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Proc. Nat. Acad. Si. USA Vol. 70, No. 5, pp. 1331-1334, May 1973 Racemization Reaction of Aspartic Acid and Its Use in Dating Fossil Bones (Olduvai Gorge/5,00-70,000-years-old range/hominids) JEFFREY L. BADA* AND REINER PROTSCHt * Scripps Institution of Oceanography and Institute of Marine Resources, University of California, San Diego, La Jolla, Calif. 92037; and t Department of Anthropology, Institute of Geophysics and Planetary Physics, University of California, Los Angeles, Los Angeles, Calif. 90024 Communicated by William A. Nierenberg, March 6, 1973 ABSTRACT In the time interval datable by radiocar- peratures might be estimated from 180/160 ratios in stalag- bon, and at the temperatures of most archeological sites, mites and stalactites. a substantial amount of racemization of aspartic acid from modern bone fragments takes place. By determination of the amount of racemiza- Preliminary results obtained tion of aspartic acid in bones from a particular location heated at elevated temperatures show that amino acids such which have been dated by the radiocarbon technique, it is as aspartic acid and alanine are racemized substantially faster possible to calculate the in situ first-order rate constant than is isoleucine (Bada, J. L., Kvenvolden, K. A. and Peter- for interconversion of the L- and D enantiomers of aspartic son, E., in preparation). These results indicate the following acid. Once this "calibration" has been calculated, the re- action can be used to date other bones from the deposit sequence of racemization rates: aspartic acid > alanine_ that are either too old to be dated by radiocarbon or that glutamic acid > isoleucine _ leucine. This pattern was also are too small for radiocarbon dating. The only assump- found in several fossil bones. tion required with this approach is that the average tem- Of particular interest is the racemization reaction of aspartic perature experienced by the "calibration" sample is repre- as sentative of the average temperature experienced by older acid; this reaction can be written samples. This "calibration" technique is used herein to date bones from the Olduvai Gorge area in Tanzania, kap Africa. L-aspartic acid -I D-aspartic acid, [1] Only L-amino acids are commonly found in living organisms. However, recent studies have shown the occurrence of D where kasp is the first-order rate constant for interconversion of isomers in fossil materials. The amino acids in fossil shells the L and D enantiomers of aspartic acid. In bone, this re- (1-3), sediments (4-6), and bones (7, 8) are partially race- action has a half-life (i.e., the time required for the ratio of mized, with the amount of racemization increasing with the D- to iaspartic acid to reach 0.333) at 200 of about 15,000- age of the fossil. This racemization reaction has obvious ap- 20,000 years (Bada, J. L., Kvenvolden, K. A. and Peterson, plications in geochronology, and it appears that the reaction E., in preparation). Thus, in the time interval datable by can be used to date deep-sea sediments (4-6) and fossil bones radiocarbon, a substantial amount of racemization of aspartic (7, 8) found in certain environments. acid will take place. This finding suggests that by determining Most studies have concentrated on isoleucine. The epimeri- the extent of racemization of aspartic acid in bones from a zation of L-isoleucine produces the nonprotein amino-acid D- particular site which have been dated by the radiocarbon tech- alloisoleucine. (For the reaction involving isoleucine, the pro- nique, it should be possible for one to calculate the in situ kep cess is more properly termed epimerization rather than racemi- value for the deposit. Once this "calibration" has been done, zation.) rrsoleucine and D-alloisoleucine are directly separable the aspartic-acid racemization reaction can be used to date on an automatic amino-acid analyzer. In contrast, in order to other samples from the area that are either too old to be dated determine the amount of racemization of other amino acids, a by radiocarbon (i.e., older than 45,000 years), or for which in- suitable diastereomeric derivative must first be synthesized. sufficient amounts are available for radiocarbon dating (only a The isoleucine epimerization reaction in bone has a half-life few grams are needed for amino-acid dating, compared to hun- (i.e., the time required for the ratio of alloisoleucine to isoleu- dreds of grams needed for radiocarbon dating). This "calibra- cine to reach 0.345) at 200 in excess of 100,000 years (8), and tion" procedure eliminates the need for evaluating the tem- evidence suggests that the reaction can be used to date fossil perature history of a bone before it can be dated by the amino- bones too old to be datable by