Derek Walton

The significance of a geochemically isolated intracrystalline organic fraction within biominerals

Abstract In studies of organic matter in fossil biominerals, there has been a widespread failure to distinguish between the organic matrix and organic matter trapped within the crystal elements. The existence of chemically isolated (intracrystalline) proteins are indicated by the persistence of amino acids after prolonged treatment with a strong chemical oxidant (NaOCl). The geochemical significance of these residual amino acids is illustrated by the re-analysis of aberrantly young d -aile/ l -ile ratios (0.142 ± 0.042, n = 4) of amino acids from a land snail ( Cepaea sp.) collected from Tattershall Thorpe in Lincolnshire. Following NaOCl treatment the d -aile/ l -ile ratio increased (0.178 ± 0.014, n = 5), while both the total amino acid concentration and the variance declined.

Fossil intra-crystalline biomolecules of brachiopod shells: diagenesis and preserved geo-biological information

Intra-crystalline macromolecules isolated from the shells of four brachiopod species, collected from successive horizons representing the last 1.47 million years, have been studied by immunoassay and amino acid analysis. Patterns in both immunological responses and free/total amino acid ratios indicated that degradation of these macromolecules proceeded rapidly within the first 0.13 Myr, followed by a plateau phase (0.13-0.62 Myr). Antigenic determinants could not be detected from the oldest (1.47 Myr) specimens studied, suggesting the presence of a third phase of degradation. Canonical variate analyses (CVA) of the amino acid determinations demonstrated separations of two plausible taxonomic groups among living and fossil samples, and revealed progressive diagenetic trends in each group. CVA also gave an explicit separation of the species examined based on the compositions of the free amino acids present in living and fossil shells, suggesting that the intra-crystalline microenvironment approximates a semi-closed system.

Long-term trends in the survival of immunological epitopes entombed in fossil brachiopod skeletons

Abstract We report the most comprehensive study of survival of peptide bonds and epitopes (antibody binding sites) in fossil shells from a semi-continuous New Zealand brachiopod sequence extending for 3 Ma. The study reveals for the first time long-term trends in proteins survival. The investigation focused on a sub-set of the total skeletal biomolecules, those protected from exposure to a strong oxidising agent (NaOCl); the so-called intra-crystalline component. The extent of peptide bond hydrolysis was compared with the declining immunological signal. The proportion of free amino acids increased very rapidly but between 5 and 10% of the amino acids remained peptide bound in all samples. The pattern of loss of immunological reactivity broadly mirrored the loss of peptide bonds, but overall loss of signal was much greater. Significant antibody response was observed in some but not all late Pliocene fossils (>3 Ma), but against a panel of antisera the pattern of reactivity was lost in samples >0.5 Ma. Alternative models of polypeptide chain scission were used in an to attempt to relate the rate of peptide bond hydrolysis to the loss of immunological determinants. The findings suggest that, despite early optimistic reports, the application of immunology to shell carbonates does not appear capable of extending into deep time.

Intracrystalline Molecules from Brachiopod Shells

Shells are composed of both organic and inorganic constituents. It is believed that the organic compounds have important functions at several stages during the formation of biominerals. In brachiopod shells the disposition of inorganic biominerals and their enclosing organic sheaths have been thoroughly investigated using both scanning and transmission electron microscopy but little is known about the biochemistry of the intracrystalline molecules i.e. those enclosed within the inorganic portion. Such information is crucial for an understanding of biominerals if, as has been suggested, these compounds (i) induce crystal nucleation by providing a surface for precipitation, (ii) form compartments that determine the shape and volume of the biocrystal and (iii) determine the pattern of growth in the mineral phase in what is termed ‘matrix mediated nuneralisation’ [1].

Biogeochemistry of brachiopod intracrystalline proteins and amino acids

Amino acids were released from a range of fossil and Recent samples, sediments and fingertips. Fingertips contain up to 100 times the concentration of amino acids in the fossils, and sediments up to 10 times the concentration. However, the distribution of amino acids in these samples were significantly different from those in fossil samples, and were easily discriminated using statistical methods. Incarcerated molecules (protein, lipids and carbohydrates) from Recent samples were released from the calcium carbonate of the shell into solution. The protein fraction of the samples were purified to homogeneity using SDS PAGE, and the separated proteins analysed by partial N-terminal sequencing and amino acid analysis. The crude extract was also fractionated by reverse phase liquid chromatography. Following the partial characterisation of these intact, extant molecules, attempts were made using similar techniques to fractionate the bulk organic extract from related fossils. This only resulted in broad bands or peaks of low molecular weight compounds at low concentrations, rather than the sharp peaks which resulted from the analysis of Recent extracts, and it was not possible to purify the molecules using these methods. It was also concluded that during the filtration stage of preparation the majority of the molecules were lost. A different method of preparation was applied to fossil samples, which did not include any concentration or filtration steps, and the free amino acids and small peptides which are present in the acid soluble fossil extracts were therefore also quantified. Amino acid analysis of these extracts from fossils revealed that the proteins which were originally present within the shells have undergone natural hydrolysis reactions (cleavage of peptide bonds as a result of time or heat) and are highly degraded, indicating why the separations by biochemical techniques could not be applied to fossil samples. By 0.2 Ma, up to 80% of the amino acids are present in the free state, indicating that the majority of the peptide bonds in the sample have been broken, leading to the production of larger numbers of small peptides. Some peptides remain, indicated by increased concentrations of amino acids following acid hydrolysis. Proteins from intracrystalline sites within fossils are therefore in a poor state of preservation, and it is unlikely that it will be possible to separate and concentrate these molecules in order to complete primary sequence analysis. The peptide bonds break rapidly in the samples in response to the action of temperature, the presence of water, and the nature of the residues present on either side of the peptide bonds. Individual amino acids also undergo degradative reactions in the fossil record, and the molecules may be grouped in terms of their stability. The decomposition of most amino acids may be described by exponential or logarithmic curves, indicating a rapid rate of decomposition in the fossil record. The amino acids are generally degraded more rapidly in the free state than when they are bound into peptides. Degradation products may be non-amino acids, non-standard amino acids or proteinogenic amino acids, depending on the original molecule which has decomposed. The advantage of utilising intracrystalline molecules is that the degradation products of both proteins and amino acids remain within the shell and are not leached out (as is the case for intercrystalline molecules) ensuring that any preserved taxonomic signal remains within the shell. Despite the severe degradation of the molecules, taxonomic information is still preserved within the samples, although at a lower level of discrimination than in the Recent, and this information may be revealed by statistical analyses. No homogenisation of the amino acids in the sample had taken place, and that subordinal level discrimination is possible to at least 0.4 Ma. Older samples showed merging of samples from similar orders, although different orders and phyla could easily be discriminated by this method. When samples are analysed together from all horizons using these statistical methods, samples could still be discriminated to the ordinal level, no matter what the age of the sample, indicating that although proteins and amino acids were decomposed rapidly, some taxonomic signal remains in the shell.