Structural analysis of the neuropeptide substance P by using vibrational spectroscopy


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.

Graphical abstract

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8


(Ag/Au) NPs:

(Silver/gold) Nanoparticles


Full width at half maximum


Limit of detection


Substance P in amidated form


Substance P in acidic form


Surface-enhanced Raman scattering


















  1. 1.

    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.

    CAS  PubMed  Google Scholar 

  2. 2.

    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.

    CAS  PubMed  Google Scholar 

  3. 3.

    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.

    CAS  PubMed  Google Scholar 

  4. 4.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Pernow B. Substance P. Pharmacol Rev. 1983;35:85–141.

    CAS  PubMed  Google Scholar 

  6. 6.

    Ananthanarayanan VS. Peptide hormones, neurotransmitters, and drugs as Ca2+ ionophores: implications for signal transduction. Biochem Cell Biol. 1991;69:93–5.

    CAS  PubMed  Google Scholar 

  7. 7.

    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.

    CAS  PubMed  Google Scholar 

  8. 8.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    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.

    CAS  PubMed  Google Scholar 

  10. 10.

    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.

  11. 11.

    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.

    CAS  Google Scholar 

  12. 12.

    Chassaing G, Convert O, Lavielle S. Preferential conformation of substance P in solution. Eur J Biochem. 1986;154:77–85.

    CAS  PubMed  Google Scholar 

  13. 13.

    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.

    CAS  PubMed  Google Scholar 

  14. 14.

    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.

    CAS  PubMed  Google Scholar 

  15. 15.

    Keire DA, Fletcher TG. The conformation of substance P in lipid environments. Biophys J. 1996;70:1716–27.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Keire DA, Kobayashi M. The orientation and dynamics of substance P in lipid environments. Protein Sci. 1998;7:2438–50.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Carey PR. Raman spectroscopy, the sleeping giant in structural biology, awakes. J Biol Chem. 1999;274:26625–8.

    CAS  PubMed  Google Scholar 

  18. 18.

    Tuma R. Raman spectroscopy of proteins: from peptides to large assemblies. J Raman Spectrosc. 2005;36:307–19.

    CAS  Google Scholar 

  19. 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.

  20. 20.

    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.

    CAS  Google Scholar 

  21. 21.

    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.

    CAS  Google Scholar 

  22. 22.

    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.

    CAS  PubMed  Google Scholar 

  23. 23.

    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.

    CAS  PubMed  Google Scholar 

  24. 24.

    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.

    CAS  Google Scholar 

  25. 25.

    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.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    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.

    CAS  PubMed  Google Scholar 

  27. 27.

    Tu AT. Spectroscopy of biological systems. Chichester: Wiley; 1986.

    Google Scholar 

  28. 28.

    Williams RW. Methods in enzymology (Hirs CHW, Timasheff SN, editors), vol. 130. Academic Press: New York. 1986. p. 311–31.

  29. 29.

    Fabian H, Anzenbacher P. New developments in Raman spectroscopy of biological systems. Vib Spectrosc. 1993;4:125–48.

    CAS  Google Scholar 

  30. 30.

    Bandekar J. Amide modes of reverse turns. Vib Spectrosc. 1993;5:143–73.

    CAS  Google Scholar 

  31. 31.

    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.

    CAS  PubMed  Google Scholar 

  32. 32.

    Biswas BB, Roy S, Miura T, Thomas G Jr. Proteins: structure, function, and engineering, vol. 24. US: Springer; 1995. p. 55–99.

    Google Scholar 

  33. 33.

    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.

    CAS  PubMed  Google Scholar 

  34. 34.

    Barth A, Zscherp C. What vibrations tell us about proteins. Q Rev Biophys. 2002;35:369–430.

    CAS  PubMed  Google Scholar 

  35. 35.

    Lee PC, Meisel D. Adsorption and surface-enhanced Raman of dyes on silver and gold sols. J Phys Chem. 1982;86:3391–5.

    CAS  Google Scholar 

  36. 36.

    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.

    CAS  Google Scholar 

  37. 37.

    Sutherland WS, Winefordner JD. Colloid filtration - a novel substrate preparation method for surface-enhanced Raman spectroscopy. J Colloid Interface Sci. 1992;148:129–41.

    CAS  Google Scholar 

  38. 38.

    Garcia-Leis A, Garcia-Ramos JV, Sanchez-Cortes S. Silver nanostars with high SERS performance. J Phys Chem C. 2013;117:7791–5.

    CAS  Google Scholar 

  39. 39.

    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.

    CAS  Google Scholar 

  40. 40.

    Pelton JT, McLean LR. Spectroscopic methods for analysis of protein secondary structure. Anal Biochem. 2000;277:167–76.

    CAS  PubMed  Google Scholar 

  41. 41.

    Bandekar J. Amide modes and protein conformation. Biochim Biophys Acta Protein Struct Mol Enzymol. 1992;1120:123–43.

    CAS  Google Scholar 

  42. 42.

    Barth A. Infrared spectroscopy of proteins. Biochim Biophys Acta Bioenerg. 2007;1767:1073–101.

    CAS  Google Scholar 

  43. 43.

    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.

    CAS  PubMed  Google Scholar 

  44. 44.

    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.

    CAS  PubMed  Google Scholar 

  45. 45.

    Remondetto GE, Subirade M. Molecular mechanisms of Fe2+-induced β-lactoglobulin cold gelation. Biopolymers. 2003;69:461–9.

    CAS  PubMed  Google Scholar 

  46. 46.

    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.

    PubMed  Google Scholar 

  47. 47.

    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.

    CAS  PubMed  Google Scholar 

  48. 48.

    Malkowski MG, Ginell SL, Smith WL, Garavito RM. The productive conformation of arachidonic acid bound to prostaglandin synthase. Science. 2000;289:1933.

    CAS  PubMed  Google Scholar 

  49. 49.

    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.

    CAS  PubMed  Google Scholar 

  50. 50.

    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.

    CAS  PubMed  Google Scholar 

  51. 51.

    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.

    CAS  PubMed  Google Scholar 

  52. 52.

    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.

    Google Scholar 

  53. 53.

    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.

    Google Scholar 

  54. 54.

    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.

    CAS  Google Scholar 

  55. 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.

    CAS  PubMed  Google Scholar 

  56. 56.

    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.

    CAS  PubMed  Google Scholar 

  57. 57.

    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.

    Google Scholar 

  58. 58.

    Sanchez-Cortes S, Garcia-Ramos JV. Anomalous Raman bands appearing in surface-enhanced Raman spectra. J Raman Spectrosc. 1998;29:365–71.

    CAS  Google Scholar 

  59. 59.

    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.

    CAS  Google Scholar 

  60. 60.

    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.

    PubMed  Google Scholar 

  61. 61.

    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.

    CAS  PubMed  Google Scholar 

  62. 62.

    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.

    CAS  Google Scholar 

  63. 63.

    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.

    CAS  PubMed Central  Google Scholar 

  64. 64.

    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.

    CAS  PubMed  Google Scholar 

Download references


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.

Author information



Corresponding authors

Correspondence to Zuzana Jurasekova or Armida Torreggiani.

Ethics declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material


(PDF 691 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

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).

Download citation


  • Substance P
  • Vibrational (Raman and infrared) spectroscopy
  • Surface-enhanced Raman spectroscopy (SERS)