Blackbody infrared radiative dissociation of bradykinin and its analogues: energetics, dynamics, and evidence for salt-bridge structures in the gas phase

Abstract

Blackbody infrared radiative dissociation (BIRD) spectra of singly and doubly protonated bradykinin and its analogues are measured in a Fourier-transform mass spectrometer. Rate constants for dissociation are measured as a function of temperature with reaction delays up to 600 s. From these data, Arrhenius activation parameters in the zero-pressure limit are obtained. The activation parameters and dissociation products for the singly protonated ions are highly sensitive to small changes in ion structure. The Arrhenius activation energy (E(a)) and pre-exponential (or frequency factor, A) of the singly protonated ions investigated here range from 0.6 to 1.4 eV and 10(5) to 10(12) s(-1), respectively. For bradykinin and its analogues differing by modification of the residues between the two arginine groups on either end of the molecule, the singly and doubly protonated ions have average activation energies of 1.2 and 0.8 eV, respectively, and average A values of 10(8) and 10(12) s(-1), respectively, i.e., the presence of a second charge reduces the activation energy by 0.4 eV and decreases the A value by a factor of 10(4). This demonstrates that the presence of a second charge can dramatically influence the dissociation dynamics of these ions. The doubly protonated methyl ester of bradykinin has an E(a) of 0.82 eV, comparable to the value of 0.84 eV for bradykinin itself. However, this value is 0.21 +/- 0.08 eV greater than that of singly protonated methyl ester of bradykinin, indicating that the Coulomb repulsion is not the most significant factor in the activation energy of this ion. Both singly and doubly protonated Lys-bradykinin ions have higher activation energies than the corresponding bradykinin ions indicating that the addition of a basic residue stabilizes these ions with respect to dissociation. Methylation of the carboxylic acid group of the C-terminus reduces the E(a) of bradykinin from 1.3 to 0.6 eV and the A factor from 1012 to 105 s(-1). This modification also dramatically changes the dissociation products. Similar results are observed for [Ala(6)]-bradykinin and its methyl ester. These results, in combination with others presented here, provide experimental evidence that the most stable form of singly protonated bradykinin is a salt-bridge structure.

Figure 1

Data for the dissociation of (a) des-Arg¹- and (b) des-Arg⁹-bradykinin fit to unimolecular kinetics at the temperatures indicated. The rate constants in order of increasing temperature are (a) 0.0058, 0.0095, 0.014, 0.023, 0.037 s⁻¹ and (b) 0.0087, 0.020, 0.035, 0.066, 0.098 s⁻¹.

Figure 2

Blackbody infrared radiative dissociation spectra of singly protonated (a) des-Arg¹-bradykinin with a reaction delay of 45 s and a cell temperature of 191 °C and (b) des-Arg⁹-bradykinin with a reaction delay of 10 s and a cell temperature of 179 °C.

Figure 3

Data for the dissociation of bradykinin fit to unimolecular kinetics at the temperatures indicated. The rate constants in order of increasing temperature are 0.0027, 0.0042, 0.013, 0.016, 0.043, 0.068, and 0.32 s⁻¹.

Figure 4

Arrhenius plot for the dissociation of singly protonated bradykinin (•), des-Arg¹-bradykinin (□), des-Arg⁹-bradykinin (▪), methyl ester of des-Arg⁹-bradykinin (▵), Lys-bradykinin (▴), and doubly protonated bradykinin (○).

Figure 5

Blackbody infrared radiative dissociation spectra of singly protonated (a) bradykinin (45 s reaction delay), (b) [Thr⁶]-bradykinin (45 s reaction delay), (c) [Ala⁶]-bradykinin (45 s reaction delay), and (d) des-Pro²-bradykinin (15 s reaction delay) measured at 191 °C.

Figure 6

Blackbody infrared radiative dissociation spectra of singly protonated (a) [Lys1]-bradykinin (45 s reaction delay at 191 °C) and (b) Lys-bradykinin (240 s reaction delay at 203 °C). R ≡ arginine, P ≡ proline.

Figure 7

Illustration of a salt-bridge for singly protonated bradykinin in which both arginines are protonated and the C-terminus is deprotonated.

Figure 8

Blackbody infrared radiative dissociation spectra of the singly protonated (a) methyl ester of bradykinin and (b) methyl ester of [Ala⁶]-bradykinin measured at 197 °C with a 15 s reaction delay.

Figure 9

Blackbody infrared radiative dissociation spectra of the singly protonated (a) methyl ester of des-Arg¹-bradykinin (420 s reaction delay at 219 °C) and (b) methyl ester of des-Arg⁹-bradykinin (480 s reaction delay at 215 °C).

Figure 10

Blackbody infrared radiative dissociation spectra of doubly protonated (a) bradykinin, (b) des-Pro²-bradykinin, (c) [Thr⁶]-bradykinin, and (d) [Ala⁶]-bradykinin measured at 191 °C with a 15 s reaction delay.

Figure 11

Blackbody infrared radiative dissociation spectra of doubly protonated (a) Lys-bradykinin (45 s reaction delay at 210 °C) and (b) methyl ester of bradykinin (180 s reaction delay at 215 °C). R ≡ arginine, P ≡ proline.

Figure 12

Molecular dynamics simulation of doubly protonated bradykinin at 450 K for 11 ns: (a) distance between the charges on the arginine residues (charge sites are represented by location of the central carbon atom on each guanidine group), (b) distance between the charge on Arg and the α-carbon of the phenyl side chain located on Phe, (c) distance between the charge on Arg the C-terminus carboxyl carbon, and (d) distance between the charge on Arg and the carbonyl oxygen of Pro.

Scheme 1