather than the wet-chemical sample preparative steps. It is proposed that the powder samples described here be used in all future publications of fossil amino acid enantiomeric ratio data.     

The pre-Holocene marine limit on Hopen, Svalbard

A pre-Holocene marine level is found at 109 m a.s.1. on Hopen. Fragments of Mya truncata and Hiatella arctica occurring on a raised coastal spit at that altitude have been radiocarbon dated to >45,000 BP. The amino acid epimerization of these shells, a clearly mixed sample, correlates with samples from Kongsøya that are of Eemian or Early Weichselian age (alloisoleucine/isoleucine ratios, hydrolysed fraction, between 0.084 and 0.213). No direct evidence, such as striations or roche moutonées, of overriding glacier ice has been found, and no erratics were found above the pre-Holocene marine limit. The existing Holocene shoreline displacement curve, with its upper limit at 60 m a.s.1., is supported by four new radiocarbon dates.     

Racemization of amino acids in marine sediments determined by gas chromatography

Ratios of d- to l-amino acids in acid hydrolysates from foraminifera of two deep-sea cores from the Caribbean Sea and Atlantic Ocean increase with depth and consequently with age over a span from 40,000 to 2,000,000 yr. The changing ratios do not seem to follow first-order reversible rate laws. Valine, leucine and glutamic acid apparently racemize (isoleucine epimerizes) at slower rates than do phenylalanine, alanine, aspartic acid and proline. The general relative order for rates of racemization of total (free and bound) amino acids may depend on the electron-withdrawing capacity of the R substituents of the amino acids and on the rates with which the amino acids are naturally hydrolyzed. In contrast to the total amino acids, the free amino acids in these samples are more extensively racemized, probably as a result of various catalytic and hydrolytic reactions.Previous related work based on ion-exchange chromatography has considered only ratios of alloisoleucine to isoleucine. With the gas chromatographic method used here, d/l ratios of all common asymmetric amino acids can be estimated. Measurement of the extent of racemization of amino acids in marine sediments seems to provide the basis for a geochronological tool covering the last few million years.     

Amino acid ratios in Quaternary molluscs and foraminifera from western Norway: correlation, geochronology and paleotemperature estimates

Isoleucine epimerization (alle/Ue) ratios in the pelecypod Mya truncata and benthic foraminifer Cibicides lobalulus from emerged marine units in western Norway allow construction of a regional relative chronostratigraphy for the Ecmian and Weichselian. Two in situ interglacial sections are considered correlative by the similar biostratigraphy and alle/Ile ratios in C. lobalulus. Overlying sediments at the two sites are of both marine and glacial origin. Neither site contains a complete Weichselian record, but allelic ratios, lithostratigraphy and fauna! changes suggest at least four stadial and three interstadial events occurred along the western Norwegian coast during Early and Middle Weichselian time. Kinetic data defining the relationship between the isoleucine epimerization rate constant and temperature for the species studied allow the estimation of paleotemperatures for samples of known age. Accepting published age estimates for the Eemian interglacial beds, the average Weichselian temperature in western Norway is calculated to have been ca. 4°C below the average Holocene temperature, whereas the last interglacial was 1 to 2°C warmer that the Holocene. The limited temperature depression over this region during the Weichselian implies that coastal western Norway was ice-covered only about 30% of this period, and that Atlantic water, although not necessarily in a warm surface current as today, entered the Norwegian Sea during much of marine isotope stage 5 and intermittently during stage 3. Interpolated amino acid ages date interstadial events at ca. 94 ka, 78 ka and 52 ka, B.P., whereas glacial events are dated ca. 103 ka and bracketed by limiting dates between 78 and 89 ka, between 52 and 63 ka and less than 36 ka B.P.     

The stereochemistry of bound and extractable pentacyclic triterpenoids during closed system pyrolysis

The distributions of hopanoic acids, ranging from C₃₀ to C₃₄, in the Messel oil shale were characterized in both the free and bound states. The bound acids were released by thermochemolysis in the presence of tetramethylammonium hydroxide (TMAH). These were compared with the distributions of the hopanoic acids and hopanes released or generated from Messel oil shale kerogen following closed system microscale pyrolysis. This comparison revealed that epimerization had occurred at C-17, C-21 and C-22 during heating. It was also clear that the residual bound hopanoic acids had undergone configurational isomerization. During the pyrolysis there is a large loss of hopanoic acids following their rapid release from the kerogen into the free fraction even at 250 °C. In these particular experiments this loss does not appear to result in exclusive formation of hopanes, by way of decarboxylation or reduction reactions, unless the resulting hopanes are either themselves rapidly transformed into other compounds or the reaction rates are a function of the total number of carbon atoms in each hopanoic acid precursor.     

