Intra-crystalline protein diagenesis (IcPD) in Patella vulgata. Part II: Breakdown and temperature sensitivity

Abstract

Artificial diagenesis of the intra-crystalline proteins isolated from Patella vulgata was induced by isothermal heating at 140 °C, 110 °C and 80 °C. Protein breakdown was quantified for multiple amino acids, measuring the extent of peptide bond hydrolysis, amino acid racemisation and decomposition. The patterns of diagenesis are complex; therefore the kinetic parameters of the main reactions were estimated by two different methods: 1) a well-established approach based on fitting mathematical expressions to the experimental data, e.g. first-order rate equations for hydrolysis and power-transformed first-order rate equations for racemisation; and 2) an alternative model-free approach, which was developed by estimating a “scaling” factor for the independent variable (time) which produces the best alignment of the experimental data. This method allows the calculation of the relative reaction rates for the different temperatures of isothermal heating.High-temperature data were compared with the extent of degradation detected in sub-fossil Patella specimens of known age, and we evaluated the ability of kinetic experiments to mimic diagenesis at burial temperature. The results highlighted a difference between patterns of degradation at low and high temperature and therefore we recommend caution for the extrapolation of protein breakdown rates to low burial temperatures for geochronological purposes when relying solely on kinetic data.

Testing the limitations of artificial protein degradation kinetics using known-age massive Porites coral skeletons

“…12b) is clearly temperature dependent; at lower temperatures the observed degree of racemization in the FAA fraction increases relative to the THAA fraction. Demarchi et al (2013a) report a similar pattern in heated Patella; as was the case for corals, for a given THAA D/L value, FAA Asx D/L was generally higher at 80 °C, than at 110 °C and 140 °C. The relationship between the extent of hydrolysis (the %FAA) and the degree of racemization also varies between temperatures (Fig.…”

“…11) and, by extrapolating this rate down to an assumed temperature history, acquired numerical ages for fossil opercula. However, as is the case for Patella ( Demarchi, 2009 ;Demarchi et al, 2013a), serine decomposition in Porites does not appear to follow an exponential relationship, which indicates that the calibration is not suitable for extrapolation. Indeed the presence of a residual bound fraction in biomineral observed in high-temperature studies on avian eggshell , terrestrial gastropods (Penkman et al, 2008a), and marine gastropods (Demarchi et al, 2013a), suggests that liberation of free Ser, and hence [Ser]/[Ala] decomposition, is unlikely to conform to a simple pseudo-first order relationship.…”

“…Therefore, the rate constant for racemization can be calculated for a fossil, if its effective burial temperature (the integrated effect of all temperatures to which the fossil sample has been exposed (Wehmiller, 1977)) is known. The rate constant can subsequently be substituted into an appropriate 'age equation' to estimates the fossil's age; for example, if the rate constants are derived assuming reversible first order kinetics (RFOK) the following equation can be applied: equation (3) constant = racemization induced during sample preparation; t = age of the sample (yrs); D/L = ratio of D to L enantiomers There are several theoretical issues associated with extrapolating from high to low temperatures that may affect the validity of this approach (Kimber and Griffin, 1987;Goodfriend and Meyer, 1991;Walton, 1998;Collins et al, 1999;Collins and Riley, 2000 ;Demarchi et al, 2013a). Firstly, the use of kinetic models that do not adequately represent the observed reaction will result in inaccuracies in the derived rate constant and Arrhenius parameters (Goodfriend and Meyer, 1991).…”

“…A limited number of studies utilizing different biominerals have sought to test these assumptions, with varying results (e.g. Goodfriend and Meyer, 1991;Miller et al, 2000;Laabs and Kaufman, 2003 ;Demarchi et al, 2013a). A "hybrid" approach is often adopted to provide more robust estimates of the temperaturesensitivity of racemization that constrains data acquired during the high-temperature isothermal experiments using rates calculated in independently dated (unheated) fossils with estimated effective burial temperatures (e.g.…”

“…Exposure of skeletal material to a strong chemical oxidant (such as bleach) isolates a 'protected' component of the organic matrix, referred to as the 'intra-crystalline fraction' (Towe, 1980 ;Berman et al, 1993). This intra-crystalline fraction has been shown to effectively operate as a closed system in a range of biominerals (Penkman et al, 2008a;Penkman et al, 2011;Demarchi et al, 2011;Demarchi et al, 2013a ;Crisp et al, 2013), although not within the freshwater bivalves Margaritifera falcata ( Orem and Kaufman, 2011) and Corbicula ( Penkman et al, 2007). The protocol used to isolate the intra-crystalline fraction in our samples follows Hendy et al (2012); all powdered coral samples (both 'naturally aged' and modern) were exposed to 12% NaOCl (50 μL/mg of sample) for 48 h, maximizing removal of the inter-crystalline proteins but minimizing bleach-induced racemization.…”