radiocarbon. The rate of the acid racemization reaction. In this paper, we report how this amino-acid racemization reaction is dependent upon tempera- approach was used to date fossil bones from the Olduvai ture. Therefore, in order to use the reaction to calculate ages Gorge area in Tanzania, Africa. which are reasonably accurate, some estimate of the tempera- ture history of a fossil bone must be available. Due to the prob- MATERIALS AND METHODS lem of temperature uncertainties, the isoleucine epimerization The bones from the Naisiusiu Beds were sent to us by M. D. reaction can apparently only be used to date fossil bones found Leakey, Centre for Prehistory and Paleontology of the Na- in certain environments (7, 8) such as caves where paleotem- tional Museum, Nairobi, Kenya. The actual location in Olduvai Gorge where the bones were collected is described * To whom reprint requests should be addressed. elsewhere (9). About 10 g of bone were used for the amino- 1331 Downloaded by guest on September 27, 2021 1332 Anthropology: Bada and Protsch Proc. Nat. Acad. Sci. USA 70 (1978) Modern Bovine Bone Fragment the residue was dissolved in double-distilled water and de- salted on unused Dowex 50W-X8 (100-200 mesh) resin, which had been cleaned with NaOH and regenerated with doubly- distilled HCl. The amino acids were eluted from the resin with D/L = 0.07 1.5 M NN40H prepared by dissolving gas into double- Ndutu Bed NH3 Sample distilled water. The effluent was concentrated in a rotary evaporator at 530 under reduced pressure. D/L = 0.72 The ratio of the D- to L- enantiomers of aspartic acid was determined by preparing diastereomeric dipeptides using the procedure described by Manning and Moore (11). Before the j lA dipeptide synthesis, the aspartic acid was separated from the DL-aspartic total amino-acid mixture by ion-exchange chromatography UCLA 1695 acid (12) on Dowex 5OW-X8, using 1.5 M HCl to elute the as- acid. This was D/L = 0.32 L-Leu- L-Leu- partic separation step necessary because the D-Asp L-Asp dipeptide synthesis of the total amino-acid mixture isolated from the bone resulted in a large number of peaks, many of which overlapped one another. After the ion-exchange separa- tion, the isolated aspartic acid was taken up in borate buffer Buf fer (pH = 10.4), the solution was cooled to 2-4o, and a small amount of L-leucine-N-carboxyanhydride (obtained from FIG. 1. Part of the amino-acid analyzer printouts for the Cyclo Chemical Co.) was added to the solution in order to various bone samples showing the diastereomeric dipeptides L- synthesize Jleucyl-D-aspartic acid and L-leucyl-L-aspartic leucyl-D-aspartic acid and L-leucyl-L-aspartic acid. A 56 X 0.9-cm acid. A Beckman-Spinco model 118 automatic amino-acid column filled with Beckman-Spinco AA-15 resin was used for analyzer was used to separate the diastereomeric dipeptides. the separation. The column was eluted with pH 3.24 buffer for The radiocarbon age of the Naisiusiu Bed bone was deter- 40 min, then the buffer was switched to pH 4.25 for the remainder mined at the Univ. of California, Los Angeles Radiocarbon of the run. The buffer change, L-Leu-D-Asp, and L-Leu-L-Asp Laboratory by use of the method described by Protsch (13). peaks come at 72, 79, and 83 min, respectively, with this elution sequence. The small peak that elutes between the buffer peak and RESULTS L-Leu-D-Asp is glycine that was not completely separated from The Olduvai Gorge sequence consists of several stratigraphic aspartic acid on the Dowex 50 (H +) column. sections, the oldest of which has an age of several million years. The uppermost part of the Gorge is upper Pleistocene this acid analysis; 412 g were used for the radiocarbon age deter- (14, 15); stratigraphic section is subdivided into a younger mination. formation, the Naisiusiu Beds, and an older formation, the The Ndutu Bed bone was provided by R. L. Hay, Univer- Ndutu Beds (9). Bones from the middle section of the Nai- siusiu Beds were sity of California, Berkeley. The sample was from a site used to "calibrate" the aspartic-acid race- located at the rim of the Gorge, between the Main and Side mization reaction for Olduvai Gorge. Radiocarbon analysis of Gorges and 150 m south of archeological site FLK-NNI and the collagen fraction isolated from the bones yielded an age of 70 m west of the recently excavated site in the Masek Beds. of 17,550 i 1,000 yr (UCLA-1695). This age agrees closely with the of At the sampled locality, the Ndutu Beds comprise 1.5 m of age (9) 17,000 yr (Lamont-no.?) determined on horizontal unstratified zeolitic eolian tuff, the lithology and ostrich eggshell found in the same stratigraphic horizon and in close to the mineral content of which indicate assignment to the upper unit proximity bones.

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