Degradation of intracrystalline proteins and amino acids in fossil brachiopods

PII: S0146-6380(97)00126-5

Org. Geochem. Vol. 28, No. 6, pp. 389±410, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0146-6380/98 $19.00 + 0.00

Degradation of intracrystalline proteins and amino acids in fossil brachiopods DEREK WALTON Division of Earth Sciences, University of Derby, Kedleston Road, Derby, DE22 1GB, U.K. (Received 6 October 1996; returned to author for revision 1 April 1997; accepted 4 December 1997) AbstractÐFour genera of Recent to Plio-Pleistocene articulated brachiopods were collected from up to 16 horizons spanning the last 3.3 Ma of sediment deposition in the South Wanganui Basin, New Zealand, and assayed for the preservation of intracrystalline proteins and/or amino acids. The proteins present in the shells of living and Recent brachiopods undergo rapid degradation through the decomposition of the peptide bond. Up to 95% of the constituent amino acids from the proteins are present in the free state by 0.12 Ma. This rate of degradation is far higher than was originally expected for intracrystalline proteins. Quantitative analysis of the concentrations of amino acids present within the shells of fossil brachiopods indicates a range of reaction rates for the subsequent degradation of individual amino acids. The degradation of these amino acids may lead to the total loss of compounds, to the generation of non-standard amino acids, or to diagenetically produced proteinaceous amino acids. These reactions do not necessarily mirror those which occur during the pyrolysis of an aqueous solution of the pure amino acids, either in their rate or products. # 1998 Elsevier Science Ltd. All rights reserved Key wordsÐintracrystalline proteins, amino acids, protein degradation, decomposition pathways, brachiopods

INTRODUCTION

The fossilised hard parts of invertebrates are rich sources of the degraded remains of organic molecules originally trapped during biomineralisation (see, for example, Curry, 1988). Although the potential signi®cance for these biomolecules in establishing molecular phylogenies for fossil material was recognised in the early stages of their study (Abelson, 1955), it was not clear to what extent compositional di€erences were due to phylogenetic di€erentiation or were re¯ecting variable degradation of the original molecules (Abelson, 1955). For phylogenetic interpretation to be meaningful, it is important to determine precisely the degradation characteristics of these biomolecules from a site protected from extraneous contamination. The discovery that molluscs (Watabe, 1963), echinoderms (Pilkington, 1969) and brachiopods (Collins et al., 1988) contain organic molecules within (intracrystalline), as well as between (intercrystalline), the shell crystallites provided a source for fossil biomolecules entombed within the biomineral which cannot be degraded or contaminated by bacteria (Sykes et al., 1995). As the term ``intracrystalline'' remains subjective, this study follows the operational de®nition given by Sykes et al. (1995). Degradation (and therefore change in relative abundance) of the molecules is due to the prevailing physico±chemical conditions, rather than microbial 389

reworking or contamination. Degradation products remain trapped within the inorganic phase until demineralisation or recrystallisation, allowing sampling of both preserved indigenous biomolecules and the degradation products of less stable components. Much of our understanding of protein decomposition in the fossil record stems from arti®cial pyrolysis experiments (for example Vallentyne, 1964, 1968). The aim of the present study was to extract preserved amino acids, peptides and proteins from intracrystalline sites within the shells of brachiopods and to compare these with the previously published simulation of amino acid degradation based on pyrolysis experiments, in an attempt to quantify protein and amino acid degradation in the geological record. Throughout this study, it has been assumed that there has been no signi®cant evolution (i.e. change in composition) of the protein and that recorded change re¯ects diagenetic alteration. This study has utilised the rich and diverse articulated brachiopod fauna of New Zealand which o€ers a rare opportunity to study changes in protein and amino acid composition as their shell crystallites contain intracrystalline proteins (Collins et al., 1988; Walton et al., 1993) and their shells are composed of diagenetically stable low-Mg calcite (Clarke and Wheeler, 1922).

390

D. Walton

mostly deposited in shallow marine conditions, with the maximum accumulation occurring to the south of the area considered here (Anderton, 1981). By comparison with Anderton (1981) and Fleming (1953), the maximum depth of burial of the part of the basin considered here is approximately 1.5 km. Interspersed throughout the sequence are a number of richly-fossiliferous shellbeds containing abundant macrofossils (Fleming, 1953), ranging in age from ca. 120 ka to ca. 3.3 Ma (Table 1). From such shellbeds up to four genera of brachiopods were collected, together with representatives of other groups, for use in demonstrating the heterogeneity of the amino acid compositions. Shell preparation Fig. 1. Approximate locations in New Zealand from where samples were collected. For precise details of the sample locations see Fleming (1953).

MATERIALS AND METHODS

Brachiopod samples Recent brachiopods were collected from locations given in Fig. 1. Fossil samples, of the same genera as those present in the Recent fauna, were collected from the Plio-Pleistocene South Wanganui Basin of North Island, New Zealand (Fig. 1). The tectonic setting of the basin has allowed rapid subsidence and the accumulation of up to 4 km of sediments,

Samples were prepared according to the methods of Walton and Curry (1994). In summary, shells which were excessively bored or fractured were excluded from further study. Sediment was scrubbed from the sample and encrusting epifauna removed by scraping. Articulated shells were disarticulated and any remaining body tissue removed before being soaked in an aqueous solution of bleach (10% v/v) for 2 h at room temperature, washed extensively with Milli ROTM water and air dried. Samples were ground using a ceramic pestle and mortar, and the powder incubated in an aqueous solution of bleach (10% v/v) under constant motion for 24 h at room temperature, then washed by repeated agitation with MilliQTM water and centrifugation (typically ten washes) and lyophilised.

