Molecular recognition properties of peptide mixtures obtained by polymerisation of amino acids in the presence of estradiol

Analytica Chimica Acta 481 (2003) 41–53

Molecular recognition properties of peptide mixtures obtained by polymerisation of amino acids in the presence of estradiol Gianfranco Giraudi∗ , Cristina Giovannoli, Cinzia Tozzi, Claudio Baggiani, Laura Anfossi Department of Analytical Chemistry, University of Torino, Via P. Giuria 5, 10125 Torino, Italy Received 18 July 2002; received in revised form 30 December 2002; accepted 14 January 2003

Abstract In this paper we show that the carbodiimide-induced polymerisation of amino acid mixtures in aqueous medium and in presence of estradiol produces the mixtures of peptides with an average molecular weight of 2–6 kDa that are characterised by possessing molecular recognition properties towards estradiol. After the removal of the templating molecule, the binding properties of the peptide mixtures were studied using spectrophotometric and immunochemical methods. The experimental results show the presence of molecular recognition behaviour for all the peptide mixtures obtained by polymerisation in presence of estradiol, with affinity constant values between 0.44 × 109 and 6.6 × 109 M−1 , while the same mixtures obtained without estradiol show lower affinity constant values between 2.2 × 106 and 1.3 × 109 M−1 . The molecular recognition behaviour was found to be highly selective, as the binding constants of peptides towards the structural homologues testosterone and progesterone are lower than three orders of magnitude. Peptide fractions separated by ion-exchange chromatography show the same molecular recognition properties, with affinity constant values between 3.2 × 106 and 7.1 × 109 M−1 . Similarities and differences between this polymerisation technique and the molecular imprinting technique are briefly discussed. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Estradiol; Peptides; Amino acids; Molecular imprinting; Molecular recognition; Template polymerisation; Binding constants

1. 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 ∗ Corresponding author. Tel.: +39-011-6707622; fax: +39-011-6707615. E-mail address: [email protected] (G. Giraudi).

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 molec-

0003-2670/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0003-2670(03)00062-X

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ular 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 [9–11]. 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.

2. 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 ␮Ci/ml. The specific activities of the markers were 85 Ci/mmol for testosterone and progesterone, 89, 93 and 75 Ci/mmol for different samples of ␤-estradiol. Labelled amino acids were from Amersham (Uppsala, Sweden): glycine and ser-

ine were in aqueous solution at the concentration of 1 mCi/ml and their specific activities were 16.2 and 28.0 Ci/mmol. Leucine and glutamic acid were in aqueous solution with 2% of ethanol at the same concentrations and their specific activities were 61.0 and 46.0 Ci/mmol. Ion-exchange DEAE–Sephacel and the low-pressure chromatographic apparatus (peristaltic pump P-1, fractions collector FRAC-100, monitor UV-M and chart recorder REC-482) were from Pharmacia (Uppsala, Sweden). The size-exclusion Macrosphere GPC 60 column was from Alltech (Milan, Italy). The high-pressure chromatographic apparatus (pump L-6200A, UV-Vis detector L-4250 and integrator D-2500) was from Merck–Hitachi (Darmstadt, Germany). The counting of the ␤-radioactivity was done by a Wallac 1410 Liquid Scintillation Counter (Turku, Finland), using Eco-lite as liquid scintillation cocktail (ICN, Costa Mesa, CA, USA). All spectrophotometric measurements were recorded on a Varian Cary 219 double beam spectrophotometer (Palo Alto, CA, USA). The rabbit polyclonal antisera raised against estradiol, progesterone and testosterone and the goat anti-rabbit ␥-globulin antiserum were kindly supplied by G. Bolelli, Servizio di Fisiopatologia della Riproduzione, Ospedale Sant’Orsola, Bologna, Italy. The IgG fractions were purified from crude antiserum by immunoaffinity chromatography on a 10 mm × 130 mm column of rabbit IgG-Affiprep 10 (Bio-Rad, Hercules, USA). The polystyrene microplates, where we performed all the immunoassays, were obtained from Biohit (Helsinki, Finland). Microtiter plate washer and Microplate incubator manufactured by Bio-Rad (Hercules, CA, USA) were used. 2.1. Amino acid polymerisation For each amino acid, a stock solution was prepared by dissolving a known amount of solid compound under continuous stirring in 50 ml of carbonate buffer, 0.2 M, pH 9.0. The polymerisation mixtures, 25 mM in amino acids (expressed as the concentration resulting from sum of all the single concentrations of the amino acids included in the mixtures) were prepared in accordance with the composition scheme reported in Table 1 by mixing suitable amounts of stock solutions and immediately diluting 1+1 (v/v) with freshly distilled DMF. Then, under rapid stirring a solution

