The thermal copolymerization of lysine with other alpha-amino acids was studied. The identity of the second amino acid influences various properties of the polymer obtained, including the proportion of alpha and epsilon linkages of lysine. A review of linkages in proteinoids indicates alpha and beta linkages for aspartic acid, alpha and gamma linkages for glutamic acid, alpha and epsilon linkages for lysine, and alpha linkages for other amino acids. Thermal proteinoids are thus more complex in types of linkage than are proteins

Fox, S. W.

Suzuki, F.

English

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iLINKAGES IN THERMAL COPOLYMERS OF LYSINE
(NASA-CR- 14614 5) LINKAGES IN THERMAL	 N76-16236
COPOLYMERS OF LYSINE (diani Univ.) 11 p HC
53.50	 CSCL 07C Unclas
G3/27 13524
Sidney W. Fox
and
Fusae Suzuki
1-13 
'1^`FFS 1976
RECEIVED	 f +,
NASA M FAOUIX a'
'^ ^ 1N^lR BNACtGd,hcl;^'
Institute for Molecular and Cellular Evolution
University of Miami
Coral Gables, Florida 33134
U.S.A.
To Appear in BioSystems
I	 .
Abstract
The thermal copolymerization of lysine with other
a-amino acids has been studied further. The identity
of the second amino ac'_;: influences various properties
of the polymer obtained, including the proportion of
a and a linkages of lysine. A review of linkages in
proteinoids indicates a and a linkages for aspartic
acid, a and y linkages for glutamic acid,a and
linkages for lysine, and a linkages for other amino
acids. Thermal proteinoids are thus more complex in
types of linkage than are proteins.
t
i	 r
1
The propensity of lysine to copolymerize by heating
with other amino acids has been reported (Harada, 1559;
Fox et al., 1962; Harada and Fox, 1965; Heinrich et al.,
1969). Some data have been presented for the proportion
of bonding of lysine through the a- and e-lysine groups.
In this paper more data are reported; these permit com-
paring the influence of some other amino acids on the
type of linkage produced.
METHODS
Copolycondensation of lysine
Free DL-lysine (1.02 g) was heated at 170° to for-i the
clear lactam. The second amino acid was added after 45 min
of totgl heating. The mixture was then heated with stirring
at 195 under nitrogen gas for 2^ h. Gas evolved vigorously.
Wat^r, 20 ml, was added to the brownish solid, and the mix-
ture was dialyzed for 5 days with 3 changes of water each
24 h. The water-soluble and water-insoluble fractions of
the contents of the sac were separated; the water-soluble
fraction was lyophilized.
In other experiments, lysine monohydrochloride was
used instead of free lysine. Since these latter reaction
mixtures were not fluid, 1.0 ml of phosphoric acid (Baker,
85% orthophosphoric acid) was added per each 0.02 mol
(0.64 g) of lysine HC1. The liquid was heated at 195°C:
under nitrogen for 10 h. The product was worked up in the
same way as the polymer from free lysine.
Amino acid composition of polymers
Copolymers were dried in the Abderhalden pistol, using
tetralin of b.p. 208°, or water, until the sample was at
constant weight. Aliquots were dissolved ^n 6 N HC1, sealed
in tubes, and heated for hydrolysis at 100 for 72 h. The
hydrolyzate was dried in a vacuum desiccator, water was
added, and drying was repeated. The solution was analyzed
on a Phoenix K-5000 analyzer.
Infrared absorption spectra
Copolymers were pressed into KBr pellets and examined
on a Baird Atomic Inc. Model KM-1 infrared spectrophotometer.
_Assay of ratio of a- and
E:-linkages (Folk, 1956)
Polymer, 0.3. g, was dissolved in 1.0 ml of water con-
taining 0.1 g of sodium bicarbonate, and 2.0 ml of 2,4-
dinitrofluorobenzene solution (5% in ethanol) was added.
The mixture was shaken mechanically for 2-3 h at room temp.
1	 ^`
2
A yellow ppt formed. Water, 2.0 ml, was added to dilute
the ethanol for extraction, and the unreacted dinitro com-
pound was extracted with 3-4 ml of ether several times.
The ether layer was separated by centrifugation.
