Extraction, analysis and interpretation of intracrystalline amino acids from fossils DEREK WALTON AND GORDON B. CURRY

LETHAIA Walton, D. & Cuny, G.B. 1994 07 15: Extraction, analysis and interpretation of intracrystalline amino acids from fossils. Lethaia, Vol. 27, pp. 179-184. Oslo. lSSN 0024-1 164.

A new protocol for the extraction and analysis of intracrystalline macromolecules has been developed that allows the rapid determination of the amino-acid composition of fossils. The technique utilizes decalcification with 2 M HCI, characterization of the soluble fraction of the biomolecules by automated amino-acid analysis, and differentiation using multivariate statistics. Compared to other methods, this technique allows sampling of indigenous degraded proteins in addition to the preserved remains of peptides, leading to the recovery of data from more reliable indigenous sources. Although the extraction method is demonstrated using fossil samples to demonstrate gross phylogenetic differences, there is much potential to use these biomolecules for a wide range of applications. OAmino acids, brachiopods, fossil biomolecules, moleculnr phylogeny.

Derek Walton, Departmentofhrth Sciences, UniversityofDwby, Kedleston Road Derby, DE22 IGB, UK; Gordon B. CUT, Department of Geology and Applied Geology, University of Glasgow, Lilybank Gardens, Glasgow, GI2 SQQ, UK; 10th March, 1993.

The presence of amino acids (the building blocks of pro- teins) in the skeletons of living and fossil organisms is well documented (e.g., Abelson 1955; and reviews in Wyckoff 1972 and Curry 1988). Such biomolecules occur in two locations in shells: either between individual shell crystallites (referred to as intercrystalline biomolecules), or entombed within the inorganic phase (intracrystalline biomolecules). Although initially the most abundant, intercrystalline bio- molecules decay rapidly and are prone to contamination. lntracrystalline biomolecules have the advantage of being preserved in a closed system where micro-organisms are excluded and, although the biomolecules may be physically and chemically degraded, any degradation products re- mained trapped until the inorganic phase is destroyed.

Because ofthe preparation techniques adopted, the amino acids reported from fossil shells have generally been recov- ered from both intracrystalline and intercrystalline sites (e.g., lope 1967a, b; Degens et al. 1967; Haugen et al. 1989). However, biomolecules such as proteins are inherently un- stable in the presence of water and may be degraded and leached from intracrystalline sites in the fossil with the loss of any preserved taxonomic information. Towe (1980) dem- onstrated that perfect physical preservation is not necessarily indicative of good biochemical preservation and concluded that intracrystalline biomolecules would provide a more reliable source of genuinely indigenous biomolecules from fossil shells. In an experimental study of a brachiopod death assemblage (Collins 1986), the shells of the Recent brachio- pod Terebratulina retusa rapidly softened (< 1 year), and the secondary shell fibres began to behave as single units, rather

than as parts of a coherent structure. Such a phenomenon reflects the rapid breakdown ofthe intercrystalline proteina- ceous sheath, which surrounds the shell crystallites during the life of the organism (Williams 1968) and which is the primary source of intercrystalline amino acids.

Following the conclusion of Towe (1980), Collins et al. (1988) demonstrated the presence, by immunology, of bio- molecules within the shells of Recent brachiopods. These biomolecules exist within their own protected microenvi- ronments within the shell and are analogous to fluid inclu- sions found in minerals (Curry 1988).

Data interpretation of the amino acids extracted from fossil samples becomes increasingly complex as the number of samples to be compared increases. Several studies of these fossil biomolecules have utilized molecular percentages (e.g., Jope 1967b) and ratios between amino acids (Kolesnikov & Prosorovskaya 1986) to establish taxonomic relationships. More complex relationships have been demonstrated by the use of statistical techniques, first applied to amino-acid data by Degens et al. (1967), who used principal-components analysis (PCA) in a phylogenetic study of the biomolecules from Recent molluscs. However, data were expressed only as factor scores, making interpretation of the results more difficult. Graphic presentation of Q-mode factor analysis was used by King & Hare (1972) to establish taxonomic similarities between the biomolecules in the tests of Recent and fossil planktic foraminifera.

