Use of collagenase to purify collagen from prehistoric bones for stable isotopic analysis. BY M. DeNiro, S.Weiner (original) (raw)
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Variability in the preservation of the isotopic composition of collagen from fossil bone
Geochimica et Cosmochimica Acta, 1988
Collagen from bone was prepared by several methods. For modem and well-preserved bone the PC and SjSN 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 6°C and 6"N varied among the different protein extracts. The yield of collagen obtained with EDTA demineralization was consistently higher than extraction procedures that used HCl.
Collagen extraction from recent and fossil bones: quantitative and qualitative aspects
Journal of archaeological science, 1995
Type I collagen is the major protein in bones. The mineral matrix protects collagen from denaturation, thus permitting the recovery of large collagen peptides from fossil bones thousands or millions of years old. Collagen peptides are more or less denatured in fossil bones, with diagenetic alteration being the major cause of such denaturation. Classical extraction methods alter the remaining large collagen peptides by extensive solubilization. A method is described here that used shorter collagen solubilization times. Resulting collagen yields are favourably compared with classical methods. The size of the large peptide (>10 kDa) fraction improves considerably. Combined with a particular concentration step, the use of this shorter solubilization technique should be useful for collagen analyses that necessitate large peptides, as in the case of palaeoimmunology.
Collagen Extraction and Stable Isotope Analysis of Small Vertebrate Bones: A Comparative Approach
Bone remains of small vertebrate fossils provide valuable information for paleoenvironmental and paleoclimatic reconstructions. However, direct radiocarbon dating of small vertebrates remains challenging as the extraction of sufficient good quality collagen is required. The efficiency of eight collagen extraction protocols was tested on seven samples, representative of different ages and burial environments, including both macro and small vertebrate taxa. First, the samples were prescreened using attenuated total reflectance–Fourier transform infrared spectroscopy (ATR-FTIR) to quantify collagen content in archaeological bones, revealing that one should be discarded for 14 C dating. Then, the quantity of protein extracted (yield) and collagen integrity were checked using conventional elemental analysis. The results show that one protocol was not able to accurately extract collagen from the samples. A soft HCl-based protocol seems more appropriate for the pretreatment of archaeological small mammal bones, whereas a harsher protocol might be more efficient to extract a higher amount of collagen from large mammals as well as amphibian bones. The influence of the tested protocols on carbon and nitrogen isotope values was also investigated. The results showed that isotopic variability, when existing, is related to the interindividual differences rather than the different protocols.
Archaeological collagen: Why worry about collagen diagenesis?
2009
DNA appears to decay by random chain scission resulting in a predictable range of fragment lengths. Collagen decay has also been modelled in this same way, although it has become increasingly evident that collagen decay does not follow this same pattern. Radiocarbon and stable isotope analysis now use ultra-filtration to isolate large fragments (>30% of original polymer length) even in Pleistocene bone. How then does collagen decay? This study contrasts experimentally degraded samples with collagen extracted from forensic, archaeological and fossil bone. In experimentally degraded bone, values for amino acid and elemental (C:N) composition, bulk δ 13 C, δ 15 N, and aspartic acid racemisation (AAR) changed very little until 99% of the collagen was lost, suggesting that the collagen triple helix and polypeptide chains remained remarkably intact. This suggestion was demonstrated directly by examining the integrity of individual polypeptide chains using cyanogen bromide (CNBr) cleavage followed by SDS-PAGE electrophoresis. In ancient samples, AAR values remain remarkably stable and the pattern of CNBr-cleavage was only replaced with a smear of smaller polypeptides in the oldest (Pleistocene) bones investigated. Smearing may reflect both modification of the methionine resides (the sites of CNBrcleavage) and/or partial hydrolysis of the collagen molecule. The findings reveal why it is not usually necessary to worry about collagen diagenesis; it is mostly intact. However, evidence of partial deterioration of the oldest bone samples suggests that alternative purification strategies may increase yields in some samples.
Analysis of Bone "Collagen" Extraction Products for Radiocarbon Dating
Radiocarbon, 2013
Archaeological bones are now routinely dated in many radiocarbon laboratories through the extraction of "collagen." Methods for "collagen" extraction vary, and several laboratories now apply an ultrafiltration step after gelatinization to extract the higher molecular weight (usually >10 or 30kDa) fraction for dating, thereby removing low molecular weight contaminants. Ultrafiltration has been demonstrated to result in products that are easier to handle and have more acceptable C:N ratios, and in some instances can result in significantly improved (generally older) 14 C dates when compared to non-ultrafiltered products from the same bone. Although it has been suggested that ultrafiltration removes potential contaminants such as short-chain degraded collagen and other peptides and amino acids, fulvic acids, and salts, there remains little published evidence to support this. This paper presents data from a pilot study investigating the most suitable techniques with which to study the products of the routine "collagen" extraction procedures employed at the Oxford Radiocarbon Accelerator Unit (ORAU) (modified Longin followed by ultrafiltration). The preliminary data demonstrates that the final product of "collagen" extraction at ORAU appears to be an aggregate consisting of a range of proteins of different molecular weights, including collagen, as well as some other organic matter and inorganic species. Ultrafiltration is removing some, but not all, of the <30kDa fraction from the samples. Further work to investigate the nature of this aggregate and how best to improve the efficiency of "collagen" extraction procedures is discussed.
