Amino Acid Racemization Dating

Sean D. Pitman M.D.

August,2004

 

All living things use proteins as building blocks in the construction of their physical forms.  In turn, proteins are composed of folded strands of 20 different smaller subunits called "amino acids". All amino acids, except for one (glycine), come in two different forms known as the levoratory (L - left) and dextrorotary (D - right) forms.  These two forms are called "enantiomers", "chirals", or "stereoisomers", which basically means that they have the same molecular and structural formula but cannot be superimposed on each other no matter how they are oriented in space.  In other words, they are like one's left and right hands, which are mirror images of each other, but cannot be superimposed onto one another. 

What is especially interesting about these two L- and D-forms, at least for the purposes of this topic, is that the vast majority of living things only use the L-form.  However, as soon as the creature dies, the L-amino acids start to spontaneously convert to the D-form through a process called "racemization". If the rate of conversion can be determined, this process of racemization might be useful as a sort of "clock" to determine the time of death.


 

 

Basic Assumptions

 

 

In order to use the rate of racemization as a clock to accurately estimate when a living thing died, one must know how various environmental factors may have affected the rate of change from the L- to the D-form.  As it turns out, this rate, which is different for each type of amino acid, is also exquisitely sensitive to certain environmental factors.  These include:

   

  1.  Temperature  

  2. Amino acid composition of the protein

  3.  Water concentration in the environment

  4.  pH (acidity/alkalinity) in the environment

  5.  Bound state versus free state

  6.  Size of the macromolecule, if in a bound state

  7.  Specific location in the macromolecule, if in a bound state

  8.  Contact with clay surfaces (catalytic effect)

  9.  Presence of aldehydes, particularly when associated with metal ions

  10.  Concentration of buffer compounds

  11.  Ionic strength of the environment

 

Of these, temperature is generally thought to play the most significant role in determining the rate of racemization since a 1o increase in temperature results in a 20-25% increase in the racemization rate.1,2 Clearly, this factor alone carries with it a huge potential for error. Even slight ranges of error in determining the "temperature history" of a specimen will result in huge "age" calculation errors.  

Calibrating for even a known temperature history also seems to be rather problematic.  Consider that the rate of racemization for various amino acids is determined by placing a protein into a very high temperature environment (between 95o and 150o C) and then extrapolating these results to low temperature environments.1

Such extrapolations have been fairly recently (1999) called into question by experiments showing that models based on high temperature kinetics fail to predict racemization kinetics at physiologic temperatures (i.e., 37o C). The authors of this particular paper went on to suggest that, "As conformation strongly influences the rate of Asu [cyclic succinimide] formation and hence Asx [aspartic acid + asparagine] racemization, the use of extrapolation from high temperatures to estimate racemization kinetics of Asx in proteins below their denaturation temperature is called into question . . . We argue that the D:L ratio of Asx reflects the proportion of non-helical to helical collagen "3   Others, such as Collins and Riley have commented in the press that, "racemization in free amino acids has, unfortunately, little bearing on observed racemization of archaeological biominerals."1

Other experiments have shown that the L- and D-forms of the same amino acid do not racemize at the same rate.  This means that the equilibrium ratio may be off from "50:50" by as much as 25%.4   

As far as pH is concerned, it seems that the temperature of a solution also affects the pH of that solution.  So, the amino acid racemization (AAR) rates not only change with the effects of temperature, but also with the concurrent effects of pH changes, which are themselves affected by temperature.1 This can only increase the potential range of error for age determinations.   The local buffering effects of bone and shell matrixes are supposed to limit this effect, but it is still something to consider as potentially significant when acting over the course of tens of thousands to millions of years. 