The aim of this paper is to describe new techniques for the extraction, analysis and interpretation of the information provided by the intracrystalline biomolecules from within

180 Derek Walton e5 Gordon B. Curry LETHAIA 27 (1994)

fossil shells, and thus to provide a more reliable basis for the various applications of ancient biomolecules, illustrated by an example of basic taxonomy using fossil samples. In this paper we use graphical presentation ofthe principal compo- nents derived from amino-acid data to discriminate between samples.

Rat ionale Amino acids are naturally occurring organic acids that are ubiquitous in living organisms, as the building blocks of proteins. Twenty types of amino acid are commonly found and all species, from bacteria to humans, contain this same set of twenty proteinaceous amino acids. The linkage of amino acids via peptide bonds gives rise to a uniform chain known as the protein backbone, and the order of the amino acids along the protein backbone defines the primary se- quence of the protein. The sequence ofthe bases on the DNA biomolecule determines the sequence of amino acids on the protein, and therefore the information contained in the DNA (the genes) is passed on to the amino acids via the proteins, and hence the relative proportions of amino acids present in the shell reflects underlying variability in the genes coding for the incarcerated proteins.

In this study, the Applied Biosystems 420H amino-acid analyzer was chosen to analyse the biomolecules, as it allows automated hydrolysis and derivatization with high degrees of reproducibility and is able to sample a wide range of amino acids present within the shell in a short time scale. In short, this apparatus had the advantage of reducing sample hand- ling whilst producing rapid, reproducible results.

Potential for contamination There is considerable potential for contamination of fossil samples by non-indigenous amino acids. Contamination can happen at any stage during preparation, by agents rang- ing from microbes and boring organisms living in or on fossil samples (at any time since it was fossilized) to labora- tory dust. To reduce the risk ofsuch contamination produc- ing misleading results, latex laboratory gloves were used in all stages of the preparation. Regular checks were made on the purity of the laboratory water and on solvent prepara- tions, and all samples were analysed several times, with different powders being decalcified each time, to ensure reproducibility.

In an earlier paper to check against contamination of biomolecules from fossil samples, Walton & Curry (1991) demonstrated that amino acids derived from fossils, sedi- ments and fingertips could be easily discriminated by the use of multivariate statistics. This method has served as a useful technique in ensuring the indigeneity of the organic material, as a rapid alternative to isotopic or racemization determinations.

Preparation methods Selection of material

Samples of the best possible physical preservation were se- lected for further study. Samples that were excessively bored or fractured were omitted from further study, as were samples that were obviously recrystallized. Recrystallization from aragonite to low-magnesium calcite may result in some biomolecules being lost or other, non-indigenous, bio- molecules being incorporated.

Cleaning of material

All sediment was scrubbed from the surface of the shells and encrusting epifauna removed by scraping. Articulated shells were disarticulated and the inside surfaces cleaned. Any remaining internal body tissue was removed and the shells soaked in an aqueous solution of bleach (10% v/v) for 2 hours at room temperature, washed (using Milli ROrM) and left to air dry. Shells were ground using a ceramic pestle and mortar and incubated in an aqueous solution of bleach (10% v/v, under constant motion) for 24 hours at room temperature, to digest the intercrystalline biomolecules. The samples were washed using repeated suspension in MilliQTM water and centrifugation, until no bleach could be detected (typically ten washes). Powders were frozen (-20°C) and lyophilized.