G. Giraudi et al. / Analytica Chimica Acta 481 (2003) 41–53 Table 1 Amino acid composition of the polymerisation mixtures Amino acid

Gly Ala Leu Pro Phe Tyr Trp Ser Met Arg His Lys Asp Asn Gln Glu Cys–Cys

Total molar fraction (%) Branched mixtures

Linear mixtures

2.7 7.9 9.1 3.4 3.3 4.6 0.3 10.6 0.7 3.9 2.9 10.0 7.0 2.0 3.4 10.1 0.6

15.0 5.4 20.5 9.1 7.5 5.9 0.6 8.9 1.1 7.3 5.7 – – 5.9 6.7 – –

of estradiol (64 mM) in methanol and a solution of DCCD or N,N -diisopropylcarbodiimide (153 mM) in DMF were added. Blank peptide mixtures were prepared without estradiol. When tritium-labelled peptide mixtures were prepared, four tritium labelled amino acids (leucine, serine, glycine and glutamic acid, 1.0 mCi/ml) or labelled estradiol were added to the polymerisation mixture. The quantities of labelled reagents were proportional to the amount of the corresponding non-labelled reagents in the polymerisation mixture and assured a affordable measure of radioactivity. All the mixtures were allowed to polymerise under continuous stirring for different reaction times (1, 3 and 30 h) at room temperature and in the dark. 2.2. Peptide purification The peptide mixtures were filtered on 0.45 ␮m sintered glass to eliminate the suspended particulate due to the N,N-dialkylurea formed during the polymerisation process. Then, the filtrate was diluted 1 + 2 (v/v) with deionised water and the pH was adjusted to 8.5 with hydrochloric acid 1 M. The peptide mixtures were purified by preparative anion-exchange liquid chromatography on a 100 mm × 26 mm low pressure column packed with DEAE–Sephacel. The column was

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equilibrated with carbonate buffer, 20 mM, pH 8.5, charged with the peptide mixture and washed with the same buffer at 1 ml/min, until the detector signal was less than 0.001 units of absorbance (280 nm). The peptides were recovered by eluting the column in reverse flow mode with phosphate buffer 50 mM, 1 M sodium chloride, 1 mM EDTA, pH 7.0. To fractionate the peptide mixture the purification procedure was essentially the same, but the elution of the peptides was performed in two steps, first by eluting the column with carbonate buffer, 20 mM, 0.1 M sodium chloride, pH 8.5 (fractions A and B), and then by eluting in reverse flow mode with carbonate buffer 20 mM, 1 M sodium chloride, pH 8.5 (fraction C). 2.3. Determination of molecular weight Size exclusion chromatography was performed by eluting the peptide mixtures on a 3.9 mm × 250 mm Macrosphere GPC 60 equilibrated with phosphate buffer, 50 mM, 0.15 M sodium chloride, pH 7.0; flow rate 1 ml/min; detection 278 nm. The column was calibrated with 20 ␮l of a standard mixture of Val-AlaAla-Phe (MW 720), human angiotensin II (MW 1050), human angiotensin I (MW 1300), neurotensin fragment 1–11 (MW 1450), [Ac-d-p-Cl-Phe1,2 ,d-Trp3 , d-Arg6 ,d-Ala10 ]-LH-RH (MW 1460), neurotensin (MW 1670), Gln-Ala-Thr-Val-Gly-Asp-Ile-AsnThr-Glu-Arg-Pro-Gly-Met-Leu-Asp-Phe-Thr-Gly-Lys (MW 2150), insulin chain A (MW 2500), ACTH fragment 1–24 (MW 2900), insulin chain B (MW 3500), bovine GH-RF (MW 5100) and ubiquitin (MW 8500). 2.4. Determination of peptide concentration The purified peptide mixtures were diluted 1 + 9 (v/v) with phosphate buffer 50 mM, 1 M sodium chloride, 1 mM EDTA, pH 7.0, and the absorbance at 274 nm was measured. Getting to know the 274 nm molar absorbitivity of the aromatic amino acids (Tyr 1440 M−1 cm−1 , Trp 5300 M−1 cm−1 , Phe 4 M−1 cm−1 , the latter considered negligible) we obtained the concentrations of these amino acids in the peptide mixtures. This value was divided by the fraction of the aromatic amino acids present in the mixture to calculate the total amino acid concentration. The peptide concentrations were finally obtained by dividing each total amino acid concentration by the