The water layer was acidified with several drops of
cone. HC1 to form additional precipitate which was washed
with ether and acetone and dried. A sample of the DNP-
polymer, 30 mg. was then hydrolyze in a sealed tube for
24 h with 2 . 0 ml of 6 N HC1 at 100 C. This solution was
extracted with several 5 ml portions of ethyl acetate to
remove any N-terminal di-DNP - lysine.
The ethyl acetate solution was washed with dilute
HC1 to extract E-DNP-lysine into the water layer com-
pletely. The combined aqueous solution was then evapor-
ated in a vacuum desiccator over sodium hydroxide. The
residue was dissolved in a small amount of water and chroma-
tographed on paper with 1 M citrate buffer of pH 6.2. Each
derivative was eluted with 1.5% sodium bicarbonate and
assayed at 360 nm in a Bausch and Lomb Spectronic 20 color-
imeter. The amounts of a- and E-DNP -lysine were estimated
from standard curves.
RESULTS
Free L lysine was successfully copyrocondensed with
L glutamic acid, 2&7aspartic acid (with phosphoric acid as
a solvent), pL- glutamine, I leucine, L-isoleucine, or
L-valine; the products gave positive biuret tests. Amino
acids that did not give positive biuret tests for the
products of polymerization with lysine were L histidine,
L arginine, k tyrosine, and L tryptophan. The thermal
cocondensation of free lysine with aspartic acid was
examined at three different ratios; she results are pre-
sented in Table 1.
Table 2 indicates the composition of thermal copo-
lymers of lysine. Included for comparison is the polymer
of aspartic acid with lysine hydrochloride as well as with
free lysine. Tot&l recovery of amino acids on analysis
ranged between 40-80 percen', incomplete recoveries being
known for the analysis of thermal lysine copolymers. This
could not be overcome by prolonged hydrolysis. The copoly
(lys HC1, asp) yielded a recovery of 35% after 24 r of hydro-
lysis, a value that progressed to 45 % at 92 h of hydrolysis.
Infrared maxima of copoly ( lys HC1, asp), copoly (lys,
glu), anq copoly (lys, gluNH ) were 3300, 3080, 1650, and
1550 cm- , which indicate pe^tide bonds (Fox and Harada,
1960; Fox et al., 1962). In copoly ( lys, asp) and copoly
3Table 1
Thermal Copolymerization of
Aspartic Acid and Free Lysine in
Varying Molar Ratics
Yield
Aspartic Molar Water- Water-
Acid Lysine Ratio insoluble soluble Total
g g g g %
1.24 0.66 2:1 0.9 tr. 49
0.93 1.02 1:1 1.2 tr. 61
0.66 1.36 1:2 1.2 tr. 59
tr = trace.
Table 2
Amino Acid Composition of Thermal
Lysine Copolymers
Composition of Polyraer
Lysine	 Other Amino Acid
Monomers	 %	 %
Lys HC1, Op	 71	 26
Lys, Asp—	 82	 13
Lys, Glu	 44	 50
Lys, G1uNH	 40	 51
Lys, Ala 2	 61	 29
a^ The water-insoluble fraction of polymer was analyzed.
For all other polymers, the water-soluble fraction :;as
analyzed.
i
.1	 I	 +	 I	
I
4
(lys HC:., as$) additional absorption was observed at 1700
and 1790 cm	 These correspond to imide bonds (Fox and
Harada, 1960).
k'
All polymers of lys with asp or glu or glu(NH.) yielded,
in 1% aqueous solution, pH of 6.0 or less. The co6oly (lys,
ala) gave a pH of 8.3, consistent with an excess o- basic
groups in this polymer.
Table 3 presents the proportions of a- and c--linkages
in three copolylysines. The proportion of a-linkage is
equivalent to the proportion of a-DNP-lysine found, while
the proportion of e-linkage is proportional to the a-DNP-
lysine recovered. The a,e-di-DNP-lysine, having been removed
before analysis, is ignored in these calculations. The re-
sults with aspartic acid copolymer are in close agreemen'.
with the findings of others (Harada, 1959; Fox, 1966).
The proportion of a-linkage is major in the case of the
glutamic acid copolymer and minor for the aspartic acid co-
polymer. ThiE indicates that the second amino acid signJ_fi-
cantly influences the mode of linkage of the one under study,
in this case lysine.
