FORMATION OF AMINO ACID CONDENSATES PARTLY HAVING PEPTIDE BONDS IN
A SIMULATED SUBMARINE HYDROTHERMAL ENVIRONMENT
Kuhan Chandru, Yumiko Obayashi, Takeo Kaneko, and Kensei Kobayashi*
Department of Chemistry, Graduate School and Faculty of Engineering, Yokohama National
University, 79-5, Hodogaya-ku, Yokohama 240-8501, Japan
Corresponding author: kkensei@ynu.ac.jp(Received: July 19, 2013; Accepted: August 9, 2013)
Abstract
Although abiotic polymerization of amino acids
is an important step in origin of life study, it has often
eluded that their formation could be limited in thehydrothermal systems. To show this we heated anaqueous solution of several amino acids in asimulated hydrothermal environment to assess thepossible formation of peptides among the heatedproducts. Peptide concentrations estimated by the
Lowry method were significantly higher when the
mixture was heated at 300°C than those at lower
temperatures, despite having more than 80% of theinitial amino acids decomposed. We also revealed
that the peptides measured here were only part of thebonds in the heated products. The major heatproducts were non-peptide amino acid condensates(NPACs) that only possess partial peptide bonds. The
role of NPACs should be examined though they were
often ignored in the classical chemical evolution
scenario so far.

glycine in the system are relatively simpler to predict
due to its simple structure than in the case of using
multiple amino acids in the system. Complication
could arise simply due to reaction among the aminoacids’ side chains. From previous experiments [5], itis well understood that amino acids exposed tohydrothermal heat (in simulators) do recover better
when they are hydrolyzed, suggesting aggregated
and/or condensed product in the products [5]. One
question that needs to be asked, however, is whether
all these product turn into genuine peptides or not
when complexity is added, particularly in this casethat different kinds of amino acids are used. A
number of studies suggested that aggregates or
globules-like structures were formed [11,12,13],when various amino acids were heated at hightemperature. For example, Yanagawa and Kojima[11] demonstrated that microsphere-like structure
were generally formed when a mixture of glycine(0.3M), alanine (0.1M), aspartic acid (0.1M) andvaline (0.3M), were heated in a hydrothermal
simulator (autoclave) at 250°C or 300°C. Similarly,
Fox and Harada [12] showed the formation ofproteinoid microspheres when 18 kinds of aminoacids with a higher concentration of dicarboxylic
amino acids (i.e. aspartic acid and glutamic acids)
were heated. These kinds of structures have been
generally envisioned to play a role in chemical
evolution [11,12,13,14].
This paper attempts to show the features ofthermal condensates of amino acids in submarinehydrothermal simulator, the Super Critical Water
Flow Reactor (SCWFR) (Figure 1) [3,5]. The
survivability of the amino acids was quantitatively
examined. We then characterized the products byinfrared spectrometry and the Lowry assay. Althoughsimilar experiments have shown peptides of glycine
in the product, no work has yet estimated the peptide
yield when plural kinds of amino acids are added in
the system.

(Keywords)
hydrothermal systems, peptides, non-peptide aminoacid condensates (NPACs)
Introduction
Deep-sea hydrothermal environments are an
important area in the study of life’s origin [1]. Due tothe possible availability of starting materials such as
CH4, C2H6 and CO with heat, the hydrothermal
environment is a plausible spot to spark the earliest
life forms on earth [2]. Many experiments wereconducted using simulators to form biologically
relevant compounds (i.e. amino acids) [3,4] and/or totest their survivability [5].
In recent years, there have been increasing
interests in the formation of polypeptides, where
researchers have shown that oligomerization ofglycine up to hexamers could be obtained underhydrothermal conditions in some simulators [3,4,6,7].
One limitation to these kinds of experiments is that; ahigh concentration of the amino acid (glycine)ranging from 100 mM - 2 M was often used toamplify the yield that are usually at trace levels[3,4,6,7]. Aubrey and co-workers [8] argues that,such high concentration is unlikely in the prebiotic
ocean and their reaction threshold is often exceeded.
They estimated that the carbon reservoir in earlyocean are limited and constitute a small value only,
for example HCN was about 2 micromolar [9] andadditionally, ammonia was about 20 micromolar [10].
Both of these compounds are important starting
material to generate abiotic amino acids.
It is also worth noting, that the heat products ofViva Origino 41 (2013) 24 - 28
© 2013 by SSOEL Japan

Material and Method
We used a mixed solution of glycine, alanine,
aspartic acid and valine at 20 mM each, being lower
concentration compared to previous works[3,4,6,7,11,12], and exposed them at room
temperature (20 °C), 200°C, 250°C or 300°C at 25
MPa. The sample was injected through the IR image
gold furnace for a 2 min of heat exposure at a flowrate of 0.5 mL/min in the SCWFR (Figure 1) (SeeIslam et al, 2003 [3] for apparatus details). We
decided to use these amino acids based on the
GADVs peptide world [15] and Trifonov’s aminoacids temporal order [16].