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respective degree of polymerisation estimated from the size-exclusion chromatography measurements. As estradiol also absorbs at the wavelength of 274 nm, we corrected the measured absorbance by subtracting the calculated absorbance of the residual template to obtain only the absorbance of the aromatic amino acids. 2.5. Spectrophotometric measurements Solutions of each amino acid (0.39 mM) and of the template (0.039 mM) were prepared in carbonate buffer, 0.2 M, pH 9.0, and the spectra were registered from 250 to 300 nm. Each spectrum was recorded 10 times and numerically averaged. The differential spectra were obtained by subtracting the spectrum of an amino acid:estradiol mixture 1:1 (v/v) from the sum of the template and amino acid spectra. Differential spectra for the peptide were measured with the same technique using branched mixtures polymerised for 3 h. The solutions of peptides and estradiol were 0.1 mM in the polymerisation buffer. The purified peptide mixture, diluted in carbonate buffer, 0.2 M, pH 9.0 to obtain a final concentration of 1 ␮M, was titrated at 274 nm with 30 ␮l-volume additions of a 5 ␮M solution of estradiol in carbonate buffer. To obtain the titration plot each absorbance measurement was repeated three times, and the averaged values, corrected for progressive dilutions, were plotted against the ratio between the steroid and the peptide mixture stoichiometric concentration.

tion of labelled estradiol (50 ␮l), diluted 1:100 in the same buffer, was added. The final concentration of the labelled estradiol in the well was 660 pM. The microplates were incubated overnight at 4 ◦ C. The wells were washed three times with the phosphate buffer without gelatin. A solution of 50 mM sodium hydroxide (0.25 ml) was added into each well and incubated for 1 h at 37 ◦ C. This step was useful to release the labelled steroid from the solid phase antibody. After this step, 0.2 ml of alkaline solution was transferred into picovials where the liquid scintillation cocktail (3.0 ml) was added. Non-specific binding of the labelled steroid was evaluated by replacing the capture antibody with dilution buffer, whereas the binding of estradiol in absence of competitors was evaluated by replacing the peptide mixtures with dilution buffer. The total radioactivity of the labelled tracer was determined by dispensing an equal volume of labelled estradiol into two picovials where the dilution buffer (0.2 ml) and the liquid scintillation cocktail (3.0 ml) were added. When labelled peptides were used, the total radioactivity was measured by dispensing 160 ␮l of the solution of the peptides, diluted 1:10, 1:100 and 1:1000, in two picovials where 40 ␮l of the dilution buffer and 3 ml of the liquid scintillation cocktail were added. All measurements were performed in duplicate. 3. Results and discussion 3.1. Synthesis and purification of the peptides

2.6. Immunocompetition experiments The immobilisation of the capture antibodies (rabbit anti-estradiol, diluted 1:40,000) in the wells of microtiter plates coated with a goat anti-rabbit IgG (10 ␮g/ml) was performed according to the experimental procedure previously reported in literature [13]. Dilutions between 1:100 and 1:40,000 of peptide mixture (average concentration 1.0 mM) were made in phosphate buffer 20 mM, 0.1 M sodium chloride, 1 mM EDTA, 0.1% (w/v) sodium azide, pH 7.0 containing 0.1% (w/v) gelatin. For the peptide fractions the dilutions were made between 1:10 and 1:40,000. For the labelled peptides the dilutions were made between 1:10 and 1:16,000. Each dilution (0.2 ml) was dispensed in duplicate into the wells and then a solu-

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 the absence of these amino acids and for the higher amount of leucine and glycine “linear mixtures”. The mixed aqueous–organic medium of polymerisation was chosen to solubilise well both estradiol and amino acids and to keep a polar environment to