Also, the actual physical structure of an intact protein significantly affects the rate of racemization of various amino acids.  In fact, in many cases this may even be a more significant factor than the temperature history.  As it turns out, the N-terminal amino acids racemize faster than the C-terminal amino acids of the same types.  Also, the surface amino acids racemize much faster than the interior amino acids.  And, interestingly enough, free amino acids have the slowest racemization rate of all.  Studies with short peptides have shown that, "replacement of the asparagine residue with aspartic acid resulted in a 34-fold decrease in the rate of succinimide (Asu) formation. In the position carboxyl to asparagine in the peptide the replacement of glycine with a bulky amino acid such as proline or leucine resulted in a 33-50-fold decrease in the rate of deamidation"1 [Emphasis added] This clearly emphasizes the rather dramatic importance that amino acid position and overall protein amino acid sequence has on the racemization rate.

  Hydrolysis, or the process of breaking down a protein into smaller and smaller fragments, clearly affects the rate of racemization. The rate itself of hydrolysis "depends on the strength of the individual peptide bonds, which in turn is determined by the characteristics of the amino acids on either side of the bond, the presence of water and the temperature."1 With increased hydrolysis, the overall rate of the whole specimen would increase since there are more terminal end and surface amino acids to undergo higher rates of racemization.  All of these are confounding factors, which, if not known exactly over extended periods of time, would play havoc with any sort of age determinations. Even the process of preparing a specimen for racemic dating can affect the D/L ratio.

In this light, consider that age determinations are usually performed on specimens for which the amino acid order of the original proteins is unknown.  For example, consider that neither the structure nor the proportion of the amino acids used for dating coral, ostrich eggshell, or snail shells is known. Again, those like Goodfriend and Hare (1995) have pointed out the "difficulties in estimating the amount of asparagine in proteins and reminded researchers of the dangers of extrapolating from the behaviour of pure Asn in solution at high temperature, to the behaviour of Asn in proteins associated with biominerals at ambient temperature . . . Using a simplified model of 'racemization kinetics' of bone collagen over geological time Collins shows that almost any value of D-Asx can be obtained by varying the rates of collagen denaturation and leaching of the denatured product."1  

The Interaction of bacteria and fungi with organic specimens may also be problematic.  Such creatures have various enzymes that can digest various types of proteins, such as collagen, that are commonly used for dating. Various studies, such as those dealing with 18th and 19th century burials, showed "unexpectedly high levels of aspartic acid racemization."  The authors "suggested that either biological or chemical degradation of the tooth collagen might have caused these results." 1 Also, certain types of bacteria and other creatures actually produce the D-form instead of the L-form of amino acids.  So, special care is obviously needed in order to particularly avoid this sort of contamination.

 

Calibrating the Amino Acid "Clock"

Clearly, all of these factors create great difficulty for amino acid racemization as a dating technique.  In fact, the difficulties are so great that this technique cannot be and is not used as any sort of "absolute" dating technique.  So, how is it thought to be at all helpful?  Well, it is thought to be helpful as a "relative" dating technique. 

To overcome the various uncertainties inherent to amino acid dating, the method must be "calibrated" based on other more reliable techniques such as radiocarbon dating (carbon 14 dating).  What happens is that a specimen from a site is chosen as the "calibration sample" and both a radiocarbon date as well as a D/L amino acid ratio is determined.  These values are used to solve for a constant or "k" in the formula used to estimate ages based on the calibration sample.  Of course, the "major assumption required with this approach is that the average temperature experienced by the 'calibration' sample is representative of the average temperature experienced by other samples from the deposit."1

Much effort has gone into transforming the data in various ways to achieve linearity between the D/L ratio and the calibrated age of the specimens in a given location.  At first "cubic transformations"' and then later "power function transformations" were used that seemed to show a "strong correlation with time, but did not explain the observed kinetics."

What this basically means is that amino acid dating is not based on any sort of understanding about how racemization takes place, but is strictly a function of correlation with other dating techniques, such as the radiocarbon technique. So, if there is any problem with the basis of the correlation (i.e., radiocarbon dating) then there will also be the same problem with amino acid dating. 

