Substance P (SP) is one of the most studied peptide hormones and knowing the relationship between its structure and function may have important therapeutic applications in the treatment of a variety of stress-related illnesses. In order to obtain a deeper insight into its folding, the effects of different factors, such as pH changes, the presence of Ca2+ ions, and the substitution of the Met-NH2 moiety in the SP structure, was studied by Raman and infrared spectroscopies. SP has a pH-dependent structure. Under acidic–neutral conditions, SP possesses a prevalent β-sheet structure although also other secondary structure elements are present. By increasing pH, a higher orderliness in the SP secondary structure is induced, as well as the formation of strongly bound intermolecular β-strands with a parallel alignment, which favour the self-assembly of SP in β-aggregates. The substitution of the Met-NH2 moiety with the acidic functional group in the SP sequence, giving rise to a not biologically active SP analogue, results in a more disordered folding, where the predominant contribution comes from a random coil. Conversely, the presence of Ca2+ ions affects slightly but sensitively the folding of the polypeptide chain, by favouring the α-helical content and a different alignment of β-strands; these are structural elements, which may favour the SP biological activity. In addition, the capability of SERS spectroscopy to detect SP in its biologically active form was also tested by using different metal nanoparticles. Thanks to the use of silver NPs prepared by reduction of silver nitrate with hydroxylamine hydrochloride, SP can be detected at very low peptide concentration (~ 90 nM). However, the SERS spectra cannot be obtained under alkaline conditions since both the formation of SP aggregates and the lack of ion pairs do not allow a strong enough interaction of SP with silver NPs.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
- (Ag/Au) NPs:
Full width at half maximum
Limit of detection
Substance P in amidated form
Substance P in acidic form
Surface-enhanced Raman scattering
Qi XF, Zhorov BS, Ananthanarayanan VS. CD, 1H NMR and molecular modeling studies of the interaction of Ca2+ with substance P and Ala7-substance P in a non-polar solvent. J Pept Sci. 2000;6:57–83.
Brown SM, Lamberts DW, Reid TW, Nishida T, Murphy CJ. Neurotrophic and anhidrotic keratopathy treated with substance P and insulin-like growth factor 1. Arch Ophthalmol. 1997;115:926–7.
Williams RW, Weaver JL. Secondary structure of substance-P bound to liposomes in organic-solvents and in solution from Raman and CD spectroscopy. J Biol Chem. 1990;265:2505–13.
Pantaleo N, Chadwick W, Park SS, Wang L, Zhou Y, Maudsley BMS. The mammalian tachykinin ligand-receptor system: an emerging target for central neurological disorders. CNS Neurol Disord Drug Targets. 2010;9:627–35.
Pernow B. Substance P. Pharmacol Rev. 1983;35:85–141.
Ananthanarayanan VS. Peptide hormones, neurotransmitters, and drugs as Ca2+ ionophores: implications for signal transduction. Biochem Cell Biol. 1991;69:93–5.
Corcho FJ, Salvatella X, Canto J, Giralt E, Perez JJ. Structural analysis of substance P using molecular dynamics and NMR spectroscopy. J Pept Sci. 2007;13:728–41.
Choo LP, Jackson M, Mantsch HH. Conformation and self-association of the peptide-hormone substance P - Fourier-transform infrared spectroscopic study. Biochem J. 1994;301:667–70.
Corcho FJ, Canto J, Perez JJ. Comparative analysis of the conformational profile of substance P using simulated annealing and molecular dynamics. J Comput Chem. 2004;25:1937–52.
Bignardi C, Cavazza A, Marini M, Roda LG. Substance P self-aggregation revised: a chromatographic and mass spectrometry analysis. J Chromatogr Sep Tech. 2012;3. https://doi.org/10.4172/2157-7064.1000140.
