Molecular recognition properties of peptide mixtures obtained by polymerisation of amino acids in the presence of estradiol
Introduction
In nature, molecular recognition plays a decisive role in the chemistry of biological phenomena. In the attempts to mimic these systems, the scientific community has developed increasingly synthetic recognition systems based on tailor-made supramolecular organic structures [1], [2]. In these systems selectivity often is enhanced if, as happen in nature, a cavity exists that has been shaped to match that of the substance to be recognised. In the molecular imprinted technique these recognition systems can be obtained in highly cross-linked organic polymers: in fact complementary shaped cross-linked cavities can be formed around a molecule that acts as a template during the polymerisation step, and when the template is removed, an imprinted cavity with reversible binding capacity remains behind in the polymer [3], [4]. The molecular imprinting of organic polymers is today a promising area that includes many research fields like chromatography [5], [6], catalysis [7], and sensor technology [8]. Anyway, all artificial systems based on this kind of technique are rigid organic polymers that work successfully in organic solvents, but are far from the natural molecular recognition systems based on protein structures. Nevertheless, some theoretical studies suggest the possibility of preparing “protein-like” polymers containing active sites capable of recognising a given target molecule and characterised by a well-defined memory effect towards the template [9], [10], [11].
We adopted a different approach to “classical” molecular imprinting to mimic the binding function of protein-based natural recognition systems. We thought that in a mixture of natural amino acids, non-covalent interactions between a template and the amino acids should be able to shape the structure of growing peptides, forcing the polymerisation process to produce a template-complementary sequence. Moreover, the large structural heterogeneity in the amino acid side chains introduces a great variety of chemical properties, which can be useful in this kind of template polymerisation in order to exploit many different interactions with the template (i.e. ionic, hydrogen-bond, charge-transfer, hydrophobic), increasing the possibility of obtaining an efficient recognition system also in a polar medium such as water; thus mimicking the binding environment of natural receptors like proteins.
The aims of our work were to obtain tailor-made synthetic polypeptides with molecular recognition properties towards low molecular mass molecules. As briefly reported previously [12], we are attempting to demonstrate the feasibility of this approach by preparing mixtures of water-soluble synthetic peptides with molecular recognition properties towards the steroidal hormone estradiol.
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Materials and methods
l-Amino acids, methanol, N,N-dimethylformamide (DMF), N,N-dicyclohexylcarbodiimide (DCCD), N,N′-diisopropylcarbodiimide, all chemicals for buffers were from Merck (Darmstadt, Germany). Estradiol and peptides used to measure molecular weights were from Sigma (Milwaukee, USA). Labelled steroids were from Amersham (Uppsala, Sweden) and were in a toluene:ethanol (9:1) solution at the concentration of 25 μCi/ml. The specific activities of the markers were 85 Ci/mmol for testosterone and progesterone,
Synthesis and purification of the peptides
The amino acid composition of the polymerisation mixtures was chosen to be similar to the bovine serum albumin, as it appears to be relatively similar to the average composition of animal proteins. To study the binding properties of both cross-linked and linear structures different amino acid mixtures were polymerised, as reported in Table 1. The first ones contained Lys, Asp and Glu amino acids, able to give cross-linked structures “branched mixtures”; the second ones were different only for
Conclusions
The experimental results reported in this paper show that by polymerising a mixture of amino acids in presence of a molecule able to act as a template, it is possible to obtain a mixture of peptides that show selective molecular recognition properties towards the template itself.
The presence of cross-linking amino acids seems to influence the resulting peptide binding properties. The same occurs by increasing the polymerisation time. As the affinity of peptide mixtures obtained by
Acknowledgements
This work was supported by the Italian Ministry of University and Scientific & Technological Research (MURST), project COFIN99-9903032732-005 “New immunometric methodologies for the detection of endocrine modulators in waters and biological matrices”.
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