In this light, it is interesting to consider what happened in 1974 when some of the major proponents of amino acid dating (Bada et al) decided to analyze the Paleo-Indian skeletal material from Del Mar, California.  Their estimated age of 48,000 years before present (BP) "stunned" the archaeological community who generally believed these bones to be less than 10,000 years old. Bada went on to date other skeletal specimens between the 35,000 and 48,000 year range with one specimen from Sunnyvale being dated at an astonishing 70,000 years BP. Then, in the 1980s, something very interesting happened.

"The Sunnyvale skeleton and the Del Mar tibia were re-dated using uranium series dating. This resulted in dates of 8,000 to 9,000 years BP for Sunnyvale and 11,000 to 11,500 for Del Mar.  Conventional plus accelerator mass spectrometry (AMS) radiocarbon dating (Taylor et al. 1983) was carried out on the Sunnyvale skeleton and results of between 3,600 and 4,850 years BP were obtained. The original amino acid extractions from the racemization studies of the Paleo-Indian remains were independently dated by the AMS radiocarbon method at the Oxford Radiocarbon Accelerator Unit of Oxford University and the NSF Accelerator Facility for Radioisotope Analysis, University of Arizona. Bada et al., (1984) published the Oxford results and Taylor et al., (1985) published a paper combining the results from both laboratories. The Oxford dates were all between 4,500 and 8,500 years BP and the Arizona dates were between 3,000 and 6,600 years BP. Bada et al., (1984) stated that the Oxford AMS results reveal no clear relationship between the radiocarbon ages of the various skeletons and the extent of the aspartic acid racemization. They did note that there appeared to be a direct relationship between the extent of racemization and the level of preservation of collagen in the bones. Those samples with the most racemization had the lowest amino acid content and this poor preservation of protein would contribute to anomalous AAR results.

Later, based on AMS radiocarbon dates, Bada (1985) calculated a new value for kasp for the Californian samples. He used the Laguna skull and the Los Angeles Man skeleton as 'calibration' samples for this. Using the revised value for kasp he recalculated the AAR dates of the other Paleo-Indian samples. They all fell within the Holocene but had much larger error estimates than those of the AMS values. Although Bada claimed consistency between AAR and AMS dates others (Pollard and Heron 1996, p. 228) argue that the dates only appear to be consistent with one another because of the unacceptably large error range associated with the AAR dates. Pollard and Heron also point out that there is poor concordance between the conventional and the AMS radiocarbon dates and there is no concordance between the uranium series dates and any of the other dates either. At best three of the four methods put the bones in the Holocene."1

 

Because of these problems AAR dating of bone and teeth (teeth in different locations in the same mouth have been shown to have very different AAR ages) is considered to be an extremely unreliable practice even by mainstream scientists. That is because the porosity of bones makes them more "open" to surrounding environmental influences and leaching.  Specimens that are more "closed" to such problems are thought to include mollusk shells and especially ratite (bird) eggshells from the emu and ostrich.  Of course, even if these rather thin specimens were actually "closed" systems (more so than even teeth enamel) they would still be quite subject to local temperature variations as well as the other above-mentioned potential problems. For example, even today "very little is known about the protein structure in ratite eggshell and differences in primary sequence can alter the rate of Asu formation by two orders of magnitude [100-fold] (Collins, Waite, and van Duin 1999). Goodfriend and Hare (1995) show that Asx racemization in ostrich eggshell heated at 80 oC has complex kinetics, similar to that seen in land snails (Goodfriend 1992). The extrapolation of high temperature rates to low temperatures is known to be problematic (Collins, Waite, and van Duin 1999). A pilot study would be necessary and a reliable relationship between racemate ratio and time could remain elusive."1 

Also, there is a potential problem with radiocarbon correlations that is quite interesting.  Note what happens to the correlation constant (k) with assumed age of the specimen in the following figures.