Dissolution of inorganic phase

To release the incarcerated biomolecules from within the calcium carbonate, the latter must be dissolved. Various solutions were tested

Ethylene-diamino-tetra-acetic acid, disodium salt (Nu,- EDTA). - Following the methods of Cusack et al. ( 1992), an aqueous solution of Na,-EDTA (20% w/v) was used to dissolve the shell powder at a ratio of 23 ml/g shell. Na,- EDTA interferes with amino-acid analysis (Walton 1992) by coeluting from the analysis system with Glu, Ser, Gly and Pro. In addition, Na,-EDTA is notoriously hard to remove from a solution byeither dialysis (Worms & Weiner 1986) or ultrafiltration (M. Cusack, personal communication,l991). Attempts to use these methods resulted in concentration of the Na,-EDTA/calcium complex and contamination of the sample. However, filtration using the MinitanlM (Millipore) Tangential flow system, using a filter with lOkDa cutoff, successfully removed all of the EDTA from the solution (Cusack et al. 1992), although all free amino acids and peptides smaller than ca. 10 kDa were lost from the filtrate during this process. These smaller biomolecules constitute a large proportion ofthe total amino acids present within fossil shells (Walton 1992), and hence this preparation technique is not ideal. It has the additional disadvantage of requiring large amounts of shell material to be effective. Such large quantities of shell powder are not always available.

LETHAIA 27 (1994) Intracrystalline amino acids fiom fossils 181

Mineral acids. - Several mineral acids were tested to find a decalcification solution that did not interfere with the analy- sis system and did not damage remaining protein. Aqueous solutions (20% w/v) of acetic acid, formic acid and hydro- chloric acid were added to shell samples at the same ratio as for EDTA. However, several days after the Na,-EDTA sample had become completely demineralized, the mineral acids had not reacted to completion. A series of reactions between different strengths of acid at different volumes was set up to attempt to find a balance between strength of acid that would not damage any remaining protein, but that would leave the remaining biomolecules in a solution that would not require any further concentration before analysis and hence retain the ability to quantify free amino acids and peptides.

Decalcification was completed using2 molar (2 M) hydro- chloric acid, which fulfilled the criteria in the followingways:

Test decalcifications with Recent samples indicated low concentrations of free amino acids, indicating that the damage to the proteirdpeptides is slight, i.e. hydrolysis of peptide bonds does not occur to any great extent (Walton etal. 1993).

Hydrochloric acid, unlike Na,-EDTA, does not produce any spurious peaks on the chromatogram, nor does it seriously affect the recovery of amino acids. This was tested by adding volumes of acid to known concentra- tions of standard amino acids (Pierce Standard H) on the analysis hit. The only effect on the chromatogram was to delay the elution of Asp, Glu and Gly from the hplc (high performance liquid chromatography) column in the separation system, by decreasing the initial pH of the buffer system. This effect was easily corrected for during the calibration process.

Hydrochloric acid could be obtained at a very high purity (intended for use in protein hydrolysis and hence free of amino acids).

For complete demineralization of the shell powder, hy- drochloric acid is required at a ratio of 11 ml/g shell. The resulting yields fiom fossils were well within the sensitiv- ity limits of the amino-acid analyser, hence eliminating the need for concentration via filtration. In this process, all small peptides and free amino acids are retained in the fraction of the sample to be analysed.

Removal of insoluble compounds Demineralization of the inorganic phase leaves an acid- insoluble residue. This residue is derived from the reaction between sugars and amino compound (Hoering 1973). The insoluble residue, known as melanoidin and similar in struc- ture to humic acids (Hoering 1973), cannot be quantified by the analyser and needs to be removed, by centrifugation, prior to loading, as the melanoidin can block the analysis system.

Confirmation of intercrystalline molecular destruction To confirm that the bleaching process effectively removed intercrystalline biomolecules, a sample of shell powder (0.70 g) was directly weighed into a newly pyrolyzed (500°C for 4 hours) glass universal bottle, 10 ml of pure water (MiUiQ'") added, and the sample incubated at 110°C for 24 hours. The sample was allowed to cool and 4.5 ml removed and concen- trated on a rotary evaporator (Howe Gyrovap) to 25 pl, and an aliquot (20 pl) was loaded onto the amino-acid analyser. The results indicated that for every milligram of powder analysed, there is 0.7 ng of amino acid. The sample was lyophilized and decalcified to release the intracrystalline biomolecules. The concentration of amino acid was 169.4 ng/mg, slightly lower than the average for Recent Terebratella sanguinea ( 180 ng/mg), but within the rangeofexperimental error. Therefore the proportion of amino acid from the intercrystalline fraction that remains undestroyed by the bleaching procedure is in the order of 0.4%. The remainder of the amino acids therefore must come from within the protected microenvironment of the shell crystallites.