Table 1. Absolute dates used in this study together with the rationale for that date. The dating of this succession is relatively poorly constrained with a number of con¯icting ages given for the same beds Horizon

Age (Ma)

Reference

Rapanui Marine Sand

0.12

Pillans (1983)

Landguard Sand

0.33

Stratigraphically between Fordell Ash (0.31 Ma, Bussell and Pillans, 1992) and Rangitawa Pumice (0.34 20.03 Ma Alloway et al., 1993)

Upper Castlecli€ Shellbed

0.36

Tainui Shellbed Pinnacle Sand Lower Castlecli€ Shellbed

0.38 0.39 0.40

All dates based on the accumualtion rates of Beu and Edwards (1984)

Kupe Formation

0.50

Based on an approximate mean of the dates of Seward, 1974 (0.45 2 0.09 Ma) and Abbott, 1992 (0.55±0.6 Ma)

Kaimatira Pumice Sand

1.05

Alloway et al., 1993

Okehu Shell Grit

1.10

Based on stratigraphic position

Tewkesbury Formation

1.67

Based on the age estimates of 1.632 0.15 Ma (Alloway et al., 1993) and 1.26 20.16 Ma (Boellstor€ and Te Punga, 1977) and stratigraphic position

Waipuru Shellbed Upper Nukumaru (4243) Nukumaru Brown Sand Upper Okiwa Group Hautawa Shellbed Lower Okiwa Group (Te Rama) Upper Waipipi Shellbed Middle Waipipi Shellbed

1.75 1.85 1.85 2.15 2.20 2.25 3.20 3.30

All dates based on stratigraphic position

Degradation of intracrystalline proteins and amino acids

391

Table 2. A summary of the data used in this study Horizon

Recent

Age (Ma)

0

Organism

Total amino acid concentration Free amino acid concentration (ng mgÿ1) (ng mgÿ1) Mean

Std Dev.

Neothyris Terebratella Calloria Notosaria

108.88 190.27 167.08 706.88

4.03 19.17 7.93 146.74

Mean 4.55 7.74* 2.04 69.08

% Free

Std Dev. * 4.07 * *

4.18 1.22 9.77

Rapanui Marine Sand (Waipipi)

0.12

Calloria Notosaria Pectenid Turritellid

267.02 469.95 278.33 58.63

13.80 47.88 23.84 7.87

202.06 449.60 109.35 42.17

24.57 18.96 18.31 6.08

75.67 95.67 39.29 71.93

Rapanui Marine Sand (Waitotara)

0.12

Terebratella Calloria Pectenid

196.92 203.23 138.52

34.74 46.06 28.09

122.72 160.01 125.11

19.67 * 1.76

62.32 78.73 90.31

Landguard Sand

0.33

Neothyris

125.85

18.50

90.41

18.39

71.84

Upper Castlecli€ Shellbed

0.36

Neothyris Calloria Pectenid

108.26 325.85 326.33

5.05 21.6 28.43

87.82 198.84 180.75

12.52 26.80 *

81.12 61.02 55.39

Tainui Shellbed

0.38

Neothyris Terebratella Calloria Notosaria Pectenid Turritellid

109.24 176.67 231.27 318.54 181.64 55.65

12.94 6.71 18.77 47.49 18.02 6.50

77.45 141.71 139.91 322.12 130.41 47.58

7.50 10.38 9.50 54.41 17.81 5.04

70.90 80.21 60.49 100.00$ 71.79 85.49

Pinnacle Sand

0.39

Neothyris Terebratella Calloria Notosaria Pectenid Turritellid

101.37 236.51 165.53 310.96 263.33 116.78

5.92 7.28 41.38 49.31 14.55 23.93

95.48 184.34 150.55 292.01 140.62 101.98

6.97 18.79 13.09 17.73 23.64 21.52

94.18 77.94 90.95 93.91 53.40 87.33

Lower Castlecli€ Shellbed (Coast)

0.4

Neothyris Terebratella Calloria Pectenid Turritellid

81.75 193.39 208.67 264.99 164.62

17.89 3.74 29.78 20.65 42.52

72.20 117.09 163.84 164.46 130.13

23.40 44.94 33.65 29.59 53.18

88.31 60.54 78.52 62.06 79.05

Lower Castlecli€ Shellbed (Waipuka Road)

0.4

Neothyris Terebratella Calloria

77.60 221.47 180.09

8.72 31.58 14.11

62.36 198.17 195.90

4.53 10.67 22.18

80.35 89.48 100.00$

Kupe Formation

0.5

Neothyris Terebratella Calloria Turritellid

75.00 227.18 259.97 73.77

16.36 20.02 59.46 4.45

74.03 215.71 220.68 42.78

4.70 * 26.29 9.17

98.70 94.95 84.89 57.98

Kaimatira Pumice Sand

0.95

Terebratella Calloria

146.57 138.43

20.24 17.57

ND 152.28

ND *

ND 100.00$

Okehu Shell Grit

1.1

Terebratella Pectenid Turritellid

173.88 125.67 55.94

72.51 1.18 0.18

178.68 ND 37.08

26.04 ND *

100.00$ ND 66.29

Tewkesbury Formation

1.67

Calloria Pectenid Neothyris

101.19 57.91 47.71

11.77 12.55 4.26

92.64 44.81 49.72

6.12 2.29 16.19

91.55 77.38 100.00$

Waipuru Shellbed

1.75

Calloria

101.49

13.24

95.46

15.19

94.06

Undi€erentiated Shellbed (4243 of Fleming, 1953)

1.85

Neothyris Calloria

53.63 123.09

6.05 28.76

58.09 102.30

5.64 20.17

100.00$ 83.11

Nukumaru Brown Sand

1.85

Calloria

155.28

30.96

132.04

35.06

85.03

Undi€erentiated Shellbed (4207 of Fleming, 1953)

2.15

Neothyris Calloria Notosaria

21.93 54.57 89.26

5.45 4.81 7.98

16.82 41.39 75.14

6.68 2.84 11.65

76.69 75.84 84.18

Hautawa Shellbed

2.2

Neothyris Calloria Notosaria Turritellid

31.73 69.70 100.23 21.32

4.47 11.69 18.58 2.25

27.83 48.83 83.91 10.95

1.15 3.01 16.17 1.17

87.69 70.05 83.71 51.37

Te Rama Shellbed

2.25

Neothyris

53.50

11.41

46.09

7.23

86.16

Upper Waipipi Shellbed

3.2

Neothyris Terebratella

40.75 96.22

6.05 21.44

28.66 77.88

7.54 9.96

70.34 80.94

Middle Waipipi Shellbed

3.3

Neothyris

79.31

9.59

59.77

11.88

75.36

*One analysis only. $Corrected to 100% (see Section 2). ND, no data recorded. Full details for individual amino acids are available from the author on request.