安徽和县猿人遗址牙化石的氨基酸生物地球化学

本文从氨基酸生物地球化学的角度,对安徽和县猿人遗址牙化石中的氨基酸进行了探讨,初步获得如下结果:1.牙釉化石的氨基酸组成中,酸性氨基酸含量极高(总量不低于30%);中性氨基酸含量也较大(总量在20%左右);碱性氨基酸含量极低,均在1%以下。 2.牙釉化石的氨基酸组成基本上取决于原生蛋白质的组成模式,但由于成岩作用的影响,使牙釉化石的氨基酸组成和含量与现生哺乳动物牙釉相比,存在较大差别。3.将测得的牙釉化石中的D-别异亮氨酸/L-异亮氨酸比值,以及根据对该地点动物化石研究而估算的平均成岩温度(12.5℃-13℃),代入有关的氨基酸测年公式,求得该猿人遗址的化石年龄为23万年。     

Molecular taphonomy of macrofossils from the Cretaceous Las Hoyas Formation,Spain

Macromolecular analysis of fossil fish scales from the Cretaceous Las Hoyas Formation (using pyrolysis-gas chromatography-mass spectrometry) revealed a dominant aliphatic composition (C₈ to C₂₂) whereas modern fish scale is proteinaceous (largely collagenous). Structural analysis of the aliphatic polymer using thermochemolysis revealed the importance of ester linkages; saturated fatty acids C₁₄ to C₁₈ (particularly C₁₆) are the most abundant. These acid components and their unsaturated counterparts are evident in the lipid composition of modern fish scales. Thus, the aliphatic composition of the fossil scales is probably a result of the incorporation of lipids (including a C₁₉ aromatic hydrocarbon) from the original indicating preservation by in situ polymerization of labile aliphatic components. Fossil arthropods and plants from the same deposit also show a dominant aliphatic macromolecular component, likely derived predominantly by crosslinking of free lipid precursors. Differences in the relative distribution of molecular components indicate likely chemosystematic differences between different fossil groups.     

A reexamination of amino acids in lunar soils: implications for the survival of exogenous organic material during impact delivery

Using a sensitive high performance liquid chromatography technique, we have analyzed both the hot water extract and the acid hydrolyzed hot water extract of lunar soil collected during the Apollo 17 mission. Both free amino acids and those derived from acid labile precursors are present at a level of roughly 15 ppb. Based on the D/L amino acid ratios, the free alanine and aspartic acid observed in the hot water extract can be entirely attributed to terrestrial biogenic contamination. However, in the acid labile fraction, precursors which yield amino acids are apparently present in the lunar soil. The amino acid distribution suggests that the precursor is probably solar wind implanted HCN. We have evaluated our results with regard to the meteoritic input of intact organic compounds to the moon based on an upper limit of < or = 0.3 ppb for alpha-aminoisobutyric acid, a non-protein amino acid which does not generally occur in terrestrial organisms and which is not a major amino acid produced from HCN, but which is a predominant amino acid in many carbonaceous chondrites. We find that the survival of exogenous organic compounds during lunar impact is < or = 0.8%. This result represents an example of minimum organic impact survivability. This is an important first step toward a better understanding of similar processes on Earth and on Mars, and their possible contribution to the budget of prebiotic organic compounds on the primitive Earth.     

Organic geochemistry of silicified wood,Petrified Forest National Park,Arizona

Lignin derivatives have been isolated for the first time from carbonaceous sections of the silicified (~90% SiO₂) conifer Araucarioxylon arizonicum. The products released by sequential high vacuum pyrolysis and identified by combined gas chromatography-mass spectrometry (GC-MS) include a wide variety of alkyl-substituted, phenolic and condensed aromatic compounds.Brauns spruce lignin was pyrolized and analyzed by GC-MS as a comparison for the fossil wood data. The primary pyrolyzates at the 300°C step were CO₂, H₂O, ethanol and propanol. The main product at 450°C was 4-methyl-2-methoxyphenol (methyl guaiacol), but at 600°C the pyrolyzates were similar both in product composition and in relative abundance to those from silicified wood. The results suggest that the fossil wood experienced a mild thermal event during which the ether bonds were ruptured and loss of oxygen occurred along with the rearrangement of the original wood into a highly stable polymer.