P. E. Hare

Isotopic fractionation of nitrogen and carbon in the synthesis of amino acids by microorganisms

Two classes of procaryotic organisms were cultured on specific inorganic and organic nitrogenous substrates. The organisms fractionated these substrates in characteristic ways during the synthesis of their cellular biochemical compounds. Blue-green algae, Anabaena sp., were raised on molecular nitrogen, nitrate and ammonia in the presence of excess carbon dioxide. The difference between δ15N of either nitrate or ammonium and the δ15N of the algae grown on either source was 13%o. The δ15N of Anabaena that fixed N2 was 2% lighter than the nitrogen gas supplied to the algae. These fractionations are associated with enzymatic incorporation of the nitrogen into the cell. A heterotrophic bacterium, Vibrio harveyi was grown on a single amino acid as its source of both nitrogen and carbon. Cells grown on glutamic acid were enriched in 15N relative to substrate, whereas those grown on alanine were depleted in 15N compared to source nitrogen. The bacterial cultures were enriched in 13C relative to the substrate. These cultures were then hydrolyzed and individual amino acids isolated and isotopically analyzed. The isotopic compositions of the amino acids have a wide range of values; most appear to have isotope fractionations associated with the metabolic pathways in their synthesis. These results and the application of the coupled separation-isotopic analysis of amino acids yield a better understanding of comparative biochemistry for these organisms. Such analyses offer valuable information for the tracing of biosynthesis and early diagenesis to help explain the fossil record.

Kinetic fractionation of stable nitrogen isotopes during amino acid transamination

Abstract This study evaluates a kinetic isotope effect involving 15N, during the transamination reactions catalyzed by glutamic oxalacetic transaminase. During the transfer of amino nitrogen from glutamic acid to oxaloacetate to form aspartic acid, 14NH2 reacted 1.0083 times faster than 14NH2. In the reverse reaction transferring NH2 from aspartic acid to α-ketoglutarate, 14NH2 was incorporated 1.0017 times faster than 15NH2. Knowledge of the magnitude and sign of these isotope effects will be useful in the interpretation of the distribution of 15N in biological and geochemical systems.

Effects of diagenesis on strontium, carbon, nitrogen and oxygen concentration and isotopic composition of bone

Abstract Paleodietary analysis based on variations in the trace element and stable isotopic composition of inorganic and organic phases in fossil bone depends on the assumption that measured values reflect in vivo values. To test for postmortem alteration, we measured 87 Sr 86 Sr , 13 C 12 C , 18 O 16 O and 15 N 14 N ratios and Sr concentrations in modern and prehistoric (610 to 5470 yr old) bones of animals with marine or terrestrial diets from Greenland. Bones from modern terrestrial feeders have substantially lower Sr concentrations and more radiogenic 87 Sr 86 Sr ratios than those from modern marine feeders. This contrast was not preserved in the prehistoric samples, which showed almost complete overlap for both Sr concentration and isotopic composition in bones from the two types of animals. Leaching experiments, X-ray diffraction analysis and infrared spectroscopy indicate that alteration of the Sr concentration and isotopic composition in prehistoric bone probably results from nearly complete exchange with groundwater. Oxygen isotope ratios in fossil apatite carbonate also failed to preserve the original discrimination between modern terrestrial and marine feeders. The C isotope ratio of apatite carbonate did not discriminate between animals with marine or terrestrial diets in the modern samples. Even so, the ranges of apatite δ 13 C values in prehistoric bone are more scattered than in modern samples for both groups, suggesting alteration had occurred. δ 13 C and δ 15 N values of collagen in modern bone are distinctly different for the two feeding types, and this distinction is preserved in most of the prehistoric samples. Our results suggest that postmortem alteration of dietary tracers in the inorganic phases of bone may be a problem at all archaeological sites and must be evaluated in each case. While collagen analyzed in this study was resistant to alteration, evaluation of the possibility of diagenetic alteration of its isotopic composition in bones from other contexts is also warranted.