 

 

 

 

 

Interestingly enough, the racemization constant or "k" values for the amino acid dating of various specimens decreases dramatically with the assumed age of the specimens (see figures).5 This means that the rate of racemization was thousands of times (up to 2,000 times) different in the past than it is today.  Note that these rate differences include shell specimens, which are supposed to be more reliable than other more "open system" specimens, such as wood and bone.   

Is this a reasonable assumption?  Well, this simply must be true if radiocarbon dating is accurate beyond a few thousand years.  But, what if radiocarbon gets significantly worse as one moves very far back in time beyond just a few thousand years?  In other words, what would it mean for one to assume that the k-values remained fairly constant over time as would seem intuitive?  Well, with the k-values plotted out horizontally on the graph, the calculated ages of the specimens would be roughly affected as follows:5

 

 

Current Fossil Age Assignment

40,000

100,000

350,000

1,000,000 

Adjusted Fossil Age Assignment with horizontal k-values

Figure 1

6,000

11,000

18,000

8,000 

Figure 2

5,000

14,000

18,000

14,00

 

 

 

Clearly this is a dramatic adjustment that seems to suggest that amino acid racemization may be more a reflection of the activities of local environmental differences than any sort of differences in relative ages.  This seems especially likely when one considers that each type of specimen and each different location have different k-values meaning that the radiocarbon-derived constant in one region or with one type of specimen cannot be used to calculate the age of any other specimen or even the same type of specimen in a different location.1 

Add to this the fact that radiocarbon dating is also dependent upon the state of preservation of the specimen. 

 

     "Stafford et al., (1991) discussed AMS radiocarbon dating in bone at the molecular level. They dated a number of fractions (ranging from insoluble collagen to individual amino acids) from each of a selection of differentially preserved mammoth and human bone. Age estimates from the fractions within a bone were consistent if it was well preserved. They concluded that a poorly preserved Pleistocene-age fossil >11,000 years in age would go unrecognised because it would yield a Holocene 14C date. Thus the final irony is that the poorly preserved Californian Paleo-Indian bones would return Holocene 14C dates even if they were actually Pleistocene. The state of preservation of the bone appears to be as important an issue for radiocarbon dating as it is for AAR dating."1

 

 

So, what do we have? In short, it seems like the claims of some scientists that amino acid racemization dating has been well established as reliable appears to be wishful thinking at best. The huge number of confounding factors and a complete inability to explain the calibrating k-values in terms of amino acid kinetics leaves those with even a tiny pessimistic bone in their bodies just a bit underwhelmed. 

 

 

 

 

  1. Judith Robins, Martin Jones and Elizabeth Matisoo-Smith, Amino Acid Racemization Dating in New Zealand: An overview and Bibliography, Auckland University, Auckland, New Zealand, March 20, 2001 (http://www.arts.auckland.ac.nz/ant/Staff%20Details%20_18072003/Staff_details_files/LisaMS/my%20papers%20in%20PDF/aar.pdf )

  1. Mike Brown, Amino Acid Dating, Molecular History Research Center and Mikes Origins Resource, Accessed July, 2004 ( http://www.creation-science-prophecy.com/amino/)

  1. Collins MJ, Waite ER, van Duin AC, Predicting protein decomposition: the case of aspartic-acid racemization kinetics. Philos Trans R Soc Lond B Biol Sci 1999 Jan 29;354(1379):51-64  (Fossil Fuels and Environmental Geochemistry (Postgraduate Institute), NRG, University of Newcastle-upon-Tyne, UK. ( m.collins@ncl.ac.uk )

  1. Meyer MW, Meyer VR, Ramseyer S, The kinetics of diastereomeric amino acids with o-phthaldialdehyde, Chirality 1991;3(6):471-5 PMID: 1812958, UI: 92256092 (Institute of Organic Chemistry, University of Bern, Switzerland)

  1. R. H. Brown, Amino Acid Dating, Origins 12(1):8-25 (1985). (http://grisda.org/origins/12008.htm )

 

 

 

 

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