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increase the hydrophobic and aromatic interactions between the template molecule and the amino acids. To isolate the peptides from the reaction medium and remove the templating estradiol a low-pressure ion-exchange chromatographic method was chosen. This technique allowed us to obtain recoveries between 94 and 97% of the initial amino acids in one easy single step. We had to consider the possibility that, under these polymerisation conditions, a portion of the template could react covalently with the amino acids (or the polypeptides) to form an ester linkage, thus impairing the separation of the polypeptide mixtures from the template in the purification step. However, such esters are unstable in basic aqueous medium, such as those in our reaction conditions. Furthermore, we could not exclude that a residual portion of estradiol covalently or even non-covalently bound to the polymers remained in the polypeptide mixtures. Therefore, before starting the spectrophotometric and binding studies, it was necessary to verify whether a residual amount of estradiol was present in the peptide mixtures even after the chromatographic purification. The residual concentration of the template present in the solution containing the peptides was measured by performing the polymerisation reaction in presence of tritium-labelled estradiol (28 nM, a concentration which assures a significant measure-

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ment of radioactivity but negligible respect to the template concentration) added to the unlabelled steroid. After the polymerisation and chromatographic separation, the radioactivity remaining in the polymer was measured, thus obtaining unambiguously the fraction of the estradiol remaining in the polypeptides, bound either covalently or non-covalently. The amount of residual template determined by this procedure was only 2% of the initial estradiol, both for branched and linear mixture. This quantity was too low to interfere significantly; in fact this quantity implies that, given the polymer concentration and the initial estradiol concentration, only 3–4% of the peptides has bound estradiol molecules. Thus, the peptide recognition properties can be unambiguously valued by spectrophotometric and binding studies without the remaining bound template affecting the peptides–estradiol interaction. When the peptide mixtures were fractionated (Fig. 1), a larger rate of residual estradiol was present in the fraction A (branched 3.1%, linear 3.2%) and in the fraction B (branched 37.1%, linear 12.1%), whereas a small quantity remained in the last fraction C (branched 0.27%, linear 0.28%). Since the percentage of peptides recovered in the first peak was only 14% of the whole peptide mixture, the amount of co-eluted residual template has an important role in

Fig. 1. Example of peptide mixture purification by ion-exchange on DEAE–Sephacel: chromatography of branched template polymerisation mixture (fraction A: residual estradiol 3.1%, peptide recovery 14%; fraction B: residual estradiol 37.1%, peptide recovery 73%; fraction C: residual estradiol 0.27%, peptide recovery 13%).

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conditioning the binding studies of the A fraction. In spite of the small quantity of peptides present in the A fraction, these were able to bind a high concentration of estradiol. Therefore, these peptides seem to show a great affinity for the template. The high concentration of residual estradiol present in the B fraction could also be explained in the same way. Moreover, as the second fraction represented 73% of the peptide mixture, we expected that the properties of this fraction would be similar to the average properties of the whole mixtures purified without fractionating. For the last fraction, the amount of template is small enough to be considered negligible even if the peptides were 13% of the whole mixture. 3.2. Peptide molecular weight The chromatograms obtained by medium pressure size-exclusion chromatography of the peptide mixtures were very similar to each other, without marked differences between linear or branched mixtures and between mixtures polymerised with or without the presence of the template. As expected, the peptides mixtures purified after 30 h of polymerisation show a more pronounced total exclusion peak than the mixture purified after 3 or 1 h. The molecular weights were found to be between 2 and 6 kDa (Table 2). The reported values are the weighted averages of the detected peaks, as each mixture is composed of different fractions of peptides which could be only partially separated with the two-step ion-exchange purification procedure. The polymerisation degrees were obtained from the molecular weight by dividing by an average amino Table 2 Mean molecular weights of the peptides obtained by HPLC-SE Polymerisation time (h)

1 1, fraction A 1, fraction B 1, fraction C 3 30

Branched mixtures, molecular weight (kDa)

Linear mixtures, molecular weight (kDa)