392

D. Walton

An aqueous solution of 2 M HCl (at 19 2 18C) at a ratio of 11 ml mgÿ1 was used to dissolve the shell powder and release the entrapped biomolecules. Once demineralisation was complete, insoluble particles were removed by centrifugation (20 g hÿ1). Amino acid analysis All samples were subjected to vapour-phase 6N HCl automated hydrolysis (Applied Biosystems 420A). Amino acid analysis was completed following the standard protocols for the 420A which are given in West and Crabb (1989) and Dupont et al. (1989). Standard proteins and peptides were used during every analysis to ensure that hydrolysis proceeded to completion and blank analyses were included to check for background levels of contami-

nation. An aliquot of the same sample was analysed without hydrolysis to ascertain the quantities of free amino acids present. Individual amino acids were derivatised using phenylisothiocyanate (PITC; Heinrikson and Meredith, 1984), and transferred to a dedicated narrow bore HPLC system for separation and quanti®cation. Amino acids were identi®ed by comparison with Pierce amino acid standard H (proteinogenic) or single commercial standards (non-standard). Analyses with hydrolysis were repeated at least three times, and those without generally twice to ensure reproducibility (Table 2). As acid hydrolysis of samples causes variable loss of amino acids (Hill, 1965) the concentrations listed in Table 2 are likely to be minimum estimates. This

Fig. 2. Absolute concentration of amino acids (free + combined, given as nanograms of amino acid per milligrams of shell material, (A) and proportion of the total amino acid which are uncombined (free, given as a percentage; (B) plotted against sample age. In (A) note the overall decline in the concentration of the amino acids and the di€erence in the rate of decline between the genera. (B) demonstrates that the original proteins have undergone natural hydrolysis (c.f. Hare, 1974; Goodfriend et al., 1992). Free amino acids are not leached out (c.f. Hare, 1974) as the degradation products remain within the shell (Sykes et al., 1995; Walton et al., 1993).

Degradation of intracrystalline proteins and amino acids

complicates determination of the proportion of the amino acids that are present in the free state, as samples without hydrolysis will not su€er the same loss of amino acids. In samples where a high proportion of amino acids are present in the free state, the loss caused by hydrolysis may therefore yield a value for free amino acids greater than that for total amino acids after hydrolysis (i.e. greater than 100%; these are corrected to 100%). RESULTS AND DISCUSSION

Bulk amino acid composition The absolute concentration and proportion of amino acids in the free state are given in Table 2. Means and standard deviations are given for multiple analyses. Figure 2(A) shows the total amino acids compared with the age of the sample. All samples contain appreciable amounts of amino acid and show an overall general decreasing trend in concentration with time, although with some scatter. In three of the species investigated (Calloria, Neothyris and Terebratella) there is an initial rise in the concentration between Recent and the youngest fossils, which di€ers in magnitude and duration and which has also been observed following arti®cial diagenesis of gastropod shells (Qian et al., 1995). In this study, the increase is attributed to the solubilisation of originally acid-insoluble proteins from the intracrystalline fraction, similar to that noted by Weiner and Lowenstam (1980) where fossil samples were dominated by the soluble fraction and Recent samples by the insoluble fraction. The proportion of amino acids which are present in the free state increases from negligible amounts in the Recent (indicating that the amino acids are all bound into proteins), to ca. 80% in less than 0.5 Ma [Fig. 2(B)], indicating rapid hydrolysis, especially during the ®rst 120 ka. The proportion of free amino acids ¯uctuates with time, although remaining greater than 60% in all fossil samples. This is higher than that reported by Abelson (1955) who determined that 1±5% of peptide bonds would be broken in 0.1 Ma, but who acknowledged a loss of soluble peptides from the shell due to leaching. This resulted in a false impression of the state of preservation, with the residual protein containing a relatively high proportion of peptide bonds to free amino acids. In brachiopods, intercrystalline pro-

393

teins largely decay in under a year (Collins, 1986), perhaps due to microbial activity between the shell crystallites (Gaspard, 1989), which con®rms the potential for loss hypothesised by Abelson (1955). Some amino acids remain peptide-bound even in the oldest of the samples analysed [Fig. 2(B)], although there is no information regarding their size nor their primary sequence. The rate of hydrolysis depends on available water, temperature and the chemical characteristics of the amino acids on either side of the bond. Inclusions of water have been observed within the shell of living and fossil molluscs (Hudson, 1967), and articulated brachiopods (B. Stern, University of Newcastle upon Tyne, U.K., pers. comm.); brachiopod shells contain up to 3% water by mass (Ga€ey, 1988). As the fossil proteins are in a highly degraded state, water has been present in relatively large quantities within the shell, and hydrolysis has proceeded largely unhindered. The proportion of free amino acids in the shells of molluscs from the same horizon as the brachiopods are broadly similar (Table 2), suggesting similar hydrolysis mechanisms. There is no evidence as to the relationship between the protein and water and it is possible that the proteins are contained in aqueous solution, which provides a ready source of water for natural hydrolysis. If, however, the protein is discrete from any large included source of water, it may be better preserved (Towe and Thompson, 1972). Decomposition of some amino acids (Ser, Thr and Glu; see Table 3) yields water, which would be available to continue the degradative process. However, it is important to note that hydrolysis does not proceed to completion, suggesting a limited availability of water and a closed or partially closed decompositional environment. A second factor which can in¯uence hydrolysis is temperature. The South Wanganui Basin has undergone successive periods of subsidence and uplift, although seismic evidence demonstrates that this burial is unlikely to exceed 1.5 km (Anderton, 1981). Samples are likely to have been heated to 20±308C, increasing the rate of hydrolysis by an order of magnitude compared to average sediment surface conditions. The third factor important in hydrolysis is the nature of the residues on either side of the bond (for example Hill, 1965; Powell, 1994; Qian et al.,

Table 3. The three letter abbreviations used for amino acids Amino acid Alanine Arginine Aspartic acid Glutamic acid Glycine Isoleucine Leucine

Three letter code

Amino acid

Three letter code

Ala Arg Asp Glu Gly Ile Leu

Lysine Phenylalanine Proline Serine Threonine Tyrosine Valine

Lys Phe Pro Ser Thr Tyr Val

394

D. Walton

Fig. 3. The concentration (free and combined) of Asx (A) and Glx (B) plotted against sample age. Note the initial rise in the concentration of Asx in Calloria, due to the solubilisation of an originally acid insoluble component in the Recent sample (see text). Notosaria is omitted from (A) as its concentration is far higher than the other samples, and obscures the detail.