Amino-acid analysis Amino-acid analysis is performed on the samples in solu- tion. AU of the common techniques used in amino-acid identification involve the reaction of one of the functional groups ofthe amino acid with some other compound. Auto- mation, coupled with an increase in the sensitivity of the machinery associated with amino-acid analysis, has led to increased reliability of results and levels of analysis. The 420H amino-acid analyser uses a process involving deri- vatization with phenylisothiocyanate (PITC; Heinrikson & Meredith 1984; Dupont etal. 1989). The sample is loaded to the sample frit and the slide rotated to the hydrolysis head, where automatic vapour phase hydrolysis using 6 M HCI takes place. After hydrolysis the slide is rotated and auto- mated derivatization takes place, and the sample is then transferred to a dedicated, on-line, narrow-bore reverse- phase hplc for identification and quantification.

The derivatization with PITC combines the almost non- UV-absorbing amino acids with a compound that absorbs UV light strongly, so aiding detection. The higher the con- centration, the higher the absorption of the light. The signal from the detector is integrated to produce a peak height that corresponds to the concentration. Amino acids are quanti- fied by comparison with a standard of known concentration analysed prior to the sample.

Stat is tical analysis Numerical taxonomy was described by Sneath & Sokal (1973, p. 4) as 'the grouping by numerical methods of

182 Derek Wulton e5 Gordon B. Curry LETHAIA 27 (1994)

taxonomic units into taxa on the basis of their character states'. Numerical methods can be taken to mean any kind of mathematical data used in the investigation of samples, ranging from simple morphometric measurements to de- rived variables from multivariate statistical analyses, and implies objectivity in analysis, although in many types of morphometric analysis this is rarely fully implemented. The relative proportions of amino acids within the shell are derived directly from information contained within the DNA. The data are thus not related to the morphology of the organism and hence are free from error introduced by eco- phenotypic variation, i.e. the amino-acid data come from the genotype of the organism, not from the phenotype.

Multivariate analysis is a relatively simple process when comparing small numbers with bivariate distribution. How- ever, as both the sample and variable size increases, the calculations required become more and more involved. It is for this reason that the statistical package Data Desk" for the Macintosh computer was used. This is an integrated multi- variate statistical package that combines several statistical techniques, two of which, principal-component analysis (PCA) and cluster analysis, were used to analyse the amino- acid data. Details of these techniques may be found in Koch & Link (1971), Davis (1973) and Sneath & Sokal(1973).

Example: Fossils from the Kupe Formation, Castlecliff Beach, New Zealand Geological history

The Kupe Formation is a sedimentary deposit, dated at approximately 0.5 Ma, forming part of the Wanganui Series of the South Wanganui Basin, one of a number of sedimen- tary basins formed around the southwest of present-day North Island, New Zealand. The Kupe Formation consists of five distinct members, with fossils distributed throughout (Fleming 1953). All deposits within the formation were formed at or close to the storm wave base. Sandy horizons indicate winnowing of fine-grained particles from the sedi- ment. The deposition of the formation followed erosion of the upper surface of the underlying bed (Fleming 1953), thought to be due to a glacial period. Most of the beds in the sequence were deposited within the high level stands of interglacials (Beu & Edwards 1984).

Methods and sample selection

Samples were collected from the coastal exposure of the Kupe Formation 4-6 km northwest of Castlecliff during the field seasons of 1990-1 99 1. A mixed fauna was collected, in order to extract as much information regarding the deposit as possible.

