Biogeochemistry of Brachiopod Intracrystalline Proteins and Amino Acids

Walton, Derek (1992) Biogeochemistry of Brachiopod Intracrystalline Proteins and Amino Acids. PhD thesis, University of Glasgow.

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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.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Gordon Curry
Keywords: Paleontology, Biogeochemistry, Geochemistry, Molecular biology
Date of Award: 1992
Depositing User: Enlighten Team
Unique ID: glathesis:1992-75197
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 21:48
Last Modified: 19 Nov 2019 21:48

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