1995). Protein sequencing (Cusack et al., 1992), immunology (Endo et al., 1994) and amino acid analysis (Walton et al., 1993) have shown that the amino acid composition and the size of intracrystalline proteins are di€erent in the various brachiopods studied here. Variations in sequence and structure may be expected to cause species level variation in the rate of hydrolysis, as they appear to do in the case of amino acid racemization (Wehmiller, 1980). Individual amino acids Acidic side chains. During preparative hydrolysis, asparagine (Asn) and glutamine (Gln) are deamidated to aspartic acid (Asp) and glutamic acid (Glu) respectively (Hill, 1965), hence no distinction is made between the amino acids with the acidic side chains and their non-charged derivatives and they are referred to as Asx and Glx respectively. Rapid and irreversible deamidation also occurs within peptides with a half life of days to years (Robinson and Rudd, 1974; Brinton and Bada,

1995), and it is unlikely that Asn and Gln would persist in the fossil record. In brachiopods, Asx and Glx both show an exponential decrease in concentration, following an initial rise in Calloria, with >80% lost by 3.3 Ma indicating some parity in the degradation of the acidic amino acids, although Glx is more stable than Asx (Fig. 3). The proportion of the Asx present in the free state varies between samples, although in each case approximately 95% is free by 0.5 Ma and complete destruction in the free state in Calloria takes place by 2.15 Ma. However, for Glx the proportion in the free state in all samples is low, generally below 40% of the total present, and it maintains this level throughout the rest of the period under study (Fig. 4). Asx may decompose by two main pathways: ®rstly by reversible deamination to produce fumaric acid and ammonia (Bada, 1971; Sohn and Ho, 1995). If the ammonia remains trapped, an equilibrium will be established, leading to the persistence

Degradation of intracrystalline proteins and amino acids

395

Fig. 4. The proportion of Asx (A) and Glx (B) present in the free state. Note the di€erences in the scale of the y-axis. Glx is present in much lower concentrations than Asx due to the formation of pyroglutamic acid. This lactam reverts back to glutamic acid on hydrolysis, resulting in a depressed concentration of Glx in the free state.

of Asx in older samples, although this reaction cannot take place when Asx is peptide-bound (Bada and Man, 1980). Secondly, decomposition occurs by decarboxylation of the a- or b-carbons to form b-Alanine (b-Ala) or Ala respectively. Evidence for deamination is dicult to obtain, as there may be many sources and sinks for the ammonia produced by deamination, and fumaric acid cannot be identi®ed on the analysis system used. Ammonia reacts with PITC to give a systems peak (phenylthiourea (PTU), Bidlingmeyer et al., 1984; Cohen and Strydom, 1988), and the size of this peak rises in the fossil record, indicating that the concentration of ammonia increases with time, although this was not quanti®ed. Decarboxylation, however, results in the formation of b-Ala which can be recognised on the system used and has an elution time identical to that of Thr. Hence a-decarboxylation of Asx would cause a rise in the concentration of ``Thr''. Of the samples analysed here, Notosaria contains the highest concentration of Asx and fossil samples might

be expected to contain b-Ala in relatively high concentrations. In Notosaria, the concentration of Thr shows a very large increase over the period 0.12± 0.5 Ma (Fig. 5; Table 4), compared to a loss in Calloria of >80%. This indicates that some diagenetic reaction product in Notosaria co-elutes with Thr, probably b-Ala, and that the a-decarboxylation reaction may take place in geological samples. A similar rise also occurs in samples of the pectenids analysed. However, as there is no direct correlation between the concentrations of Asx and ``Thr'' not all Asx degrades by decarboxylation. Samples such as Calloria which have a lower initial concentration of Asx do not have a rise in ``Thr'', but it is likely some conversion to b-Ala is occurring, but only after 120 ka (Fig. 5). Pyrolysis has indicated that decarboxylation reactions are rare (Bada, 1971) and less than 0.2% decarboxylation occurs during pyrolysis (Bada and Miller, 1969, 1970). However, these estimates were based on reactions at elevated temperatures of the pure compound alone, and in this case may not be

396

D. Walton

Fig. 5. Histogram showing the change in concentration (free and combined) of Asx and ``Thr'' in Notosaria. Note the increase in the concentration of ``Thr'' and the decrease in the concentration of Asx over the time period, probably due to the production of b-alanine from the decarboxylation of Asx. This production does not take place until after 120 ka. There is no dramatic increase in the concentration of ``Thr'' in the other samples which have a much lower initial concentration of Asx, possibly indicating that there is a relatively low proportion of b-alanine production through this mechanism.

directly applicable to fossil biomolecules (c.f. Cowie and Hedges, 1994, although this study does not distinguish between chemical and biologically mediated decomposition). If a-decarboxylation occurs it is possible that b-decarboxylation will also occur, resulting in Ala, which may explain some of the increase in Ala concentration found in fossils [see Fig. 6(A)]. Glx may undergo degradation by two reaction pathways. Firstly, by g-decarboxylation to produce g-aminobutyric acid (Hare and Mitterer, 1967), and secondly by lactam formation to produce pyroglutamic acid (Wilson and Cannan, 1937). g-decarboxylation in brachiopods is indicated by gaminobutyric acid, although there is no direct correlation between the age of the sample, the decrease in concentration of Glx and the increase in size of the peak at the position of g-aminobutyric acid. The proportion of Glx in the free state is low (ca. 40%), which may be due to either the selective preservation of poly-Glx, or the formation of pyroglutamic acid by lactamisation. The low proportion of