24Viva Origino 41 (2013) 24 - 28

Figure 1: Schematic diagram of the Super Critical Water
Flow Reactor (SCWFR)

A portion of the samples were then hydrolyzed
with 6 M HCl for 24 hours at 110°C. Bothhydrolyzed and non-hydrolyzed portions weredesalted using a solid phase extraction spin column
Monospin-SCX provided by GL Sciences andanalyzed using an ion exchange HPLC, which ismade up by two HPLC pumps. (Shimadzu LC-10), a
cation exchange column (Shimpak ISC-07/S1504, 4mm i.d. x 150 mm), a post-column derivatization unit,
and a Shimadzu RF-535 fluorometric detector.
N-acetyl-L-cysteine (N-AcCys) and o-phthalaldehyde(OPA) were used as derivatizing agents in borate
buffer. Procedure blanks was done in every steps ofthe experiments (simulation, hydrolysis, desalting
and analysis).
The products (non-hydrolyzed) at all
temperature were analyzed using a Fourier transform
infrared spectrometer (JASCO FT-IR-6200) tocompare their structure. Freeze-dried products werepressed between two KBr plates before analysis.
An aliquot of the samples was also used todetermine the occurrence of peptide bonds using the
Lowry assay protocol [17,18,19]. The method waschosen because of it’s sensitively to low
concentration to peptides. Conventionally, bovine
serum albumin (BSA) has been used as a standard in
most protein calorimetric assay. In this study,
however, pentaglycine was preferred instead tocompensate BSA’s tyrosine and tryptophan residues
and -SH groups [20]. These amino acids are known
to enhance the colour yield of the Lowry solution,
which could underestimate the concentration of
peptide bonds in the products. Absorbance wasrecorded
using
a
JASCO
V-630
UV-spectrophotometer at 750 nm.
Additionally, scanning electron microscopy(SEM) was utilized to access any physical traits in
the heated products, after trapped on a
0.2-micrometer pore size polycarbonate membranefilter or on clean aluminum foil.
All experiments were done using Milli-Q waterand analytical grade chemicals. Amino acids, BSA,
pentaglycine and Lowry solution’s chemicals(sodium tartrate dihydrate, CuSO4•5H2O, NaOH,
Na2CO3) were purchased from Wako pure chemicals.
Only Folin and Ciocalteu’s Phenol Reagent was
obtained by Sigma-Aldrich. All glassware were
baked at 500°C before using for all experiments.

Figure 2: Recovery ratio (%) of amino acids after heatedin the SCWFR at different temperature. Amino acids ineach sample were determined before and after hydrolysis.
Table 1: Estimation of peptides concentration and theircontribution to the total combined amino acids in theheated products.

aPercentage of free amino acids in the total recovered
amino acids after heatingbConcentration difference between hydrolyzed and nonhydrolyzed portions (based from Figure 1) to produce theyield of combined amino acidsc
Peptide concentrations estimated by using pentaglycine asa standard (based on Figure 3) in the non-hydrolyzed
portiond
Peptide bonds corrected by subtracting free amino acids’interferencea in the non-hydrolyzed portionse
Percent contribution of peptide bond to the total
combined amino acids based on the corrected valuesd

consistent with previous works [3,5], where therecovery ratio decreased with the increment of
temperature. The difference between hydrolyzed andnon-hydrolyzed portion were referred to as combined
amino acids’ yield (Table 1). The 300°C samples
exhibit the lowest yield indicating an overall 20% orResults and Discussion
Figure 2 shows the recovery of amino acids
before and after hydrolysis. The present results were25

Viva Origino 41 (2013) 24 - 28

less recovery of amino acids, with no free aminoacids in their non-hydrolyzed portion (Figure 2).
Additionally, glycine and alanine showed higher
recovery than valine and aspartic acid; aspartic acidwas almost depleted at this temperature.
The FT-IR analyses showed that the 200°C and
250°C spectra were somewhat similar to that of room
temperature (control) (Figure 3), indicating theinterference of coexisting free amino acids in theheated samples. The 300°C spectrum, however, wassignificantly different from the others. Absorbance of
amide I (1600-1700 cm-1) and II (1510-1580 cm-1)
regions were found in the 300°C sample (Figure 3),
which might be due to the presence of peptides in theheated products.
The Lowry assay, used to quantify the peptides
showed that the absorbance of the room temperature(control) sample was 0.02 (Figure 4), inferring the

