Effects of hydrolysis on the δ13C values of individual amino acids derived from polypeptides and proteins

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Abstract
This study investigates the effects of hydrolysis on the δ13C values of individual amino acids (IAAs) derived from polypeptide standards, and modern and ancient bone collagen. All IAAs were derivatised to their trifluoroacetyl/isopropyl (TFA/IP) esters for δ13C determination using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). Firstly, authentic single poly amino acid standards (SPAAs; n = 5) were hydrolysed for 4, 10, 24 and 48 h. As expected, IAA yields increased as a function of hydrolysis time. Significantly, it was only after 24 h of hydrolysis that IAA δ13C values were statistically identical to bulk SPAA values for all five standards. The accuracy of IAA δ13C values was thus shown to be a function of yield; however, poly phenylalanine demonstrated accurate IAA δ13C values with yields of only 1.4 and 4.3%, after 24 and 48 h of hydrolysis time, respectively. Authentic mixed poly amino acid standards (MPAAs; n = 5) comprising two different amino acids were then hydrolysed for 24 h. Percentage recoveries ranged from 36–95%. Estimates of bulk MPAA δ13C values calculated from measured IAA δ13C values agreed within experimental error with measured bulk MPAA values for three out of the five standards. Finally, the experimental procedure was applied to modern rat (MBCs; n = 20) and ancient ovi-caprine and bovine (ABCs; n = 27) bone collagen samples where the δ13C values of 12 out of its 18 constituent amino acids were determined. Estimated bulk MBC and ABC δ13C values were calculated from constituent amino acid δ13C values using mass balance. With the exclusion of three ABC samples, calculated bulk bone collagen δ13C values (δ13CBCcal) were shown to correlate extremely well with measured bone collagen values (δ13CBCmes) for both modern and ancient samples, where R2 = 0.91 and 0.84, respectively. Significantly, the variation between calculated and measured bone collagen values (Δ13CBCcal-BCmes) exhibited similar ranges for both MBC (from −2.6 to +1.2‰) and ABC (from −2.7 to +2.2‰) samples, providing evidence for the preservation of intact collagen in the ancient samples. These results demonstrate that the experimental procedures employed in the acid hydrolytic cleavage of peptides or proteins to their constituent amino acids does not involve significant isotopic fractionation. Copyright © 2003 John Wiley & Sons, Ltd.
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