Glx in the free state is here attributed to lactam formation, rather than the preservation of poly-Glx. This reaction, however, does not explain the decrease in the concentration of Glx, which either occurs via decarboxylation to form g-aminobutyric acid, or via the decomposition of pyroglutamic acid. Lactam formation may only take place when Glx is in the free state, hence the protein must have undergone hydrolysis prior to lactamisation. Pyroglutamic acid may be converted back to Glx through protein hydrolysis. Aliphatic hydroxyl side chains. Serine (Ser) and threonine (Thr) initially decay rapidly, with ca. 80% of the original concentration lost by 1 Ma, and their concentration remains at a similar level in older samples (Fig. 7). The proportion of Ser present in the free state rapidly increases to a maximum (approximately 90% free) and then decreases until none remains in the uncombined state (Fig. 8). Any Ser which remains in the sample after approximately 0.7 Ma is bound via an HCl sensitive bond. Most of the decomposition therefore takes place in

Table 4. The changing concentrations (free and combined) of Asx and Thr over time. Note the decrease in the concentration of Asx and a corresponding increase in Thr which is attributed to the co-elution of b-Ala formed from decarboxylation of Asp Concentration (ng mgÿ1) Sample Recent Rapanui Marine Sand Tainui Shellbed Pinnacle Sand Upper Okiwa Group Hautawa Shellbed

Age (Ma) 0 0.12 0.38 0.39 2.15 2.20

Aspartic acid/asparagine

Threonine

223.64 109.19 42.65 41.72 1.61 1.98

7.86 4.49 53.78 34.21 23.27 22.29

Degradation of intracrystalline proteins and amino acids

397

Fig. 6. The concentration (free and combined) (A) and the proportion in the free state (B) of Ala plotted against sample age. In (A) the very large increase in the concentration of this molecule is due to diagenetic production and the concentration in the oldest samples is approximately the same as in the Recent. Note that the maxima of molecules in the free state correlates to the maxima in the diagenetic increase.

the free state, although some may decay whilst remaining peptide-bound (Akiyama, 1980). Decomposition of Ser and Thr may occur via three pathways (Vallentyne, 1964; Bada et al., 1978): ®rstly, by dehydration of the hydroxyl group, forming Ala (from Ser) or a-aminobutyric acid (a-ABA, from Thr). Secondly, by aldol cleavage resulting in the formation of Gly and formaldehyde (from Ser) or Gly and acetaldehyde (from Thr). Thirdly, by decarboxylation, resulting in the formation of ethanolamine (Ser) or propanolamine (Thr). Deamination is not a major pathway (Sohn and Ho, 1995). Dehydration is the prevalent reaction when Ser is in the free state and aldol cleavage is dominant when the molecule remains bound (Bada and Man, 1980). Aldol cleavage of Thr occurs more rapidly when the reactions are catalysed by metal ions, or when it is in peptides (Vallentyne, 1964). Although the 0.37-life gained by pyrolysis for Thr at 108C is in the region of 30 Ma (Vallentyne, 1964), greater than is shown here

(where the 0.37-life would be ca. 0.5 Ma), the pyrolysis experiments were completed on solutions of pure amino acids and may not be directly comparable. The increase in concentration of Ala in the brachiopods [Fig. 6(A)] is suggestive of dehydration reactions, although there is no direct correlation between the concentration of Ser and Ala, and the increase in concentration of Ala cannot be explained solely by this decomposition. Decomposition by aldol cleavage is negligible, as there is only a small rise in the corresponding concentration of Gly: most of the Ser has decomposed to Ala. Complete destruction of Thr occurs within 1 Ma (Bada et al., 1978), consistent with the results presented here. a-ABA has been identi®ed in the fossil species examined in this study, but not quanti®ed, indicating some decomposition by dehydration, although Bada et al. (1978) demonstrate that only

398

D. Walton

Fig. 7. The concentration (free and combined) of Ser (A) and Thr (B) plotted against sample age. Notosaria is omitted from (B) as the rise in the concentration of b-alanine, co-eluting with Thr, obscures the detail. Some of the Thr for the remaining samples may also be due to b-alanine (see also

approximately 10% of the Thr decomposes by this mechanism. Basic side chains. Arginine (Arg) proceeds to complete destruction in less than 1 Ma (with the exception of two results of older samples of Calloria and one from Terebratella). In contrast, the concentration of lysine (Lys) is variable (Fig. 9) showing an initial increase and then a decrease with time. The third amino acid with a basic side chain, histidine, was not quanti®ed, although it is only found in small concentrations in brachiopods (Walton et al., 1993). The proportion of free Arg shows a rapid increase, until almost 100% is free by 0.38 Ma. This proportion decreases rapidly to 0% free by 1.5 Ma (Fig. 10), indicating that any Arg which remains in older samples must be bound via an HCl sensitive bond. The proportion of Lys present in the free state is highly variable between species. Pyrolysis of Arg (Vallentyne, 1968) indicates that the 0.37-life is ca. 100 yr at 208C, and decomposition yields urea, ornithine, ammonia, proline and

an unidenti®ed compound which is associated both with ornithine and with Arg (Murray et al., 1965; Vallentyne, 1964, 1968; Sohn and Ho, 1995). Ornithine occurs in Mercenaria (Hare and Mitterer, 1967) and brachiopods (Fig. 11). Although the height of the peak is variable, it does show a relationship between the decrease in concentration of Arg and the increase in ornithine. Low concentrations of ornithine are identi®ed from Recent samples which may be caused by the hydrolysis of peptides and proteins containing Arg, although at the temperatures and time of hydrolysis used there would be a negligible degradation (Murray et al., 1965). The concentration of ornithine rises rapidly in the ®rst fossil shells, re¯ecting the decrease in Arg, but then begins to decline, probably re¯ecting decay via decarboxylation to putrescine (Murray et al., 1965). Lys is one of the least stable of the amino acids (Vallentyne, 1964). This is not unexpected, as it contains a secondary amino group susceptible to deamidation and is also prone to condensation with