Template

Blank

Template

Blank

3 4 3 3

3.2 3.5 3 3

3 4.1 3 3.5

3 4 3 3.9

3 5

2.9 3.5

3 5

2.8 2.8

acid molecular weight (130 Da). The results were between 15 and 46, with an average value of 30. 3.3. Spectophotometric characterisation of peptides A preliminary evaluation of the presence of non-covalent interactions between template and amino acids or polymerised peptides was performed by differential spectrophotometry. Spectral variations in the wavelength range considered were observed in the presence of several amino acids, such as leucine, lysine, serine, tyrosine and above all tryptophan, which gave a very marked effect (Fig. 2). This behaviour confirmed that estradiol could interact with the monomers and potentially drive the template polymerisation towards the formation of structures that are shaped to recognise the template. In fact, the differential spectra of the peptide mixtures (Fig. 3) showed, in the same wavelength range (250–300 nm), a very different behaviour of the template mixture versus the blank one. The strong change in absorbance present in the template mixture spectrum seems to be the result of a significant interaction between the peptides and the estradiol. The decrease in the molar absorption coefficient, though limited, was used to perform a spectrophotometric titration of the peptides with estradiol (Fig. 4). Although data is slightly scattered around the linear portions of the curves it is evident that the template peptide mixtures bind about 1 mol of estradiol per mol of peptides. Similar data cannot be obtained from the titration of the blank mixture, as the scattering of data is higher than the differences in the slope of the linear portions (i.e. we cannot observe two linear portions). From the titration data an estimate of the equilibrium association constant between estradiol and peptides cannot be obtained due to the absence of curvature in the equivalence region of the curve. The reason can be the high equilibrium constant or the scattering of experimental data or both. As a strong interaction is not observed with the blank polymer, the titration curve clearly indicates a greater interaction of estradiol with the template mixture. As a consequence, the results obtained from the spectrophotometric titration of template peptides indicate that the added estradiol binds the template polymer in a ratio of 4:5 approximately. Moreover, the differential UV spectra reveal

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Fig. 2. Differential spectra of amino acids (leucine, lysine, serine, tyrosine and tryptophan) with estradiol.

a defined templating effect, as the absorbance change appears greater than that observed with the blank polymer, thus indicating a stronger interaction. Thus, the templating effect can be unambiguously detected by the spectrophotometric titrations, after that the quantification of the remaining estradiol (only 3–4% of the template peptides has bound the steroid) showed that it could not affect the observed peptides–estradiol interaction.

3.4. Affinity for estradiol of peptide mixtures Based on the spectrophotometric results, we decided to perform immunocompetition studies where the peptides competed with an immobilised antiestradiol antibody for tritium-labelled estradiol, in order to evaluate the binding affinity of the peptides. The measurement of estradiol bound to the antibody in the presence of the peptides allowed us to determine the

Fig. 3. Differential spectra of branched peptide mixtures polymerised for 3 h with estradiol (continue line: template mixture, dashed line: blank mixture).

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Fig. 4. Spectrophotometric titration of the template linear mixture polymerised for 30 h, [estradiol] and [peptides] are the stoichiometric concentrations of the steroid and of the peptides.

average binding constants, K, of the peptide mixtures, by fitting a proper mathematical equation through the experimental data (see Appendix A, Eq. (A.13)). This equation defines the ratio between the binding of the tritium labelled estradiol with the antibody in the presence of and without the peptides as a function of several parameters: the binding constant of the antibodies for the estradiol, the concentration of antibodies on the solid-phase, the tritium-labelled estradiol concentration, the concentration of peptides and the binding constant of the peptides for the estradiol. The first two parameters were determined in separate experiments as reported in the literature [13], so by knowing the molar concentration of tritium-labelled estradiol and of peptide mixtures we could calculate the binding constants of the peptide mixtures for the estradiol from the competition curves (examples of which are reported in Fig. 5). All these results are not conditioned by any contribution of residual estradiol, as only 3–4% of the template peptides has bound estradiol molecules. As a higher concentration of estradiol was present in the peptide fractions, for the treatment of this data the mathematical equation was corrected by including a parameter that allowed us to keep in mind the presence of some residual estradiol (see Appendix A, Eq. (A.12)). In order to verify that there were no interactions between the rabbit anti-estradiol antibodies or the goat

anti-rabbit ␥-globulin antibodies and the peptides, we checked the interactions of tritium-labelled peptides with the immobilised antibodies. In both cases we registered no significant binding to the solid phase. So, the inhibition of the binding between the labelled estradiol and its antibody is caused only by the binding properties of peptide mixtures. Observing the results reported in Table 3, we can note the very high affinity of the peptide mixtures for the templating molecule. We must underline that the affinity of the human serum albumin is 104 to 105 M−1 for this steroid [14]. The difference between the binding constants of the template and the blank mixtures shows the importance of the templating effect on the amino acid mixtures and the efficacy of these systems as competitors of the antibodies themselves. For all mixtures the binding constants increase with the polymerisation time and this effect can be explained with the formation of increasingly complex structures which are able to interact better with the estradiol. The ratio between template and blank constant decreases with the increasing of the polymerisation time, probably because in the blank mixtures a longer reaction time increases the formation of more complex peptide structures able to rearrange themselves around the steroid molecule to obtain a greater interaction. Instead, in the template mixtures, this effect is less important as the binding properties are mainly due

