Amino Acid Analysis in Radiocarbon Dating of Bone Collagen

7 Amino Acid Analysis in Radiocarbon Dating of Bone Collagen AFIFA A. HASSAN

Downloaded by CORNELL UNIV on September 2, 2016 | http://pubs.acs.org Publication Date: June 1, 1978 | doi: 10.1021/ba-1978-0171.ch007

Department of Geology, Washington State University, Pullman, WA 99164 P. E. HARE Geophysical Laboratory, Carnegie Institution of Washington, Washington, DC 20008

Erroneous radiocarbon dates on bone collagen may result from impurities remaining after sample processing. Amino acid analysis and a determination of the nitrogen/carbon ratio can quickly test the purity of the extracted collagen to indicate if further purification is required. Collagen is separated from bone by dialysis in dilute HCl and in distilled water. The collagen is then converted to gelatin. The results indicate the presence of non-collagen organic materials in fossil and in modern bones. When these materials are indigenous to the bone and not contaminants, they do not affect the radiocarbon dates. The amino acid composition of "collagen" residue from some fossil bones indicates that the residue is not collagen. Radiocarbon dates on non-collagen material extracted from fossil bone should be interpreted with caution.

" D one tissue is composed of inorganic and organic material. The main constituent of the organic fraction is the protein collagen, and the inorganic fraction is composed of the bone mineral apatite. Collagen, i n contrast with the bone mineral, does not exchange carbon with the bone diagenetic environment. Thus, collagen is a suitable dating material if it is uncontaminated. M a n y investigators have devised methods to separate collagen (1-11). I n most of these methods, however, the purity of the separated collagen is questionable. The method of selective liquid chromatography of amino acids (2,9) seems to eliminate the problem of 0-8412-0397-0/78/33-171-109$05.00/l © 1978 American Chemical Society Carter; Archaeological Chemistry—II Advances in Chemistry; American Chemical Society: Washington, DC, 1978.


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impurities; however, the separated amino acids could be from other sources besides collagen. It would be safe to date the amino acids that exist only i n collagen, but that is not practical since it requires an enormous amount of bone sample. Also the amount of collagen left after sample preparation is often insufficient for radiocarbon dating because of the breakdown and loss of collagen as HCl-soluble material. If the HCl-soluble fraction of collagen could be recovered, it would be easier to obtain sufficient quantity for radiocarbon dating. Dialysis of bone i n dilute HC1 retains the HCl-soluble fraction of collagen inside the dialysis tube, thus improving the collagen yield (11). Conversion to gelatin (8) removes the water-insoluble non-collagenous material, but it is still necessary to check the separated collagen for the presence of water-soluble non-collagenous material of high molecular weight ( I I ) . In this study, the modern and fossil bone collagen separated by dialysis is analyzed quantitatively for amino acids and the nitrogen/carbon ratio to determine if the separated material is pure collagen or if it contains impurities which might affect radiocarbon dating. The fossil bone specimens with associated ages are: Bison antiquus (10,000-11,000 B. P.) Folsom site, N M ; Mammtithus columb. femur and rib (mammoth III) (11,170 ± 360 B.P.) Blackwater Draw, Clovis level, N M ; Mammuthus imperator (11,045 ± 647 B.P.) Domebo site, Clovis level, O K ; Mammut americanum (16,540 =b 170) Boney Springs, M O ; Mammut americanum (34,300 db 1,200 B.P.) Trolinger bog, M O ; and whale bone ( ~ 100,000 B.P.) Baffin Island, Canada, Early Wisconsin. Fossil bones and their expected ages, except the whale bone, were obtained from C . Vance Haynes, Jr. of the University of Arizona at Tucson. The whale bone was obtained from Gifford Miller of the University of Colorado. The modern bones belong to an elephant that died i n 1964 and was buried for six years prior to re-excavation. The elephant bones were obtained from Charles M c N u l t y of the University of Texas at Arlington. Experimental Nitrogen content of the different bone samples was determined by a carbon-nitrogen-hydrogen analyzer (Hewlett-Packard model 185). N i trogen percentages which reflect the amino acid content of the samples are used to calculate the weight of the different samples required to obtain a sufficient amount of collagen. The collagen is separated by a method developed by Hassan ( I I ) i n which the crushed, sized, and calcite-free bone samples are placed i n dialysis tubes of 12,000 m o l wt, cut off, and filled with distilled water. The dialysis tubes, with their top end untied to permit the generated C 0 to escape, are placed i n long cylinders filled with 0.3N HC1. The H C 1 solution i n the cylinders is changed frequently until the bone apatite is dissolved, after which the HC1 is replaced with distilled water. The distilled water is changed fre2

Carter; Archaeological Chemistry—II Advances in Chemistry; American Chemical Society: Washington, DC, 1978.

