1. Tracing peptidization in the equichiral and inequichiral α-amino acid solutions with aid of turbidimetry and the circular dichroism (CD)
A. Godzieka, A. Łągiewkaa, I. Schislerb, Z. Drzazgab, T. Kowalskaa, and M. Sajewicza aInstitute of Chemistry, University of Silesia, 9, Szkolna St., Katowice, Poland bDepartment of Medical Physics, University of Silesia, 1A 75 Pułku Piechoty St., Chorzów, Poland a) b) c)
Introduction
From our earlier experiments [1-2], it came out that spontaneous non-linear peptidization of the monomeric α-amino acids in abiotic solutions (like, e.g., water-methanol, or water-acetonitrile) results in non-linear turbidity changes of a given liquid system in the course of storage period, most probably due to precipitation of insoluble peptide particles, followed by the sequential decay thereof. At the initial stages of sample ageing, precipitation of insoluble peptide particles remains invisible to naked eye and due to that, turbidimeter operating in continuous registration mode proves an invaluable measuring tool, properly suited for the task. In this study, a comparison is made between the dynamics of the spontaneous oscillatory peptidization of the selected α-amino acids in the monocomponent and the binary equichiral mixtures (L, D, L-L, or D-D) on the one hand, and in the respective binary inequichiral mixtures (DL, L-D, or D-L) on the other. As the dynamics of peptidization results in the formation of insoluble peptide particles, it is traced with aid of turbidimetry in the continuous registration mode (for 14 days at 25oC).
Aim
The aim of this study is to trace the turbidity patterns of spontaneous peptidization in the monocomponent and binary systems assembled in the equichiral and inequichiral configurations using turbidimetry with continuous registration and the spectroscopy of circular dichroism (CD) is used to verify our hypothesis as to the formation of the α-helical structures in the amino acids solutions.
Experimental
Analyzed samples: L-Ala, D-Ala, L-Phe and D-Phe; for all the experiments, concentrations of the investigated α-amino acids were 1.0 mg mL-1 for the monocomponent systems and 0.5 mg mL-1 for the binary systems; dissolved in 70% aqueous methanol;
Turbidity: Turbidity measurements were carried out with use of the turbidity sensor (TRB-BTA, Vernier Software & Technology, Beaverton, OR, USA); the 15 mL aliquots of the amino acids solutions were freshly prepared and placed in the instrument vial.
CD: The CD spectrometer model J-815 (Jasco Electronics Holdings, Johannesburg, South Africa); was used to registered the CD spectra of the analized samples and the blank sample was registered too.
Scheme 1. Molecular mechanisms of the processes running in the monocomponent L-Ala system dissolved in an aqueous medium: (a) Chiral conversion of L-Ala to D-Ala (intermediary non-chiral structures are marked with black ovals); (b) homopeptide formation (L-Ala-L-Ala); and (c) peptidization (Ala-Ala)n running in the parallel.
Figure 1 Turbidity (in nephelometric turbidity units, NTU) for water (H2O), methanol (MeOH), and 70% aqueous MeOH registered for 24 h.
(b)
(d)
(e) (f)
Figure 3 CD spectra recorded for the solutions of (a) L-, D-, and DL-Ala, (b) L-, D-, and DL-Phe, and (c) L-Ala-D-Phe and D-Ala-L-Phe after the four months samples ageing, and for the fresh and aged solutions of (d) L-Ala-L-Phe, (e) D-Ala-D-Phe, and (f) DL-Phe (after the four months samples ageing).
Results and conclusions
The non-linear turbidity changes with the monocomponent and binary Ala and Phe solutions are due to the oscillatory peptidization process which results in formation of insoluble higher molecular weight peptides, extensively discussed elsewhere. In this study, similarity of turbidity patterns is in the main focus of our attention. This similarity is demonstrated in Figs 1A and B and tentatively ascribed to equichirality of all α-amino acid units in a molecule of a given peptide and consequently, to a possible formation of the peptide α-helix structure. Analogously, dissimilarity of turbidity patterns is demonstrated in Fig. 1C and it is tentatively explained by inequichirality of α-amino acid units in the respective peptides.
The CD spectroscopic results confirm spontaneous formation of α-helical secondary structures in the equichiral L, D, L-L, and D-D systems on the one hand, and an inability to form α-helices in the inequichiral DL system (Fig. 2), In this sense, the obtained CD results support our assumptions derived from turbidimetric data
 However, formation of peptide α-helices is regarded as a working hypothesis only, conceived by equichirality of the monomeric units involved and the other molecular level mechanisms leading to the secondary peptide structures cannot be excluded, which might eventually complement or replace the α-helix concept.
Figure 2 Patterns of turbidity changes (in nephelometric turbidity units (NTU)) in the function of time for the solutions: (A)(i) L-Ala, (ii) L-Phe, (B)(iii) D-Ala, (iv) D-Phe, (v) L-Ala-L-Phe, (vi) D-Ala-D-Phe, (C)(vii) DL-Ala, (viii) DL-Phe, (ix) L-Ala-D-Phe, and (x) D-Ala-L-Phe.
References
[1] A. Godziek, A. Maciejowska, E. Talik, R. Wrzalik, M. Sajewicz,T. Kowalska, Curr. Protein Pept. Sci., 17(2), (2016)
[2] A. Maciejowska, A. Godziek, M. Sajewicz, T. Kowalska, Acta Chromatogr., DOI: /