Degradation of intracrystalline proteins and amino acids

399

Fig. 8. The proportion of Ser (A) and Thr (B) present in the free state. Notosaria is omitted for the reasons given in the caption to Fig. 7. Note that some Ser remains in all samples (Fig. 7; except 2 of Neothyris) throughout the period under study, but that it is not present in the free state, only when

reducing sugars via the Maillard reaction. Vallentyne (1968) used column chromatography to detect the presence of ammonia in the pyrolysed solutions, indicating that either primary or secondary deamination was occurring. Under di€erent conditions, Sohn and Ho (1995) found little ammonia produced by thermal degradation. There is no evidence in this study to indicate which decay pathway Lys follows, although the production of ammonia may be recorded in the increase in the size of the systems peak PTU in the current study. Aromatic side chains. The concentration of both tyrosine (Tyr) and phenylalanine (Phe), after an initial increase in Calloria, show a rapid decrease in concentration over time to greater than 80% loss (Fig. 12). Tryptophan, a third amino acid which contains an aromatic group in its side chain, is completely destroyed during the acid hydrolysis of peptides (Hill, 1965) and is therefore not quanti®ed here. The proportion of Tyr and Phe present in the uncombined state rises up to 100% by 0.5 Ma in all species studied (Fig. 13), but is very variable.

Pyrolysis of Tyr has not been studied in detail, although Gly is formed in small quantities (Vallentyne, 1964). Tyr is almost totally destroyed by 3.3 Ma, but it is not apparent by which reaction pathway. The main pathway of decomposition for Phe during pyrolysis is decarboxylation to form phenethylamine, which is then further decomposed to benzylamine (Vallentyne, 1964), following ®rst order kinetics, with a 0.37-life of 100 Ma at 108C, much slower than that found in the present study. The decomposition of Phe in the pyrolysis of mixtures of amino acids is more rapid than the single compound (Vallentyne, 1964), and it is this phenomenon which may cause such rapid decomposition in fossils. As the initial increase in the concentration of Ala [Fig. 6(A)] cannot be accounted for by the dehydration of Ser alone, other decomposition reactions must also have an in¯uence on the increase in concentration of this amino acid. Tyr, with the potential for cleavage of the ring from the remainder of the molecule must be considered for the origin of

400

D. Walton

Fig. 9. The concentration (free and combined) of Arg (A) and Lys (B) plotted against sample age. Note

some of the diagenetic Ala. Other reactions, such as decarboxylation and deamidation could proceed in Tyr, although further work is required to determine potential products and to con®rm reaction pathways. Aliphatic side chains. The six amino acids with aliphatic side chains show variable decomposition pathways and will be grouped accordingly. Concentrations of valine (Val) are variable through time, although there is a trend representing 50±60% destruction [Fig. 14(A)]. All samples show a large (up to 100%) initial increase in Val concentration between Recent and young fossil samples, with a maxima at 0.5 Ma, after which there is a decrease. The proportion of Val present in the free state rises to greater than 80% by 0.12 Ma [Fig. 14(B)], but then shows a slight decrease indicating that the rate of degradation is higher in the free than in the bound state, and that once the susceptible peptide bonds are broken, the rate of release slows dramatically. Pyrolysis experiments have not conclusively identi®ed the decomposition products of Val, although Gly has been tentatively identi®ed, with a

very high 0.37-life (Vallentyne, 1964). Decarboxylation of Val produces 2-methylpropylamine (Meister, 1965), which was not identi®ed. Leucine (Leu) and Isoleucine (Ile) decompose rapidly in the fossil record until approx. 80% of that found in the Recent samples of the same species has decomposed by 3.3 Ma (Fig. 15). Most samples show an increase between the Recent and the youngest fossil samples. The proportion of free amino acids rises rapidly to around 80% by 0.5 Ma for both Leu and Ile, and this level is maintained for most of the period under study, although in older samples the proportion of free amino acids decreases. Such levels of free amino acid indicate that some Ile remains bound by an HCl sensitive bond in the fossils (Fig. 16). Pyrolysis of Ile did not yield identi®able products (Vallentyne, 1964), although Leu released ammonia on decomposition. The formation of ammonia only represented 25±40% conversion when 85±98% of the Leu was lost, indicating that deamination was not the sole pathway of decomposition (Vallentyne, 1968). The data presented in this study identi®es a

Degradation of intracrystalline proteins and amino acids

401

Fig. 10. The proportion of Arg (A) and Lys (B) present in the free state. Note that unlike other amino acids, the proportion in the free state rapidly declines. A value of 0% free in older samples corresponds here to complete destruction of the amino acid (see also Fig. 9).

much more rapid rate of decay of Leu than is indicated by the pyrolysis reactions. Such rapid decomposition of this amino acid (when compared to pyrolysis experiments) has broad implications for

Fig. 11. Graph to show the changing concentration (free and combined) of Arg and its decomposition product ornithine in Calloria. A similar pattern is observed in the other samples, but Calloria is shown as this is the genus with the highest number of samples.

previous studies, some of which (for example Bada and Man, 1980) have represented the data as being Leu equivalents, or have used the concentration of Leu as a constant on the basis of the stability in pure aqueous solution (Bada et al., 1978). Leu is very stable when pure solutions of the amino acid are pyrolysed, but this does not appear to be the case when the amino acids are peptide-bound, in free mixtures or when associated with an inorganic phase. Comparisons with the results of pyrolysis of pure compounds need to be drawn with caution in these samples, and this is con®rmed by the pyrolysis of oyster shell powders (Totten et al., 1972), where Leu is also rapidly decomposed. Glycine (Gly) is one of the most thermally stable amino acids (Abelson, 1954). However, although Calloria, Neothyris and Terebratella demonstrate an initial increase, there is considerable degradation over the period studied, resulting in a highly variable loss [Fig. 17(A)]. All samples show a general decreasing trend over time, with up to 80% of the molecules being degraded by 3.3 Ma.