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Fig. 5. Immunocompetition of reticulated mixtures polymerised for 1 h (circles), 3 h (squares) and 30 h (triangles) in presence (filled points) and in absence (open points) of estradiol. Curves were obtained by fitting the mathematical equations reported in Appendix A through the experimental data.

to the templating effect. However, it is also true that such high binding constants for the blank mixtures could originate from an unwanted templating effect of the carbodiimide used as a condensing agent, which has a ring shaped structure. To verify this hypothesis we performed the polymerisation using a different condensing agent, N,N -diisopropylcarbodiimide. The results gave the same values of affinity constants reported; for this reason we could exclude an influence of the condensing agent on the binding properties of the peptides. We also evaluated the effect of pH and ionic strength on the binding constant of the branched template mixture polymerised for 1 h. The methods were the same as those described in the Section 2 for the immunocompetition but we changed the dilution buffers: a

carbonate buffer (50 mM, 1 M sodium chloride, 1 mM EDTA, 0.1% (w/v) gelatin, pH 9.0) was used to evaluate the pH effect, and a phosphate buffer (20 mM, 1 M sodium chloride, 1 mM EDTA, 0.1% (w/v) gelatin, pH 7.0) was used for the ionic strength. We observed that the peptides–estradiol interaction is not influenced by the variation of ionic strength (K = (9.0 ± 0.24) × 108 M−1 at 0.1 M versus K = (8.8 ± 0.29) × 108 M−1 at 1 M), ruling out possible polar interactions between peptides and estradiol, whereas there is a decrease of affinity when the pH moves from neutral to basic medium (K = (1.6 ± 0.19) × 108 M−1 at pH 9.0 versus K = (9.0 ± 0.24) × 108 M−1 at pH 7.0). This decrease can be attributed to the deprotonation of the oxydrilyc group on the aromatic ring A of the estradiol, which should reduce the ␲–␲ interaction with

Table 3 Binding constants of the peptide mixtures Peptide mixtures

Polymerisation time (h)

Ktemplate (×108 , M−1 )

Kblank (×108 , M−1 )

Ktemplate /Kblank

Branched

1 3 30

9.0 ± 0.24 12 ± 0.17 66 ± 1.2

0.090 ± 0.0037 0.15 ± 0.020 13 ± 3.1

100 80 5.1

Linear

1 3 30

4.4 ± 0.15 5.8 ± 0.13 14 ± 0.81

0.022 ± 0.0020 0.028 ± 0.0091 3.6 ± 0.11

200 207 3.9

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the peptides. This is an evidence of the key role of the aromatic interactions between estradiol and peptides. 3.5. Affinity for estradiol of peptide fractions To study the binding properties towards estradiol of the fractionated peptides, immunocompetitions were performed using the fractions obtained from the mixtures polymerised for 1 h. The study of the fractions A and B were more complicated for the presence of the residual estradiol and the discussion of their experimental results had to keep in mind the interference caused by the residual template. The influence of the residual estradiol was stronger for the fraction A than for the fraction B. In fact, the peptide concentration determined by spectrophotometry must be decreased to consider the residual estradiol (about 24% for the fraction A and about 16% for the fraction B), resulting with a concentration of 9.3 ␮M (fraction A) and of about 70 ␮M (fraction B). The concentration of the fraction C was not conditioned by the residual estradiol and was 15 (linear) and 78 ␮M (branched). The binding constants reported in Table 4 were obtained by fitting the experimental data through the equation that takes into account the residual estradiol, because it represents a significant proportion of the initial amount, concerning, above all, the first and second fractions. Fraction A, which had the greater molecular weight, is the part of the peptides with a higher binding strength. That seems reasonable, since bigger peptide structures should be able to rearrange themselves easily around the molecule, thus creating a well-shaped binding site. Anyway, as the corresponding blank fractions show a relatively high binding strength, the templating effect can be considered less significant than for fraction B.