Variability in the preservation of the isotopic composition of collagen from fossil bone

Abstract Collagen from bone was prepared by several methods. For modern and well-preserved bone the δ13C and δ15N of collagen replicas obtained after HCl or EDTA demineralization were similar to those obtained with a gelatinization procedure. However, in more poorly preserved fossil bone the δ13C and δ15N varied among the different protein extracts. The yield of collagen obtained with EDTA demineralization was consistently higher than extraction procedures that used HCl. The δ13C of individual amino acids separated from the collagen of modern and fossil whale bone varied up to 17%., and the δ15N from the same amino acids ranged over 47%. The δ13C and δ15N of most amino acids clustered closely to the average of the HCl insoluble collagen. The δ13C of the major amino acid in collagen, glycine, differed from the average HCl insoluble collagen by approximately 8%. in the fossil whale and 14%. in the modern whale. The δ15N of glycine differed from the average HCl insoluble values by approximately 4%. in the fossil whale and 7%. in the modern whale. Thus, diagenetic changes that alter the ratio of glycine to other amino acids in bone can be expected to perturb the values for carbon and nitrogen isotopes.

Amino acid adsorption by clay minerals in distilled water

Abstract The adsorption of 15 protein amino acids from dilute (~ 10 μM) distilled water solutions onto organic-free kaolinite and montmorillonite clay minerals (1 wt% suspensions) was determined at room temperature over a 48 hour period. The systems came to steady state within 2 hours. Basic (positively charged) amino acids were strongly adsorbed (40–80% removal) by both clay minerals. Neutral (uncharged) amino acids were taken up appreciably (10–15%) by montmorillonite, but little if any (

Racemization of Amino Acids in Marine Sediments

Isoleucine, one of several amino acids isolated from a suite of welldated deep-sea cores, shows a progressive increase in the degree of racemization with the age of the sediment. Amino acids in sediments show an initial rate of racemization almost an order of magnitude faster than the rate observed for free amino acids at a comparable pH and temperature. The observed kinetics depend on a variety of diagenetic processes, but it appears that the ratio of alloisoleucine to isoleucine is a reliable indicator of age for samples less than 400,000 years old; for older samples the results are more ambiguous. Isoleucine is racemic in samples older than about 15 x 106 years.

Dating pleistocene archeological sites by protein diagenesis in ostrich eggshell.

Eggshells of the African ostrich (Struthio camelus), ubiquitous in archeological sites in Africa, have been shown by laboratory simulation experiments to retain their indigenous organic matrix residues during diagenesis far better than any other calcified tissue yet studied. The rate of L-isoleucine epimerization to D-alloisoleucine follows reversible first-order kinetics and has been calibrated for local temperature effects and used to estimate the age range of stratified archeological sites. Age estimates are consistent with radiocarbon dates from several stratified archeological sites. With adequate calibration, this technique can provide accurate ages to within 10 to 15 percent for strata deposited within the last 200,000 years in the tropics and the last 1,000,000 years in colder regions such as China.

Aspartic acid racemization and protein diagenesis in corals over the last 350 years

Abstract D/L aspartic acid values from a 350-year time series of annual growth bands of a living colony of the coral Porites australiensis show a very regular pattern of increase with age. The initial rate of racemization is extraordinarily rapid (0.6% per year) but slows in older growth bands to 0.04% per year (4% per century). The skeletal proteins show progressive hydrolysis with increasing age, with free aspartic acid comprising 16% of the total aspartic acid in the 350-year-old band. The proteins are unusually rich in aspartic acid (nearly 50 mol%). The relative weakness of the peptide bonds formed by aspartic acid moieties is probably responsible for the rapid hydrolysis and consequent rapid racemization of aspartic acid. Racemization analysis provides a means for checking for sections of missing bands in corals and for screening of prospective samples for U-Th dating.

Amino acid composition of the test as a taxonomic character for living and fossil planktonic foraminifera

Sixteen species analyzed for 17 amino acids, multivariate analysis indicates that amino acid composition varies in a systematic way which parallels morphology, Miocene, Holocene, Atlantic Ocean

Racemization of amino acids in marine sediments determined by gas chromatography

Abstract 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.