Samples were processed using the methods outlined above. Data were processed to convert the raw data into molecular percentages, before being input into the statisti- cal package Data Desk". The amino acids His, Met and Cys, which are known to be strongly affected by the hy- drolysis technique (Dupont et ul. 1989), were disregarded from the study.

Results

All samples were found to contain amino acids of differing relative concentration (Table 1). This finding, coupled with the analysis of the water-soluble amino acids from the sedi- ment, indicate that no homogenization of the amino-acid content has taken place through the deposit, and hence it is likely that the amino acids sourced represent those that are derived from the original proteins of the organism.

The relative-abundance data in Table 1 were analysed via PCA in order to summarize the data variation in a form whereby changes and relationships are easily observed. Table 2 shows the eigenvalues for the data set. A total of 75.4% of the total variation in the data set is contained within the first three eigenvalues and hence within the eigenvectors. The first principal component contains 42.2% of the total varia- tion of the data set and is governed primarily by changes in Gly (0.357),Arg (-0.338), Ile (-0.361) and Phe (-0.345). The second principal component contains 19.1% of the varia- tion, reflecting differences in Asp (0.541), Ala (0.576) and Tyr (-0.353). The third principal component has variation which accounts for 14.6% ofthe total present in the data set, mainly due to Ser (0.557), Thr (-0.372) and Tyr (0.481). The signs ofthe values represent the direction of the eigenvectors. It should be noted that all ofthe amino acids analysed have an influence on the distribution.

When the first three principal components are plotted on a three-dimensional plot (Fig. 1, which may be rotated on the computer screen to show relationships), separation of the groupings is obvious. Three genera of brachiopods (Neo- thyris, Terebrutellu and Wultoniu) are members of the same family and plot as one major group. Notosuriu, a member of a different order to the remaining brachiopods, plots close to the other brachiopods but is well differentiated. Within the Mollusca, samples were not identified to the same taxo- nomic level, but all groups are well separated, and this differentiation within the phylum is as expected.

This method for rapid determination of the amino-acid composition of fossils should allow greater use of this infor- mation source for a variety of applications from within the fossil record.

Acknowledgements.- We would like to thank Dr. M. Cusack for her invalu- able input during the development of this method and also for her com- mentson an early draft ofthis manuscript. Sandra Miller prepared the shell samples. This work was completed during the tenure of a UK NERC studentship to DW (GT4/89/GS/42) in the Department of Geology and Applied Geology at the University of Glasgow. GBC was supported by a Royal Society University Research Fellowship.

LETHAIA 27 ( 1994) Intracrystalline amino acids fiom fossils 183

Table 1 . Relative proportions (weight percentages) of intracrystalline amino acids extracted from the named sample, used to calculate principal components. These samples are used for illustration only and are not intended to be used for systematic palaeontology.

Amino acid D E S G R T A P Y V i 1 F K

Brachiopods Notosaria 0.16 Notosaria 0.15 Terebratella sanguinea 3.68 Terebratella sanguinea 4.07 Waltonia 3.77 Waltonia 4.77 Neothyris 3.72 Neothyris 3.62 Neothyris 3.62

Molluxs Bivalve (1) Bivalve (1) Bivalve (2) Bivalve (2) Oyster Oyster Oyster Oyster Pectenid Pectenid Gastropod Gastropod Turritellid Turritellid

2.47 3.67 3.64 5.66

10.82 9.49

14.13 8.30

14.47 15.96 2.02 2.65 3.79 3.97

2.04 2.01 5.15 5.85 6.75 7.49 7.66 7.14 7.54

5.55 0.00 5.82 6.50 5.38 5.32 5.77 4.88 8.07 6.90 8.88 9.68

12.79 12.72

0.36 51.30 0.35 50.84 0.33 54.92 0.36 54.14 0.35 50.35 0.35 49.20 1.11 51.26 0.70 55.98 0.89 53.81