The binding constants of fraction B are quite similar to the constants of the whole mixture; this result is expected as these fractions represent about 73% of the whole peptide mixtures. For linear fraction C, where the binding properties of template and blank peptides are practically the same, the templating effect is negligible, whereas in the case of the branched fraction C, a greater affinity towards the estradiol is present and the templating effect cannot be ignored. This is the only significant difference that could be observed between linear and branched fractions C by examining the experimental data. These results seem to confirm the presence of heterogeneous peptide structures in the mixtures, which bind the estradiol with different affinities. 3.6. Peptide binding selectivity An important parameter to consider is the specificity of binding. We held an immunocompetition using the template branched peptides (1 and 30 h) to evaluate the binding constant of these mixtures for structural homologues, like the steroids testosterone and progesterone. The experimental procedures were always the same reported in the previously described experiments. In the first immunocompetition the capture antibodies were directed against testosterone (rabbit anti-testosterone, diluted 1:30,000) and a tritium-labelled testosterone was used. In the second immunocompetition we used capture antibodies directed against progesterone (rabbit anti-progesterone, diluted 1:10,000) and tritium-labelled progesterone. For template branched peptides polymerised for 1 h, the binding constant for the testosterone was found to be (1.2 ± 0.37) × 105 M−1 versus (9.0 ± 0.24) ×

Table 4 Binding constants of fractions obtained from peptide mixtures polymerised for 1 h Peptide mixtures

Fraction

Branched

A B C

Linear

A B C

Ktemplate (×108 , M−1 )

Kblank (×108 , M−1 )

Ktemplate /Kblank

71 ± 3.5 11 ± 0.80 2.3 ± 0.75

1.0 ± 0.097 0.082 ± 0.011 0.015 ± 0.0024

71 134 153

31 ± 1.1 3.7 ± 0.56 0.032 ± 0.0029

1.4 ± 0.37 0.013 ± 0.0029 0.029 ± 0.0047

22 285 1.1

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108 M−1 for the estradiol, whereas for the blank mixture the binding constant was found to be (3.3±0.10)× 104 M−1 versus (9.0 ± 0.37) × 106 M−1 for the estradiol. The binding constants obtained for the branched peptides polymerised for 30 h (template: (7.7±0.67)× 105 M−1 versus (6.6 ± 0.12) × 109 M−1 , blank: (4.8 ± 0.62) × 106 M−1 versus (1.3 ± 0.31) × 109 M−1 ) confirm the hypothesis that longer reaction times produce structures which can rearrange themselves to better match the steroid molecule without a loss of selectivity, thus indicating that the imprinting effect is operative in all cases. For the progesterone it was possible only to estimate an upper limit for the binding constant of the template peptides (∼1.0 × 104 M−1 ). This is further evidence of the high affinity and high specificity of recognition towards the imprinting molecule.

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described in this paper does not seem conditioned by the degree of reticulation and it is generated in a highly polar medium. Moreover, the molecular imprinting technique produces insoluble materials, in the shape of beads, amorphous monoliths or thin layers of polymers, while the template polymerisation of amino acids produces low-mass water-soluble polymers. The experimental data was obtained by using estradiol as a templating molecule. At present our experimental work is addressed to define whether the memory effect is a peculiar property of the amino acids/estradiol system or it could be observed with different templating molecule, and could be considered a general consequence of the presence of intermolecular non-covalent interactions in these systems.

Acknowledgements 4. 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 polymerisation without the templating molecule increases with the same trend, the maximum neat templating effect could be observed for shorter polymerisation times. A certain degree of heterogeneity is present in the peptide mixtures and this is reflected by different affinities for the templating molecule showed by distinct peptidic fractions separated by ion-exchange chromatography. On our opinion this data is the evidence that the presence of a templating molecule during the polymerisation step could generate a “memory effect” in the resulting peptide mixture, with a striking analogy to the well known molecular imprinting effect. But it is important to stress that there are marked differences between this kind of memory effect and the molecular imprinting. In fact, the molecular imprinting is typical of highly cross-linked, rigid polymers, and generally it could be obtained only in weak polar or non-polar solvents, whereas the “memory effect”

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”.