DISCUSSION
The behavior of lysine in these studies of thermal
polycondensation with other amino acids is consistent with
earlier observations (Harada, 1959; Fox et al., 1962;
Harada and Fox, 1965; Heinrich et al., 1969).
The catalog of the tendency of lysine to copolymerize
thermally with other amino acids to yield peptidic products
is further extended by the data presented here. Although
lysine undergoes thermal condensation with several a-amino
acids it does not do so with some others. This reveals a
tendency that is less than that observed for aspartic acid
with comonomeric amino acids, or that of glutamic acid with
additional amino acids, excepting proline (Harada and Fox,
1958). The ability of all common proteinous amino acids to
condense thermally with lysine (Fox et al., 1962) is thus to
be explained by the accessory action of aspartic acid, glu-
tamic acid, and perhaps other amino acids during the same
polymerization.
The results of Table 3, moreover, indicate that the
linkage involved when a trifunctional amino acid like
lysine is pyrocondensed is influenced by the other amino
acids involved (Table 3) and by the temperature (Fox, 1966).
Similar effects of neighboring amino acid residues in the
hydrolysis of aspartoylimide to yield either a- or s-link-
ages have been predicted on the basis of results obtained
5Table 3
Proportion of a- and a-Linkages of Lysine
ResidueF in Three Thermal Copolymers
Linkage
Copolymer	 a, %	 e, %
j
(Lys, asp)	 29	 71
(Lys, glu)	 55	 45
(Lys, gluNH2 )	 56	 44
r}
6in the hydrolysis of various imides (Hoagland and Fox, 1973).
a-Linkages have been reported for thermal copolyaspartic
acids, and the finding has been questioned. Andini et al.(1975)
failad to find, by NMR, a-asparty3 linkages in homopolyaspartic
acid, in contrast to the observations of Kovacs et al. (1961)
who earlier reported a-aspartyl linkages in a number of prepar-
ations (Andini et al.do not cite this paper). Comparative
examination of the results of each group of workers shows that
each studied small fractions (.<5%) of the polyaspartic Ecid
that each made; they obtained these fractions by very dif^erent
techniques. Kovacs et al., for example,used copper salt to
fractionate the crude polyaspartic acid. For formation of the
polyaspartic acid, Andini et al. followed the procedure of
Rohlfing (1967), who studied copolymers of aspartic acid, but
not the homopolymer on which Andini et al. report. The heating
ccnditio$s of Andini et al., patterned after Rohlfing, were
thug 175 at 760 mm for 3^ h, whereas those for Kovacs were
200 at 0.1 mm for 120 h. Rohlfing's polymers have a-linkages
as indicated by biuret tests on polymers of amino acids that
are predominantly difunctional glycine, a structure that per-
mits little linkage other than a (Saunders and Rohlfing, 1972).
While Andini et al. acknowledge that methods of hydrolysis other
than the kind thel used might give a-peptide linkages they, as
stated earlier, do not cite the most thorough study in which
such report has already appeared (Kovacs et al., 1961).
Copolymers of glutamic acid having N-terminal pyroglutamyl
residues must in their nature also have some a-linkage of the
original trifunctional amino acid (Phillips and Melius, 1974).
Additional evidence for a-linkages in thermal copolyamino
acids is found in the isolation of alanylalanine from copoly
(ala, lys) as described by Harada and Fox (1965). The linkage
between the two alanine residues of the dipeptide must of course
be an a-peptide bond.
Andini et al. refer to linkages not commonly found in con-
temporary proteins as "errors". The antievolutionary views
resultant from so labelling variant structures were pointed out
long ago (Fox, 1953).
As a consequence of the above experimental reports, we
must conclude that no evidence has been presented to indicate
that a-linkages of aspartic acid, glucamic acid, or lysine
cannot, or have not, been obtained in thermal copolyamino
acids in addition to w-linkages. It is of course trae that
proteinoids are not proteins; many of them are in 1:,rge part
peptides, and they are polyamino acids. In an evolutionary
context, proteinoids should also not be regarded as prebiotic
proteins, but rather as preproteins.
