Towards an Optimal Method of Archaeological

,h""'lrlll,,II-7!/II of cillcium. Shards were demin 35 kJ mol I: Zhang and Nancollas, 1990) than transport-controlled dissolution. It should be noted that the dissolution mechanisms for apatite are complex and are believed to include both diffusion and surface processes (e.g. Margolish and Moreno, 1992). Putnis et {If. (1995) investigating the temperature dependence of the dissolution of barite by diethylenetriaminepentaacetic acid (DTPA), observed higher actiVation energies than we have observed with EDTA (40-5S kJ mol f). Although dissolution of bone mineral by EDTA (pH 8.0) is much slower than in acid dissolution and the reaction more temperature-dependent, the esti~ mated activation energies, E" (6-26 kJ mol 1) are in the range expected of a diffusion controlled reaction (Table JI). Putnis cI (/1. (1995) noted that dissolution of barite was more rapid when the concentrations of OTPA were reduced. This WaS atlributed to the lower rate of removal of the organa-metal complex from the surface when it is present at higher concentrations, thereby passivating the sltrface. We investigated this for EDTA, but although the rates were proportionally lower than the increase in concentration, dissolution was more rapid in the more concentrated EDTA solutions.

BONE COLLAGEN PREPARATION

The failure to observe a similar effect nlay be due to the difference in geometry of the surface bound OTPA and EOTA ligands but is more probably simply due to the much greater surface area of the bioapatite. The much slower EOTA demineralization of more coarsely crystalline archaeological bone powders and the elevated activation energies of the archaeological powders and the modern shards (compared with powders) indicates that these samples had lower available surface areas and the dissolution was more strongly influenced by surface processes. There is evidence that the efficiency of concentrated solutions as solvents diminished as the apatite becomes more crystalline. Archaeological bone powders and modern shards had similar activation energies but different concentration dependence on the rate of reaction ~ the former only doubling in rate vvith a tenfold increase in concentration, whereas the latter sustained a fourfold increase in rate (Table II). This observation is consistent with the OTPA passivation observed by Putnis f'f al. (995). The most striking feature of EOTA dissolution was its slowness relative to acid dissolution, a featme of both the mechanism of dissolution (EOTA requiring the formation of a surface complex) and the rate of diffusion through the bone composite of the respective ions (acid demineralization) and metal chelates CEOTA demineralization). This slow rate of EDTA demineralization means that it is necessary to powder samples (to counteract the slow rate of diffusion into the mineral matrix). Add demineralization is much more rapid. The very low E., of mineral add dissolution experiments are lower than studies of organic add dissolution of powdered enamel apatite (e.g. Nelson ct al., 1983) and indicate that the reaction is diffusion controlled. An increase in temperature will not significantly increase the rate of what is already a fast reaction, an increase from 4~C to 37·C will only increase the rate of reaction by 23%. Although powders do demineralize more rapidly, the short deminerali-

215

zation time of both preparations offsets the need for pulverization. Furthermore, because the low pH will cause proLeins to precipitate (Linde et ai., 1980), there is no need to add enzyme inhibitors. Archaeological powders and shards demineralize more rapidly than their modern counterparts, despite the fact that the bones have higher crystallinity. It would appear that the lack of macro porosity (again lowest in modern bone) and the presence of the organic matrix (e.g. Nelson et ai., 1983), combine to slow the rate of diffusion of product ions out of the bone, proteins coating the mineral will also reduce the available surface area for reaction (Voegel ct af., 1981). Whilst bone shards are sufficiently small for the rate of dissolution to remain relatively unaffected by diffusion limitation, this does become a problem when larger samples are used. Under identical conditions a whole cow tibia was not fully demineralized after tw"o days at 4"C (results not shown). The kinetic data itself should be treated with some caution, as the rates are for dissolution of bone mineral, a protein/mineral composite. In this material the internal porosity is too small to contribute to a difhlsion controlled reaction as estimated internal pore sizes (c. Nielsen-Marsh, personal communication) are much smaller than the typical Nernst layer thickness for poorly stirred solutions (1Q...-2cm: Helfferich, 1962). The surface area of the Nernst layer for a settled powder is small but will be significantly increased if the sample is agitated and the pellet disaggregated. A form of agitation is therefore strongly recommended even if it is simply a rocking table, as used in this study. This increase in the surface area of the Nernst diffusion layer caused by gentle agitation and hence mixing of particles should not be confused with accelerated rates of diffusion into the bulk solution caused by a decrease in thickness of the layer itself which to have a significant effect requires very rapid hydrodynamic flow (e.g. Margolish and Moreno, 1992).