Appendix A. Immunocompetition data treatment The experimental results of the immunocompetition experiments were treated with a mathematical equation obtained as described afterwards. In the wells of the microplates, the competition between peptides and antibodies for a limited quantity of labelled estradiol is defined by two equilibrium constants: C ∗ ∗ Ab + E∗
AbE∗ , KAb (A.1) = AbE CAb CE∗ and P + E∗
PE∗ ,

KP∗ =

CPE∗ CP CE ∗

(A.2)

∗ is the equilibrium binding constant of where KAb the reaction between the labelled estradiol and the antibody, KP∗ the equilibrium binding constant of the reaction between the labelled estradiol and the pep∗ tide, CAbE the concentration of the antibody-labelled estradiol complex, CPE∗ the concentration of the

52

G. Giraudi et al. / Analytica Chimica Acta 481 (2003) 41–53

peptide-labelled estradiol complex, CAb , CP and CE∗ the concentrations of the free antibody binding sites, the free peptide and the free labelled estradiol, respectively. We must add these equilibrium constants to the ones of the equilibria where the residual estradiol is present: CAbE Ab + E
AbE, KAb = (A.3) CAb CE and CPE P + E
PE, KP = (A.4) CP CE where KAb is the equilibrium binding constant of the reaction between the residual estradiol and the antibody, KP the equilibrium binding constant of the reaction between the residual estradiol and the peptide, CAbE the concentration of the antibody–residual estradiol complex, CPE the concentration of the peptide–residual estradiol complex and CE the concentration of the free residual estradiol. The mass action laws are: Ab0 = CAb + CAbE∗ + CAbE (A.5)

We measured the binding of labelled estradiol to the solid phase antibody (B), then B = CAbE∗ . Furthermore we worked at peptide concentrations in which CP (CPE + CPE∗ ), so we could substitute P0 to Cp . Obtaining CAbE∗ from Eq. (A.1), CE∗ from Eq. (A.7) and CAb from Eq. (A.9), we obtain a quadratic equation in B:
 ET 1 KAb 1 + ∗ B2 E0 d
   PT ET 1 −B 1+KAb Ab0 + KP +KAb 1+ ∗ E0∗ d E0 d

PT P0 = = CP + CPE∗ + CPE d

from which B can be obtained:

B= +

(A.6)

mixture, PT and ET , corrected for the dilution factor d). By substitution of CAbE∗ and CPE∗ derived from the Eqs. (A.1) and (A.2), CAbE and CPE derived from the Eq. (A.3) and (A.4) into Eqs. (A.5), (A.7) and (A.8), and rearranging Eq. (A.5) we obtain:
 ET 1 CAb = Ab0 − CAbE∗ 1 + ∗ (A.9) E0 d

+KAb Ab0 E0∗ = 0

(A.10)

1 + KAb Ab0 + KAb E0∗ + [(KP PT + KAb ET )/d] 2KAb (1 + (ET /E0∗ )(1/d))

[{1 + KAb Ab0 + KAb E0∗ + [(KP PT + KAb ET )/d]}2 − 4KAb Ab0 [KAb E0∗ + (KAb ET )/d]]1/2 2KAb (1 + (ET /E0∗ (1/d)))

E0∗ = CE∗ + CAbE∗ + CPE∗

(A.7)

ET (A.8) = CE + CAbE + CPE d where Ab0 and E0∗ are the total concentrations of the E0 =

(A.11)

The immunocompetition data, i.e. the ratio between labelled estradiol bound to the antibody in the presence of the peptides (B) and without the peptides (B0 ), versus the dilution factor can be fitted with the equation:

1 + KAb Ab0 + KAb E0∗ + [(KP PT + KAb ET )/d] B = B0 2KAb B0 (1 + ET /E0∗ (1/d)) +

[{1 + KAb Ab0 + KAb E0∗ + [(KP PT + KAb ET )/d]}2 − 4KAb Ab0 [KAb E0∗ + (KAb ET )/d]]1/2 2KAb B0 (1 + (ET /E0∗ )(1/d))

antibodies on the solid phase and of the labelled estradiol, respectively. P0 and E0 are the total concentrations of the peptides and of the residual estradiol, respectively (equal to the concentrations in the purified

(A.12)

When we treated the experimental data of the blank mixtures, in which the estradiol is absent in the polymerisation mixtures, the value of ET = 0 and thus this equation becomes:

G. Giraudi et al. / Analytica Chimica Acta 481 (2003) 41–53

53



B 1 1 KP = + Ab0 + E0 + P0 B0 2B0 KAb KAb 
2 1 KP − + Ab0 + E0 + P0 KAb KAb 1/2  −4Ab0 E0

(A.13)

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