402

D. Walton

Fig. 12. The concentration (free and combined) of Tyr (A) and Phe (B) plotted against sample age. Both show very rapid decomposition, in some cases after an initial increase (Calloria and Neothyris; see

The proportion of Gly present in the free state is similar in all species examined, with a rapid rise to greater than 75% free, followed by a maintenance of this level [Fig. 17(B)]. The oldest samples show a slight decrease in the proportion of free amino acids, indicating that Gly decays whilst in the free state. The major pathway for the degradation of Gly under pyrolysis is via decarboxylation to produce methylamine (Vallentyne, 1964), although this product was not positively identi®ed in this study. In most samples, there is a rise in the concentration of Gly, indicating formation as a decomposition product from Val, Ser, Thr or Tyr. These reactions are considered in the relevant sections. Where Gly is the decomposition product, the ``new'' molecule will decompose in the same way as the original Gly, and hence the overall concentration of this amino acid will decrease once production from other sources has ceased or declined. The diagenetic production of this amino acid will distort the pattern of its occurrence in fossil shells.

Alanine (Ala) is one of the group of amino acids which are thermally very stable (Abelson, 1954; Vallentyne, 1964). The concentration within the shells is an essentially random spread with respect to age, with a large increase between the Recent and the fossil, with the maximum at approx. 0.5 Ma [Fig. 6(A)]. This is followed by a decreasing trend, although in all cases the concentration of Ala in the oldest samples is similar to, or greater than, the initial concentration found in Recent samples. The maxima of Ala concentration correlates with the highest proportion in the free state. There is a rapid increase in the proportion of free amino acids with greater than 90% being free in most cases by 0.5 Ma [Fig. 6(B)]. A high proportion of free Ala remains throughout the samples studied, indicating that the increase in concentration occurs in the free state, rather than by degradation of peptide-bound amino acids. Decay of Ala is via decarboxylation to form ethylamine (Abelson, 1954), although pyrolysis is very slow under nitrogen, indicating that thermal decomposition may not be important

Degradation of intracrystalline proteins and amino acids

403

Fig. 13. The proportion of Tyr (A) and Phe (B) present in the free state. The irregular pattern is caused

(Abelson, 1954). In the presence of oxygen, however, the reaction rates would increase dramatically (Conway and Libby, 1958). The rate of decay of Ala appears, from both this and previous studies (Hare and Mitterer, 1967, 1969), to be much more rapid than that recorded for pyrolysis of a solution of the pure amino acid. This phenomenon could have a number of causes, notably the presence of oxygen, surviving peptide bonds within the shell and the e€ect of mixtures of amino acids (Vallentyne, 1964). The concentration of Ala present in the Recent samples is almost identical to that recovered from samples dated at 3.3 Ma. The intervening period, however, shows major variations in concentration, with the increase being due, at least in part, to its diagenetic formation. There is no di€erence between the original and diagenetic Ala in terms of decomposition pathways, hence some formed diagenetically will also decompose. As there is no direct correlation between the decomposition of Ser and the production of Ala, it is likely that other diage-

netic reactions may produce Ala, for example the bdecarboxylation of Asx. Proline (Pro) has an aliphatic side chain which is bonded to both the nitrogen and the a-carbon atoms. Pro is the most stable of the amino acids tested by pyrolysis at temperatures below 2128C (Vallentyne, 1968), although it shows rapid decay in the fossil record to a level representing greater than 80% loss over the 3.3 Ma of the study (Fig. 18). The proportion of Pro present in the free state rapidly increases to greater than 80% in 0.2 Ma, and maintains that proportion throughout the rest of the period under study. In pyrolysis experiments, Pro decomposed to form ammonia, indicative of decay by deamidation (Vallentyne, 1968), although calculated 0.37-lives indicate that no decomposition would be expected at the temperatures the fossils have been subjected to. Again, the data presented here contradicts this and shows that there is an increased rate of decomposition in fossil samples, not mimicked by pyrolysis of the pure compound. Other factors, such as the

404

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Fig. 14. The concentration (free and combined) (A) and proportion in the free state (B) of Val plotted against sample age. Note in (A) the initial increase in concentration and the relatively slow rate of decomposition, demonstrating that Val is one of the most stable of the amino acids and the slowing in

inorganic component of the shell or the e€ect of mixtures of amino acids, clearly have a major e€ect on the rate of decomposition. Sulphur-containing side chains. Cysteine and methionine contain sulphur within their side chains. During the hydrolysis of the samples in preparation for analysis, these amino acids undergo variable degradation, and these molecules were therefore not quanti®ed. The e€ect of carbohydrates on the destruction of amino acids The reaction between carbohydrates and amino compounds is well documented (for example Hoering, 1973; Collins et al., 1992). The reactions begin with interactions between the reducing groups of sugars and amino groups of other compounds to form glycosylamines. The end product of the reaction is a dark heteropolymer referred to as melanoidin (Hoering, 1973). Carbohydrates are present within the shells of brachiopods (Collins et al., 1991), but it is not certain whether they are

attached to the protein in these samples (M. Cusack, University of Glasgow, U.K., pers. comm.). However, at least some of the proteins in the brachiopod Terebratulina retusa are glycosylated (J. Laing, University of Derby, U.K., pers. comm.). Although not examined in this study, carbohydrates have an e€ect on amino acid decomposition. Pyrolysis reactions involving amino acids and glucose (Vallentyne, 1964) indicate that the higher the concentration of glucose in a standard solution of Ala, the faster the decomposition rate of the amino acid. The 0.37-life of the Ala at 1678C without glucose is just over 10 yr, but is reduced to approx. 2 h when 0.05 M glucose is present. This interaction of carbohydrates with amino acids could explain some of the accelerated degradation reactions detected in this study. The use of pyrolysis as a predictive tool Pyrolysis experiments have been the only method by which amino acid degradation over an equivalent time scale has been examined prior to this

Degradation of intracrystalline proteins and amino acids

405

Fig. 15. The concentration (free and combined) of Leu (A) and Ile (B) plotted against sample age. Both decompose rapidly in contrast to the ®ndings of previous studies.

