Carbon and nitrogen isotopic fractionation in bone collagen during chemical treatment

Abstract

In measuring radiocarbon ages of fossil bone samples, it is necessary to extract pure bone protein from the samples by chemical treatment. To evaluate the effect of the chemical treatment including XAD-2 chromatography on carbon and nitrogen isotopic values, we measured C / N ratio, δ 13C and δ 15N in decalcified fraction, gelatin, hydrolysate, and XAD-treated hydrolysate extracted from fossil bones together with modern samples. The C / N ratios of the collagen fractions progressively decreased as chemical treatment proceeded. The δ 13C values of the fractions became more positive as purification proceeded. There was an overall + 0.5∼+ 2.0‰ difference in δ 13C and a slight trend of decrease in δ 15N during chemical processing and around + 0.3‰ difference in δ 13C and + 0.2‰ in δ 15N during XAD-2 treatment only. The large change in C / N ratio and δ 13C of the hydrolysates following XAD-2 chromatography in the Bovine Achilles tendon collagen standard could be explained by the removal of lipids unextracted before the XAD-2 treatment. There was no difference in δ 13C between hydrolysates and XAD-treated hydrolysates in fossil bones because the latter contains negligible preserved lipids. Fossil bones and lipid-extracted collagen standard showed the similar C / N change and isotopic fractionation during sequential chemical treatment. Individual amino acid standards showed little to no increase in δ 13C and δ 15N values during XAD-2 treatment, except for aspartic acid and glutamic acid, which showed pronounced increase in δ 15N values. Furthermore, regarding amino acid compositions separated by XAD-2 treatment, alanine and glycine tend to be enriched, while valine, threonine, isoleucine, leucine, and serine compositions tend to be depleted. The carbon and nitrogen isotopic fractionation during sequential chemical treatment might reflect variation in the amino acid composition of the extracted fractions due to degradation, such as decarboxylation, rather than removal of contaminants. The variation during XAD-2 treatment is due to both degradation by HCl and isotopic fractionation related to the XAD-2 resin.