Figure 4: Absorbance of the heat products by the
Lowry assay

positive interference of free amino acids in thesolution [21]. The 250°C samples showed slightly
higher absorbance 0.08 and the 300°C samples
exhibited a very prominent absorption of about 0.27
(Figure 4). Our results suggested that the possible
formation of prebiotic peptides could be morefavorable in hydrothermal conditions, especially at
300°C. As mentioned above, we used pentaglycine toestimate the peptide concentration: The estimatedconcentrations were 0.69 mg/L (200°C), 6.84 mg/L
(250°C) and 75.4 mg/L (300°C), respectively. After
subtracting positive interferences by free amino acids[21] corrected concentrations of peptide bond were
almost 0 mg/L (200°C), 6.4 mg/L (250°C) and 75.4
mg/L (300°C) (Table 1). These contributed about
0.3% (250°C) and 9.3% (300°C) of the total
combined amino acids in the heated products at eachtemperature. The contribution of the peptide bonds inthe amino acids condensates produced at 300°C wassignificantly higher than those produced at lower
temperature. The majority of the products were what
we would define as non-peptide condensates(NPACs) for the all heated sample. Since aspartic
acids were mostly depleted (Figure 2), we predict that
only glycine, alanine and valine were involved in thethermal condensates when heated at 300°C.
The present findings that NPACs were major
products derived from plural amino acids in thesimulation experiments suggested that such complex
molecules could also be major components in theactual prebiotic setting. One would assume that using
singular amino acids (i.e., glycine) does not show thecomplete picture of oligomerization in hydrothermal
environment. Compounds such as NPACs, often
ignored in the classical scenario, could possess somechemical functions relevant to origins of life.
Although we have not shown their functionality here,a similar experiment was done by Oba andco-workers [19], who performed a series ofexperiments using a heat-drying and wet cycle
technique (on a mixture of glycine, alanine, aspartic
acid and valine) to simulate peptide formation basedon the GADV’s protein world hypothesis [15]. After
30 cycles of heat-drying and wet, their products that
also exhibit peptide bond occurrences (by Lowry
Method) could perform hydrolytic activity in thepresence of BSA solutions. Since we applieddifferent process to simulate prebiotic amino acid

Figure 3: FT-IR absorbance spectra of the heat products.
Pentaglycine and BSA are also shown as references. r.t. =room temperature.

26

Viva Origino 41 (2013) 24 - 28

condensation from their heat-drying and wet cycles,
their findings might not be directly transferable toours. Therefore, studies on NPACs’ functionality
should be done in future.
It is also noteworthy that any sort of
microsphere-like structures [11,12] were not detectedby SEM in our heated samples (data not shown). As
mentioned earlier, Yanagawa and Kojima [11]demonstrated that their microspheres were generally
formed when the four amino acids were heated at
250°C or 300°C. However, the amide II band peakwere not detected in their IR spectra; and the role ofsilica from glass tube was also emphasized to form
such structures. In this study, we used the same fouramino acids as their experiment but at lower
concentration. We also avoided using glass tube
during the heating process hence eliminating thepossible influence of silica. However, despite thelack of microsphere-like structures, the 300°C sample
was showing similar attributes to proteinoid
microsphere [12] demonstrated by Fox and Harada,
such as being Biuret positive [17] (Lowry method)
and the presence of the amide I and II peak indicating
peptide bonds. This
study
confirmed
thatformations of morphological structures are often
associated with high concentration of starting
compounds and/or the involvement of other inorganic[14] compounds like silica.

We thank the following individuals for theircontribution to the manuscript: Dr. Pierre deMarcellus (University of Paris-Sud, France), Dr.
Palash Kumar Sarkar (Independent University,
Bangladesh), Dr. Hironari Kurihara (Yokohama
National University), Dr. Akira Naito (Yokohama
National University), Mr. Yukinori Kawamoto(Yokohama National University), Mr. Yuichi Kondo(Yokohama National University), Mr. Riddhideep
Mandal (Yokohama National University), Mr.
Veeramani Krishnan (Keio University), Miss.
Christel Halliez (Individual), Mr. Shinji Ishihara(Yokohama National University) and Dr. Shigetoshi
Aono (Institute for Molecular Science, Japan)
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Conclusion
In this investigation, the aim was to quantify
the thermal condensate in relation to peptides using
multiple amino acids. The combinations of these data
reveal that amino acids did not only degrade withheat, but also form condensation products as well. A
part of the bonds in these products were peptides.
However, the contribution of peptide bonds in the
combined amino acids was less than 10%, even in the
300°C sample, which showed the highest
contribution of peptides. The major constituents ofthe product were non-peptide amino acid condensates(NPACs) for all the heated samples. A limitation ofthis study is that we did not identify the products
structure of NPACs and peptides; due to the fact thatthe products might be too enormous and yield ofindividual species would be too low to accurately pin
point the compounds. However, we have shown thatyield of peptides is rather low in comparison to theoverall product. This research has thrown up manyquestions in need of further investigation. One ofthem is, whether NPACs have any function or not inchemical evolution and/or the origin of life. Complex
organic compounds like NPACs were often ignoredin the classical chemical evolution so far, since theimportance of origin of life research is often focus inbiologically relevant compounds (peptides) only. We
recommend
furtherexperimentsonthenon-biologically relevant compounds like NPACs to
examine their role in chemical evolution on earth andextraterrestrial bodies.
Acknowledgments

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