:-"1./. COLLINS A0JD P. GALLEY

216

Acid Hydrolysis of Peptide Bonds

The principal reason for avoiding the use of HCi is thi1t the low pH is detrimental to the protection of proteins and other biopolymers; aspari1gine and glutamine deamidation, -r-carboxyglutamic acid decarboxylation and peptide bond hydrolysis are all sensitive to low pH. The aim of any sample preparation must be to minimize the damage caused by extraction, in the case of collagen extraction, this means preventing gelatinization (i.e. peptide bond hydrolysis) during demineralization. The role played by acid in the cleavage of peptide bonds was an area of intense investigation in the early 1960s, \vhen early sequencing of internal regions of proteins \vas aided by selective chemical cleavage (e.g. Hill, 1965). Surprisingly fe\'\' studies have specifically addressed the temperature dependence of hydrolysis, but investigations all report similar acti\~ation energies, the activation energy does not appear to be pH dependent, and ranges from 83-99 kJ mol I (Table Ill). From these data, the effect of different incubation temperatures on the rate of lwdrolysis of collagen can be estimated (Table Fig. 7). Approximately ten times more peptide bonds are cle8ved at room temperature (20 C) than at 4~C, and more than 40 times more at 3TC (note that DNA depurinMion is even more temperature dependent than peptide bond hydrolysis: Table III). This enhanced rate of hydrolysis leads to the almost complete gelatini-

III;

T,\BLE III

zation of collagen at 37 C after 100 h (Fig. 5). At low pH collagen swells, becoming less hydrophobic and increasing in solubility. ThiS. is another process to be avoided, but short detn111eralization steps at low temperipf-i for s 3, an altern 2 mm) shards. This effect is both dramatic and significant; much more damage is being done to the collagen by grinding than by prolonged exposure to 0.5 M HCl (the decline from the original O.6M is due to the dissolution of the apatite). Methods which attempt to optimize collagen yields but which powder the samples (e.g. Serna! and Orban, 1995)

o

20% _v-v\?

,if3-,,-

..

Q.'

10%

'i1 'V~ ~~2mm, 710-63pm and 2mm). Samples of powder and shards were stored at -7Q"'C prior to use. Comparison was made with chemically precipitated apatite (Biorad BiogeD \-vhich has a mesh size of 8-180 ~Lm. Bone mineral has a very high surface area (200m 2 g I), and therefore the chemically precipitated apatite was ground and

sieved to < 63 pm, and subsequently acid etched. The surface area of the treated Biogel apatite was subsequently estimated by nitrogen BET isotherm at 50 m l g I, still only 25% of that of bone mineral. Archaeological Bone A sample of cattle metapodia from \vaterlogged burial conditions at the Roman fort in Carlisle was used to compare with modern bone. Collagen yields ranged from 12% to 16'7, of modern bone. The samples, hereafter called 'archaeological bone', were in all respects treated in the same way as modern bone. Hydroxyproline Assay The proportion of collilgen in the soluble and insolublephases w(lsdetermined usinga hydroxyproline (Hyp) assay. Hydroxyproline represents 12.57% by weight of the total amino acid content of collagen but is very uncommon or absent in the non-collagenous protein (NCP) fraction; small amoLlnts of Hyp which helve been reported in the NCP fraction are probably mainly derived from soluble collagen. Thus we have used the presence of Hyp in the soluble fraction to determine if collagen has been depolymerized by the extraction method. A similar approach is less suitable for archaeological samples as Hyp is common in a variety of plant proteins and glycoproteins (Levy and Staehelin, 1992). The Hyp assay was based upon the method of Huszar et Ill. (1980). Briefly this method involves hydrolysis at 120 C in 4 M NaOH, followed by oxidation of the free hydroxyproline using chloramine T (sodium N-chloro-p-to]uenesulfonamide), and its subsequent condensation with Ehrlich's reagent (p-dimethylaminobenzaldehyde) to form a coloured product. The blood red colour was fully developed at room temperature ~. Cllrr. 0l'iu. Cdl Bini. 5, R56-R62.

Lind,lh!, T. and Nyberg, B. (1972) Rate of depurinMion of n~tive dcoxyribonuclcic Jeid. Biorlielilis/I'Y 11, 3610-3618. Lindo:, A., Shown,]I.j. ntin utilizing techni'!ut>s to avoiu artifilct~.I. Bio/. Cla'IlI. 255, 5931-5942.

Longin, R. (1971) New method 01 coHagen extraction (Dr r.l(liocarboll dilting. Nlllllre 230, 2,11-242. M using HPLC techniqUl' to determine the degrt>e of AAKI. FmCIIS. Sci. 39, 1'12..'i-1431. Nelson, D.G-A., Feather"tune, ).D.B., DUllcan.. l.F. ~nd ClItre;;!', T.W. (1983) Effegrad,ltion. 4. PnthW,lYS, b.int>tics ,md mech,mi~m of degril(t1tion of an ,lbpartyl rt>sidue in a model hexapt'ptid .... I'lmmmcvL Re~. 10, 95-102. Putnis, A., Putnj~, CY. and ('llt ill the dis~o\lltion of barium sulphate sCilll1 deposib. Proc. Sf'L lill. 5.111111" Oilfidd Ci1em. !"Iper SPE ()2909
Qian, Y.R., Engel, M.H., Macko, 5.A., Carpentl'r, S. and Deming, I.W. (1993) Kinetic" of peptide hydroly~is ,lI\d Jmilllhlcid dt>compobitioll at high-l ... mperilturc. Geochilil. C(hIIlDChilll. Adil 57, 3281-3293. Ritz,S, Schiitz, IJ.-W. ,md Peper, C. (19