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

Abstract

High-temperature isothermal heating of biominerals has commonly been used to artificially accelerate protein degradation in order to extrapolate kinetic parameters to the lower temperatures experienced in vivo and in the burial environment. It is not easy to test the accuracy of these simulations due to the difficulty of finding samples of known age held at a known temperature. We compare protein degradation in the intra-crystalline organic matrix of heated (80 °C, 110 °C, and 140 °C) massive Porites sp. coral to that directly measured in the skeleton of colonies growing at ∼26 °C and deposited over the last five centuries. This provides the opportunity to critically evaluate the underlying assumption that high-temperature experiments accurately mimic degradation processes and kinetics occurring in a 'naturally aged' biomineral. In all samples the intra-crystalline protein fraction was isolated and the L-and D-concentration of multiple amino acids measured using reverse-phase high-performance liquid chromatography (RP-HPLC). There was no evidence of a failure of the closed system in the high-temperature experiments (assessed by X-ray diffraction, thermogravimetric analyses and determination of leached amino acid concentration). We compared conventional methods for estimation of kinetic parameters with a new 'model-free' approach that makes no assumptions regarding the underlying kinetics of the system and uses numerical optimisation to estimate relative rate differences. The 'model-free' approach generally produced more reliable estimates of the observed rates of racemization in 'naturally aged' coral, although rates of hydrolysis (as estimated from the release of free amino acids) were usually over-estimated. In the amino acids for which we were able to examine both racemization and hydrolysis (aspartic acid/asparagine, glutamic acid/glutamine and alanine), it was clear that hydrolysis was less temperature sensitive than racemization, which may account for the differences in degradation patterns observed between the 'naturally aged' coral and high-temperature data. It is clearly important to estimate the individual temperature dependence of each of the parallel reactions. Highlights► We compare protein degradation in heated modern, and naturally aged Porites coral. ► Heating to 140 °C does not compromise the integrity of the closed-system. ► We use a new approach that fits relative rates to estimate temperature dependence. ► Protein degradation patterns change over the temperatures investigated (26-140 °C). ► We observe different temperature sensitivities for hydrolysis and racemization.

An aminostratigraphy for the British Quaternary based on Bithynia opercula

“…Many attempts have been made to refine the temporal resolution and generate numerical dates from kinetic experimental data (see Clarke and Murray-Wallace (2006) for a review), but recent studies have shown that high temperature kinetic experiments may not accurately mimic low temperature protein diagenesis in some biominerals (Demarchi et al., 2012; Tomiak et al., 2012). It has even been proposed that temporal resolution at the level of isotopic substages can be achieved in the Middle Pleistocene using amino acid data (Westaway, 2009, 2010).…”

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

“…A similar pattern has been observed in a range of biominerals that retain a closed system of proteins (e.g. Miller et al., 1992; Crisp et al., 2013; Tomiak et al., 2013). Due to the progressive slowing of the apparent rates of hydrolysis, a logarithmic model is not appropriate to mimic the patterns of “late” diagenesis (Fig.…”

“…However, our study on bleached Patella and recent work carried out on the closed-system (bleached powders) proteins isolated from ostrich eggshell (Crisp et al., 2013), Porites corals (Tomiak et al., 2013) and other marine molluscs (NEaar laboratory, unpublished data) has highlighted that this divergence between low- and high-temperature data is a phenomenon that can be observed across a range of biominerals.…”

“…We stress that whilst our model-free approach generally is useful for estimating rates at burial temperature (see also Tomiak et al., 2013), it cannot be used to predict low-temperature rates of reaction. Therefore, it is important that the validity of high-temperature experiments over a range of temperatures be tested by checking their ability to mimic natural diagenesis; for example plots of % FAA versus D / L , of the extent of racemisation in both the THAA and FAA fractions, as well as the extent of decomposition, are all valuable indicators that can be used simultaneously to check the patterns of protein breakdown within the closed system.…”

“…This can be explained in terms of different relative rates of hydrolysis and racemisation at high and low temperatures, which reflects the complexity of the temperature dependence of each reaction. This pattern has also been found in other bleached biocarbonates (Crisp et al., 2013; Tomiak et al., 2013). It is possible that our observations may be limited to bleached carbonates heated in water, because high-temperature studies on unbleached substrates of different biominerals (e.g.…”