7In overview, these interpretations are explained el4P-
where (Fox, 1974), essentially as follows: Basic proteinoids
functioned early in evolution to yield protoribosomes at the
surface of which true contemporary proteins might have had
their origin. The proteinoid (more properly referred to as
preprotein than as preb.iotic protein or protoprotein) did not
bequeath its contained information directly to proteins.
Rather, the synthesis of true protein would have begun in a
protoribosome under influence of nucleic acids.* The fact
that proteins and proteinoids exhibit compositional and other
similarities (Mikelsaar, 1975) is understandable on the basis.
that they were produced under similar steric and other con-
straints. These constraints would have acted in a parallel
manner at various stages of evolution, rather than in a serial
manner in which proteinoid structure dictated later protein
structure. The significant aspect of prebiotic proteinoids
would thus have been their functional capabilities in yielding
the first cells and the first ribosomes. A detailed identity
of proteinoid to protein would be neither functionally nor
structurally meaningful, nor even compatible with rudiments
of such an evolutionary theory.
Acknowledgments
This work was aided by Grant no. NGR 10-007-008 of she
National Aeronautics and Space Administration. It is from
a thesis submitted by Fusae Suzuki to the University of Miami
in partial fulfillment cf the requirements for the M.S. degree.
Contribution no. 292 of the Institute for Molecular and Cellular
Evolution. We thank Mr. C. R. Windsor for amino acid analyses.
We thank Drs. Kaoru Harada and Tadayoshi Nakashima for helpful
advice.
*The relationship is illustrated by a synthesis of phenyl-
alanine peptides from ATP and phenylalanine in the presence
of particles composr.d of lysine-rich proteinoid and poly-
adenylic acid. Thu;; appear peptides having necessarily
a-linkages, but requiring phase-separated particles formed
from lysine-rich proteinoid which may not have required a-
linkages (Fox et al., 1974).
8REFERENCES
Andini, S., E. Benedetti, L. Ferrara, L. Paolillo, and P. A.
Temussi, 1975. NMR studies of prebiotic polypeptides, Origins
of Life, 6, 147.
Folk, J.E., 1956, Reactions of glucose with lysine, Arch.
Biochem. Biophys. 61, 150.
Fox, S. W., 1953, A correlation of observations suggesting a
familial mode of molecular evolution as a concomitant of bio-
logical evolution. Amer. Naturalist 87, 253.
Fox, S. W., 1966 1
 Prospectus for chemical synthesis of pro-
teinaceous foodstuffs, in: ed. Ames Research Center, The
Closed Life-Support System (NASA SP-134, Washington) 189.
Fox, S. W., 1974, The origins of biolcgical information and
the genetic code, Mol. Cell-Biochem. 3, 129.
Fox, S. W. and K. Harada, 1960, The thermal copolymerization
of amino acids common to protein, J. Amer. Chem. Soc, 82, 3745.
Fox, S. W., K. Harada, and D. L. Rohlfing, 1962, The thermal
copolymerization of a-amino acids, in: Polyauino Acids, Poly-
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Fox, S. W., J. R. Jungck, and T. Nakashi.ma , 1974, From proteinoid
microsphere to contemporary cell: formation of internucleotide
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Harada, K., 1959, Ph.D. thesis, Osaka University.
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Harada, K. and S. W. Fox, 1965, Characterization of thermal
polymers of neutral a-amino acids with dicarboxylic amino
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Heinrich, M. R., D.L. Rohlfing, and E. Bugna, 1969, The effect
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Experientia 29, 962.
9Kovacs, J., H. N. Kovacs, I. Koenyves, J. Csaszar, T. Vajda,
and H. Mix, 1961, Chemical studies of polyaspartic acids, J.
Org . Chem. 26, 1084.
Mikelsaar, H. _l., 1975, A concept of amino acid archaeorelation:
origin of life and the genetic code, J_ Theor. Biol. 50, 203.
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of amino acids, Int. J. Peptide Protein Res. 6, 309.
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proportions of aspartic acid, Nature 216, 657.
Saunders, M. A. and D. L. Rohlfing, 1972, Polyamino acids: pre-
paration from reported proportions of "prebiotic" and extraterres-
trial amino acids, Science 176, 172.


Linkages in thermal copolymers of lysine

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