Rolka K, Erne D, Schwyzer R. Membrane-structure of substance P. 2. Secondary structure of substance P, [9-leucine] substance P, and shorter segments in 2,2,2-trifluoroethanol, methanol, and on liposomes studied by circular dichroism. Helv Chim Acta. 1986;69:1798–806.
Chassaing G, Convert O, Lavielle S. Preferential conformation of substance P in solution. Eur J Biochem. 1986;154:77–85.
Sumner SCJ, Gallagher KS, Davis DG, Covell DG, Jernigan RL, Ferretti JA. Conformational analysis of the tachykinins in solution: substance P and physalaemin. J Biomol Struct Dyn. 1990;8:687–707.
Teleman O, von der Lieth CW. Molecular dynamics simulation provides a possible structure for substance P-like peptides in aqueous solution. Biopolymers. 1990;30:13–23.
Keire DA, Fletcher TG. The conformation of substance P in lipid environments. Biophys J. 1996;70:1716–27.
Keire DA, Kobayashi M. The orientation and dynamics of substance P in lipid environments. Protein Sci. 1998;7:2438–50.
Carey PR. Raman spectroscopy, the sleeping giant in structural biology, awakes. J Biol Chem. 1999;274:26625–8.
Tuma R. Raman spectroscopy of proteins: from peptides to large assemblies. J Raman Spectrosc. 2005;36:307–19.
Torreggiani A. In: Kozyrev D, Slutsky V, editors. Handbook of free radicals: formation, types and effects: Nova Science Publisher, Inc.; 2009. p. 377–419.
Corredor C, Teslova T, Cañamares MV, Chen ZG, Zhang J, Lombardi JR, et al. Raman and surface-enhanced Raman spectra of chrysin, apigenin and luteolin. Vib Spectrosc. 2009;49:190–5.
Podstawka E, Borszowska R, Grabowska M, Drąg M, Kafarski P, Proniewicz LM. Investigation of molecular structures and adsorption mechanism of phosphonodipeptides by surface-enhanced Raman, Raman and Infrared Spectroscopies. Surf Sci. 2005;599:207–20.
Podstawka E, Kafarski P, Proniewicz LM. Structural properties of I-X-I-met-I-ala phosphonate tripeptides: a combined FT-IR. FT-RS, and SERS spectoscopy studies and DFT calculations. J Phys Chem A. 2008;112:11744–55.
Podstawka E, Proniewicz LM. The orientation of BN-ralated peptides adsorbed on SERS-active silver nanoparticles: comparison with a silver electrode surface. J Phys Chem B. 2009;113:4978–85.
Aliaga AE, Osorio-Roman I, Garrido C, Leyton P, Cárcamo J, Clavijo E, et al. Surface-enhanced Raman scattering study of L-lysine. Vib Spectrosc. 2009;50:131–5.
Seballos L, Richards N, Stevens DJ, Patel M, Kapitzky L, Lokey S, et al. Competitive binding effects on surface-enhanced Raman scattering of peptide molecules. Chem Phys Lett. 2007;447:335–9.
Jurasekova Z, Tinti A, Torreggiani A. Use of Raman spectroscopy for the identification of radical-mediated damages in human serum albumin. Anal Bioanal Chem. 2011;400:2921–31.
Tu AT. Spectroscopy of biological systems. Chichester: Wiley; 1986.
Williams RW. Methods in enzymology (Hirs CHW, Timasheff SN, editors), vol. 130. Academic Press: New York. 1986. p. 311–31.
Fabian H, Anzenbacher P. New developments in Raman spectroscopy of biological systems. Vib Spectrosc. 1993;4:125–48.
Bandekar J. Amide modes of reverse turns. Vib Spectrosc. 1993;5:143–73.
Prestrelski SJ, Byler DM, Liebman MN. Comparison of various molecular forms of bovine trypsin: correlation of infrared spectra with X-ray crystal structure. Biochemistry. 1991;30:133–43.
Biswas BB, Roy S, Miura T, Thomas G Jr. Proteins: structure, function, and engineering, vol. 24. US: Springer; 1995. p. 55–99.