study. Several authors have considered the pyrolysis of shell powders, in addition to the pure solutions of amino acids (for example Jones and Vallentyne, 1960; Vallentyne, 1964; Hare and Mitterer, 1969; Totten et al., 1972). Some of the amino acids most stable to pyrolysis as solutions of the pure amino acids, such as Phe, Lys and Asx were some of the least stable of the amino acids when fossilised (Jones and Vallentyne, 1960). The decomposition of Ala was studied by the pyrolysis of shell powders, where it occurred more rapidly than in an aqueous solution of the pure compound, a ®nding con®rmed by this study. This is likely to be due to one of three possibilities (Jones and Vallentyne, 1960; Vallentyne, 1964). Firstly, the act of heating does not mimic the e€ect of time accurately. Implicit in this statement is the e€ect of pressure on the decomposition of solutions of pure amino acids. The reaction vessels were sealed before heating, and the evaporation of the water from the aqueous solution would increase the pressure. No measure of this pressure was made, although Vallentyne (1964)

noted that the vessels frequently shattered during heating, indicating the great increase in pressure. The possibility of the increased pressure increasing the rate of reaction has not been considered in any of the pyrolysis experiments except in the consideration on the rate of hydrolysis (Qian et al., 1995). The burial history of the horizons containing the fossils will have a marked e€ect on the rate of decomposition reaction. Pyrolysis data (Vallentyne, 1964, 1968) shows that the rate of reaction will increase by an order of magnitude between 20 and 408C. Such an increase in temperature represents burial of approximately a kilometre, a possibility which exists for the samples under study (Fleming, 1953; Anderton, 1981). The results of pyrolysis experiments cannot therefore be directly applied to fossil amino acids unless a detailed burial history for each bed is known. Without this, the pyrolysis results cannot be used as a predictive technique for assessing the age or the molecular state of preservation of fossil biomolecules.

406

D. Walton

Fig. 16. The proportion of Leu (A) and Ile (B) present in the free state. Some molecules remain in pep-

Secondly, the stabilities of the amino acids may be a€ected by factors other than temperature. For example, metal ions catalyse oxidative deamination, by chelation of the amino acid (Ikawa and Snell, 1954). Ca2+ or Mg2+ ions present within the shell carbonate could act as a chelation site for the amino acids. Thirdly, interactions between carbohydrates and amino acids which have already been discussed. The molecular state of preservation The proteins of these fossil brachiopods are almost completely decomposed, with only a limited number of peptide bonds surviving fossilisation. The presence of relatively unstable molecules such as Asx in peptide-bound compounds is likely to be due to the stabilising e€ect of the peptide bond, the stability of which is a function of the nature of the residues on the other side of the bond (Hill, 1965). All samples have a proportion of free amino acids which rapidly (within 0.5 Ma) rises to greater than 80%. The rate of hydrolysis then slows and may decline, corresponding to the destruction of the

most labile peptide bonds and the preservation of less labile ones. Individual amino acids also undergo degradative reactions, the majority of which take place when the molecule has been released from the protein and is present in the free state. Degradative reactions produce a range of reaction products, including the diagenetic generation of other amino acids which may be either proteinogenic or non-standard and which may distort any taxonomic relationship through time (Walton, 1998). For example, Jope (1967) found that the insoluble fraction of the intercrystalline protein from fossil brachiopods showed marked di€erences from the nearest living relatives. This study also found raised Asx in the insoluble fraction, possibly indicating either post-mortem alteration or contamination. Both the present study, and that of Jope (1967) contrast with the results of Kolesnikov and Prosorovskaya (1986), who recognised ``very familiar'' compositions between Recent and fossil brachiopods. Such results need to be treated with caution as this study has shown that, at least in the soluble fraction, the amino acids are un-

Degradation of intracrystalline proteins and amino acids

Fig. 17. The concentration (free and combined) (A) and the proportion in the free state (B) of Gly plotted against sample age. Although in (A) there is a general decreasing trend, the concentration is highly variable, representing the diagenetic formation of Gly. Older samples have a lower proportion

Fig. 18. The concentration (free and combined) of Pro plotted against sample age. Although pyrolysis of the pure compound indicates that this is one of the most stable of amino acids, the data here suggest that this is not the case.

407

408

D. Walton

stable to di€ering degrees which results in changing amino acid ratios over time. Fossil samples will have di€erent amino acid ratios to those of extant species. The advantage of using intracrystalline biomolecules is therefore to be certain of in situ degradation, where the products of degradative reactions are contained within the shell and not lost. Although the bulk amino acid composition of samples represents an oversimpli®cation of the nature of the proteins, it is likely to be the sole method of analysis in older samples, where the proteins have been totally degraded. If the biomolecules are undergoing in situ decay, then the only cause of this loss will be from chemical degradation, rather than di€usion of original amino acids out of the shell or microbial degradation, as is the case for intercrystalline biomolecules. CONCLUSIONS

These results indicate that pyrolysis experiments of pure compounds can act as a useful guide for the study of the relative rates of decomposition of amino acids in fossils, but they are not representative of the natural system. Therefore pyrolysis of shells must also be undertaken to discern the e€ects of the carbonate, and other factors associated with the shell. The burial history of any given sample will have a great e€ect on the degradation of the intracrystalline biomolecules. As the samples are successively buried and exhumed, temperature and pressure regimes will change, either increasing or decreasing the rate of reaction. Pyrolysis of pure compounds should not be applied directly to the fossil record, but only in combination with shell pyrolysis experiments and information on the burial history of the fossils. Due to the level of both natural hydrolysis and amino acid decomposition that has taken place in the fossil samples, it is highly unlikely that proteins from these samples will survive in a state whereby they may be routinely separated and analysed for primary sequence data. The identi®cation of pathways and products is required to reconstruct the original amino acid composition. Associate Editor Ð R. L. Patience

AcknowledgementsÐThis work was conducted during the tenure of a U.K. NERC studentship (GT4/89/GS/42) in the Department of Geology and Applied Geology, University of Glasgow, which is gratefully acknowledged. I wish to thank Drs Maggie Cusack, Matthew Collins, Heather Clegg and Phil Jackson for advice throughout the work and two anonymous referees who added enormously to the manuscript. I am indebted to Sandra McCormack for technical assistance in sample preparation. This work was written during a sabbatical funded by the University of Derby.

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