7.

HASSAN AND

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quently until all the C a from the apatite is removed from the dialysis tube. The high-molecular-weight fractions of collagen (HCl-insoluble and HCl-soluble) are retained. Small organic molecules also are removed from the dialysis tube while higher-molecular-weight materials such as humates, proteins, and some polysaccharides are retained. The two fractions, HCl-soluble and HCl-insoluble, are separated by centrifugation, are converted separately to gelatin (100°C, p H 4, 2 h r ) , and are filtered through 0.i20-/x cellulose filter paper. Conversion to gelatin and filtration removes solid contaminants such as water-insoluble humates. After evaporation to dryness in a vacuum desiccator over N a O H the gelatin samples are weighed and the nitrogen and carbon content of the gelatin determined. A n aliquot of the gelatin sample is hydrolyzed to free amino acids [6N H C 1 for 15 min at 155°C (12)]. Standard amino acid mixtures also are carried through the same procedure. A sensitive amino acid analyzer based on the ion-exchange method of Spackman, Moore, and Stein (13,14) was used to determine quantitatively the amounts of amino acids. The system is highly stable, sensitive, and capable of 1% reproducibility at the nanomole level on consecutive runs (12). The basic amino acids can be analyzed separately if desired on a single ion-exchange column using p H 4.4 citrate buffer at 50°C.


2 +

Table I.

Collagen, Expressed as Weight % of Dry Bone and Its Nitrogen/Carbon Ratio 0

% Collagen

Nitrogen/Carbon

Bone Sample

HClinsol.

HClsol.

HClinsol.

HClsol.

Modern elephant Folsom bison Blackwater D r a w mammoth Blackwater D r a w mammoth III Domebo mammoth Boney Springs mastodon Trolinger bog mastadon Whale bone

27.0 4.5 1.9 0.1 1.7 0.2 0.6 22.5

2.7 2.4 0.5 0.4 2.6 0.2 1.7 5.0

0.37 0.35 0.05 0.13 0.34 0.13 0.29 0.37

0.34 0.36 0.11 0.10 0.17 0.06 0.22 0.36

a

Collagen is separated from bone by dialysis in HC1 then is converted to gelatine.

Results The weight of the separated collagen and its nitrogen/carbon ratios are recorded i n Table I. These results indicate an increase i n the percentage of the HCl-soluble fraction of collagen with fossilization. The results also indicate lower values of nitrogen/carbon ratios i n the H C l soluble fraction of collagen compared with those of the HCl-insoluble fraction. The nitrogen/carbon ratios are also lower i n the fossil collagen compared with those of the modern collagen. Table I shows that the

Carter; Archaeological Chemistry—II Advances in Chemistry; American Chemical Society: Washington, DC, 1978.

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nitrogen/carbon ratio of the separated collagen generally is proportional to the percentage of the collagen i n the bone samples; however, there are exceptions. F o r example the amount of the HCl-insoluble "collagen" separated from the Blackwater D r a w mammoth bone sample is more than expected based on the nitrogen/carbon ratio which may indicate the presence of non-collagen contaminants. This sample was covered i n the field with Alvar preservative which was scraped off during sample pretreatment. BASIC


LYS HIS

ACIDIC

NEUTRAL

O H a IMIN0

ARG ASP GLU THR SER

oFP PRO PRO

SULFUR AROMATIC

GLY ALA VAL ISO LEUCYS MET TYR PHE

400 -

-400

380 •

-380

360

;

-360

1

340-

-340

Amino Acid Analysis in Radiocarbon Dating of Bone Collagen

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