Vass E, Hollósi M, Besson F, Buchet R. Vibrational spectroscopic detection of beta- and gamma-turns in synthetic and natural peptides and proteins. Chem Rev. 2003;103:1917–54.
Barth A, Zscherp C. What vibrations tell us about proteins. Q Rev Biophys. 2002;35:369–430.
Lee PC, Meisel D. Adsorption and surface-enhanced Raman of dyes on silver and gold sols. J Phys Chem. 1982;86:3391–5.
Leopold N, Lendl B. A new method for fast preparation of highly surface-enhanced Raman scattering (SERS) active silver colloids at room temperature by reduction of silver nitrate with hydroxylamine hydrochloride. J Phys Chem B. 2003;107:5723–7.
Sutherland WS, Winefordner JD. Colloid filtration - a novel substrate preparation method for surface-enhanced Raman spectroscopy. J Colloid Interface Sci. 1992;148:129–41.
Garcia-Leis A, Garcia-Ramos JV, Sanchez-Cortes S. Silver nanostars with high SERS performance. J Phys Chem C. 2013;117:7791–5.
Aroca RF, Alvarez-Puebla RA, Pieczonka N, Sanchez-Cortez S, Garcia-Ramos JV. Surface-enhanced Raman scattering on colloidal nanostructures. Adv Colloid Interf Sci. 2005;116:45–61.
Pelton JT, McLean LR. Spectroscopic methods for analysis of protein secondary structure. Anal Biochem. 2000;277:167–76.
Bandekar J. Amide modes and protein conformation. Biochim Biophys Acta Protein Struct Mol Enzymol. 1992;1120:123–43.
Barth A. Infrared spectroscopy of proteins. Biochim Biophys Acta Bioenerg. 2007;1767:1073–101.
Navarra G, Tinti A, Leone M, Militello V, Torreggiani A. Influence of metal ions on thermal aggregation of bovine serum albumin: aggregation kinetics and structural changes. J Inorg Biochem. 2009;103:1729–38.
Navarra G, Giacomazza D, Leone M, Librizzi F, Militello V, San Biagio PL. Thermal aggregation and ion-induced cold-gelation of bovine serum albumin. Eur Biophys J. 2009;38:437–46.
Remondetto GE, Subirade M. Molecular mechanisms of Fe2+-induced β-lactoglobulin cold gelation. Biopolymers. 2003;69:461–9.
Rašković B, Popović M, Ostojić S, Anđelković B, Tešević V, Polović N. Fourier-transform infrared spectroscopy provides an evidence of papain denaturation and aggregation during cold storage. Spectrochim Acta A Mol Biomol Spectrosc. 2015;150:238–46.
Ly TN, Hazama C, Shimoyamada M, Ando H, Kato K, Yamauchi R. Antioxidative compounds from the outer scales of onion. J Agric Food Chem. 2005;53:8183–9.
Malkowski MG, Ginell SL, Smith WL, Garavito RM. The productive conformation of arachidonic acid bound to prostaglandin synthase. Science. 2000;289:1933.
Lord RC, Yu NT. Laser-excited Raman spectroscopy of biomolecules: I. native lysozyme and its constituent amino acids. J Mol Biol. 1970;50:509–24.
Torreggiani A, Domenech J, Orihuela R, Ferreri C, Atrian S, Capdevila M, et al. Zinc and cadmium complexes of a plant metallothionein under radical stress: desulfurisation reactions associated with the formation of trans-lipids in model membranes. Chem Eur J. 2009;15:6015–24.
Overman SA, Thomas GJ. Raman markers of nonaromatic side chains in an α-helix assembly: ala, asp, Glu, Gly, Ile, Leu, Lys, Ser, and Val residues of phage fd subunits. Biochemistry. 1999;38:4018–27.
