Circular Dichroism Studies of Substance P and Its C‐terminal Sequences

Abstract

CD spectra of substance P (SP) and its C‐terminal partial sequences have been measured in diluted aqueous solution including variation of hydrogen ion concentration. In the far u.v. region there is an overlapping of the amide CD absorption by the CD of the phenylalanine residue aromatic side chains (217–220 nm). This complex CD absorption is reduced during changes from acidic to alkaline pH, especially in those cases where a phenylalanine residue is at the N‐terminus of the peptide chain. These CD changes dependent on pH are due more to charge effects on the aromatic chromophores than to substantial conformational changes. However, solvation effects on the conformational features of SP peptides caused by the deprotonation of the amino groups have to be taken into account. CD and potentiometric titrations indicate that the N‐terminal α‐amino groups of the SP peptides in general are freely accessible to the solvent. Our studies did not give any evidence of the occurrence of ordered structures of SP peptides in diluted aqueous solution.

Substance P in Solution: Trans-to-Cis Configurational Changes of Penultimate Prolines Initiate Non-enzymatic Peptide Bond Cleavages

“…At longer incubation times (e.g., 150 min in Figure 1), two new peaks in the mass spectra can be observed at m/z = 548 and 254. These peaks can be assigned to products associated with cleavage of the Pro 2 -Lys 3 The subP (3)(4)(5)(6)(7)(8)(9)(10)(11) product also contains a penultimate proline residue. As the system continues to incubate, (e.g., 2880 min.)…”

“…The peptide subP (≥95% purity) was obtained from Sigma Aldrich (St. Louis, MO, U.S.A.). The truncated subP (3)(4)(5)(6)(7)(8)(9)(10)(11) peptide, and several subP analogues involving a range of Pro→Ala substitutions, were synthesized in house, using standard FMOC solid-phase peptide synthesis carried out on an Applied Biosystems 433A Peptide Synthesizer (Applied Biosystems, Foster City, CA). [35] Each purified peptide was dissolved into pure ethanol solutions to concentrations of 500 µM and stored at −22 °C.…”

“…These studies revealed that efficiency of ionizing and detecting cRP and cKP was low, relative to the larger peptides, as can be observed in the mass spectra presented below. For the kinetics plots shown below, in order to estimate the solution abundances of the different species that are present, we multiply the [subP (3)(4)(5)(6)(7)(8)(9)(10)(11) Methods for investigating proline configurations.…”

“…Substance P (subP), an undecapeptide with the sequence Arg 1 -Pro 2 -Lys 3 -Pro 4 -Gln 5 -Gln 6 -Phe 7 -Phe 8 -Gly 9 -Leu 10 -Met 11 -NH 2 , was discovered in 1931 by Euler & Gaddum [1] and was among the first known neurotransmitters. This neuropeptide is found throughout the nervous system as well as in the smooth-muscle tissue of the gastrointestinal tract.…”

“…At mM concentrations subP can form oligomers that are associated through hydrophobic residues near the C terminus. [6,7] SubP is susceptible to methionine oxidation, which decreases biological activity. [8] Additionally, when stored for long times, subP degrades to form cyclic diketopiperazine (DKP) forms of the dipeptides Arg 1 -Pro 2 and Lys 3 -Pro 4 .…”