0.54 14.84 0.83 13.39 0.59 15.77 0.73 12.04 0.56 35.23 0.79 36.41 0.59 32.79 0.58 38.16 0.97 28.40 0.64 23.09 0.00 17.22 0.00 15.38 0.69 12.83 0.00 14.91

0.18 19.97 16.09 3.47 0.19 19.86 16.37 3.53 0.52 0.18 15.14 7.09 0.54 0.17 14.94 6.87 0.63 0.00 15.89 6.10 0.60 0.00 15.87 5.99 1.08 0.00 13.70 5.95 0.85 0.00 13.63 5.71 0.99 0.00 13.73 5.76

1.13 3.27 1.06 7.10 1.47 1.07 1.28 1.51 0.81 0.59 0.78 0.47 0.73 0.69 0.71 0.55 0.39 0.66 0.34 0.86 1.59 0.00 2.31 0.00 1.71 0.96 0.76 1.08

14.35 32.61 12.31 29.52 17.65 22.03 15.03 22.07 24.08 6.37 25.63 6.46 22.42 6.75 25.08 6.74 29.34 3.42 28.93 3.93 21.31 23.39 20.25 22.58 18.21 13.94 17.76 14.63

0.89 2.24 1.00 2.33 1.63 6.32 1.74 6.19 2.14 7.02 2.37 6.79 3.08 5.88 2.69 5.06 2.84 5.39

3.37 6.45 2.51 8.07 1.77 10.15 3.25 9.68 1.62 6.44 0.91 6.04 1.74 6.12 1.26 6.14 0.24 4.12 0.37 5.28 0.00 7.33 0.00 7.90 2.08 6.09 1.87 6.18

0.35 0.35 1.57 1.61 1.31 1.24 1.91 1.48 1.70

1.24 2.02 3.21 3.52 1.31 1.15 1.24 1.15 1.38 2.04 2.49 2.42 2.41 2.42

0.76 0.80 1.77 1.79 2.77 2.61 2.44 1.67 2.00

2.57 3.91 6.33 6.03 2.42 2.50 2.51 2.52 5.37 6.99 9.25 9.51

13.92 13.98

1.56 1.58 0.97 0.99 1.40 1.32 I .29 0.90 1.04

2.57 5.03 3.43 3.51 1.32 1.35 1.38 1.36 0.81 1.06 4.13 4.34 5.85 5.63

0.57 0.57 0.72 0.74 1.50 1.39 0.93 0.57 0.69

4.90 4.05 5.88 7.12 3.03 2.71 3.15 2.56 2.36 3.60 2.41 2.97 4.73 4.10

Table2. Principal-components analysis of the data in Table 1. High num- bers reflect the relative importance of that amino acid in the variability of that eigenvector. Signs indicate the direction of the variation.

.~

D E S g r T A P Y V I L F K

V1

0.035 -0.2 15 0.034 0.357

0.180 0.0 13

-0.322 -0.041 -0.319 -0.361 -0.328 -0.345 -0.334

-0.338

Eigenvectors v2

0.541 0.212 0.110

-0.113 -0.082 -0.258 0.576

-0.238 -0.353 -0.055 0.0 18 0.159

0.068 -0.136

v3

0.220 -0.072 0.557 0.108

-0.006 -0.372 -0.122 -0.018 0.48 I 0.274 0.138

-0.265 -0.250 0.109

Eigenvalues Values Variance proportion

el 5.914 42.2% e2 2.675 19.1% e3 2.049 14.6%

Total 75.99’0 ~.

a u1 I

Notosaria A Neothyris 0 waltonia + Terebratella CB Ostrea

+ +

u2 u3

/a # 0 0 3

+ pectinid o turritellid x bivalve (1) 0 bivalve (2)

Fig. 1. Three-dimensional scatterplot of the principal-components data for fossil samples from the Kupe Formation, Wanganui, New Zealand. Note theclosegroupingsofthedata points representingthe brachiopodsand the molluscs. Different orders of brachiopods may be readily distinguished.

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