Di Foggia M, Taddei P, Torreggiani A, Dettin M, Tinti A. Interactions between oligopeptides and oxidised titanium surfaces detected by vibrational spectroscopy. J Raman Spectrosc. 2011;42:276–85.
De Gelder J, De Gussem K, Vandenabeele P, Moens L. Reference database of Raman spectra of biological molecules. J Raman Spectrosc. 2007;38:1133–47.
Garrido C, Aliaga AE, Gomez-Jeria JS, Clavijo RE, Campos-Vallette MM, Sanchez-Cortes S. Adsorption of oligopeptides on silver nanoparticles: surface-enhanced Raman scattering and theoretical studies. J Raman Spectrosc. 2010;41:1149–55.
Aliaga AE, Garrido C, Leyton P, Diaz FG, Gomez-Jeria JS, Aguayo T, et al. SERS and theoretical studies of arginine. Spectrochim Acta A Mol Biomol Spectrosc. 2010;76:458–63.
Maiti NC, Apetri MM, Zagorski MG, Carey PR, Anderson VE. Raman spectroscopic characterization of secondary structure in natively unfolded proteins: α±synuclein. J Am Chem Soc. 2004;126:2399–408.
López-Tobar E, Hernández B, Gómez J, Chenal A, Garcia-Ramos JV, Ghomi M, et al. Anchoring sites of fibrillogenic peptide hormone somatostatin-14 on plasmonic nanoparticles. J Phys Chem C. 2015;119:8273–9.
Sanchez-Cortes S, Garcia-Ramos JV. Anomalous Raman bands appearing in surface-enhanced Raman spectra. J Raman Spectrosc. 1998;29:365–71.
Guerrini L, Jurasekova Z, Domingo C, Perez-Mendez M, Leyton P, Campos-Vallette M, et al. Importance of metal-adsorbate interactions for the surface-enhanced Raman scattering of molecules adsorbed on plasmonic nanoparticles. Plasmonics. 2007;2:147–56.
Cañamares MV, Garcia-Ramos JV, Gomez-Varga JD, Domingo C, Sanchez-Cortes S. Comparative study of the morphology, aggregation, adherence to glass, and surface-enhanced Raman scattering activity of silver nanoparticles prepared by chemical reduction of Ag+ using citrate and hydroxylamine. Langmuir. 2005;21:8546–53.
Kaminska A, Inya-Agha O, Forster RJ, Keyes TE. Chemically bound gold nanoparticles arrays on silicon: assembly, properties and SERS study of protein interactions. Phys Chem Chem Phys. 2008;10:4172–80.
Frost RL, Kloprogge JT. Raman spectroscopy of the acetates of sodium, potassium and magnesium at liquid nitrogen temperature. J Mol Struct. 2000;526:131–41.
Paccotti N, Boschetto F, Horiguchi S, Marin E, Chiadò A, Novara C, et al. Label-free SERS discrimination and in situ analysis of life cycle in Escherichia coli and Staphylococcus epidermidis. Biosensors. 2018;8(4):131.
Podstawka E, Ozaki Y, Proniewicz LM. Part II: surface-enhanced Raman spectroscopy investigation of methionine containing heterodipeptides adsorbed on colloidal silver. Appl Spectrosc. 2004;58:581–90.
This work was supported by the Scientific Grant Agency of the Ministry of the Education of Slovak Republic (APVV-15-0485) and the Ministerio de Economía y Competitividad from Spain under the grant FIS2017-84314-R.
Conflict of interest
The authors declare that they have no conflicts of interest.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
About this article
Cite this article
Jurasekova, Z., Garcia-Leis, A., Sanchez-Cortes, S. et al. Structural analysis of the neuropeptide substance P by using vibrational spectroscopy. Anal Bioanal Chem 411, 7419–7430 (2019). https://doi.org/10.1007/s00216-019-02097-2
- Substance P
- Vibrational (Raman and infrared) spectroscopy
- Surface-enhanced Raman spectroscopy (SERS)