Two alternative methods of bone preparation were examined, calcium chelation by ethylenediaminetetraacetic acid (EDTA, pH 8) and dissol!ution in strongmineral acid (HCl. The rate of EDTA demineralization of modern bone powders (< 63 um) was slow andindependent of temperature, A weak temperaturedependence (20 kJ moll) was observed for both bone shards (1-3mm) and archaeological powders which was attributed to (i) the slow rate of diffusion of EDTA through the mineral matrix of the shards, and (ii) the higher crystallinity of archaeological powders. Hydrochloric acid (HCl; 0.6 M) demineralization was rapid and could be accelerated by gentle agitation which increased the surface area of the Nernst diffusion layer. The degree of collagen degradation which occurred during HCI demineralization was contrasted with the damage caused by powdering the bone. At 4 C very little gelatinization was seen during HCI demineralization, even after prolonged incubation, but at 37 C complete gelatinization occurred after less than 100 h.However, the amount of acid soluble collagen present in the supernatant prior to .,dd demineralization was six times greater for powdered samples than for shards, equivalent to 14% of the total bone collagen. These data indicate that at low temperatures more damage is caused by grinding than by prolonged exposure to add. It is suggested that to avoid damage to proteins and other bone biopolymers, samples should be demineralized in mineral acid at a low temperature and excessive grinding of samples should be avoided.
Toward a versatile protocol for radiocarbon and proteomics analysis of ancient collagen
Journal of Archaeological Science, 2019
Ancient bone proteins provide a plethora of information regarding the identity, period, and diet/environment of animal species through proteomic analysis, radiocarbon dating and isotopic analysis respectively. However, each technique imposes specific constraints for bone protein extraction. Despite the sample preciousness and heterogeneity, these analyses are not routinely performed on the same aliquot. Protocol for radiocarbon dating are the most restrictive in terms of sample preparation and their effects have seldom been evaluated at the molecular level. Here, several extraction protocols were tested using modern and archaeological bones. Molecular characterization of the extracts was performed using electrophoresis and proteomics. Protocol-induced biases in peptide sequences and their effect on species identification were evaluated using database searching and partial de novo sequencing. This work shows that extraction protocols corresponding to mild bone decalcification conditions and using ultrafiltration are the most suitable for species identification.
Establishing collagen quality criteria for sulphur isotope analysis of archaeological bone collagen
Archaeological and Anthropological Sciences, 2009
Sulphur isotope measurements of bone collagen from archaeological sites are beginning to be applied more often, yet there are no clear criteria to assess the quality of the collagen and therefore the validity of the sulphur isotope values. We provide elemental data from different methods (DNA sequences, amino acid sequences and mass spectrometric measurements) which are used to establish a reliable system of quality criteria for sulphur isotope analyses of bone collagen. The difference in the amount of sulphur from fish and mammalian collagen type I led to the suggestion to use different criteria to assess the in vivo character of the collagen between these two categories. For establishing quality ranges, the bone collagen of 140 modern animals were analysed. The amount of sulphur in fish and mammalian bone collagen is 0.63 ± 0.08% and 0.28 ± 0.07%, respectively. Based on these results we define for mammalian bone collagen an atomic C:S ratio of 600 ± 300 and an atomic N:S ratio of 200 ± 100, and for fish bone an atomic C:S ratio of 175 ± 50 and an atomic N:S ratio of 60 ± 20. These quality criteria were then applied to 305 specimens from different archaeological contexts.
A new procedure for extraction of collagen from modern and archaeological bones for 14C dating
Analytical and Bioanalytical Chemistry, 2011
Bones are potentially the best age indicators in a stratigraphic study, because they are closely related to the layer in which they are found. Collagen is the most suitable fraction and is the material normally used in radiocarbon dating. Bone contaminants can strongly alter the carbon isotopic fraction values of the samples, so chemical pretreatment for 14 C dating by accelerator mass spectrometry (AMS) is essential. The most widespread method for collagen extraction is based on the Longin procedure, which consists in HCl demineralization to dissolve the inorganic phase of the samples, followed by dissolution of collagen in a weak acid solution. In this work the possible side effects of this procedure on a modern bone are presented; the extracted collagen was analyzed by ATR-IR spectroscopy. An alternative procedure, based on use of HF instead of HCl, to minimize unwanted degradation of the organic fraction, is also given. A study by ATR-IR spectroscopic analysis of collagen collected after different demineralization times and with different acid volumes, and a study of an archaeological sample, are also presented.