Acid-base equilibrium of the Schiff base in bacteriorhodopsin

S. Druckmann, M. Ottolenghi, A. Pande, J. Pande, Robert Callender

Research output: Contribution to journalArticle

134 Citations (Scopus)

Abstract

Aqueous suspensions of dark-adapted bacteriorhodopsin (bR560) in the purple membrane of Halobacterium halobium are exposed to rapid jumps to high pH. Optical and resonance Raman measurements are carried out by using flow and stationary methods. Above pH ≃ 11.5 bR560 starts to be reversibly converted to a species absorbing at 460 nm (bR460) characterized by an unprotonated Schiff base chromophore. Above pH ≃ 13.0 bleaching takes place, first reversibly and subsequently irreversibly, to a species absorbing around 365 nm (bR365). This process competes with the formation of bR460. The pKa corresponding to the equilibrium bR560 + H2O ⇌ ka kd bR460 + H3O+ is determined as 13.3 ± 0.3. The value of the corresponding association rate constant determined from the reverse jumps (from pH 12.67 to pH 10 and 9.2) is ka = (3.5 ± 0.5) × 1011 M-1 s-1. Thus, starting with bR at pH 12.67 the reprotonation process is diffusion controlled as observed for homogeneous acid-base equilibria. The observed rate of dissociation when jumping from pH 6.5 to 12-13 is slower than that predicted by including the equilibrium bR560 + OH- ⇌ ka kd bR460 + H2O The results imply that the Schiff base is titratable in the dark, but its accessibility to external OH- ions is limited. The limitations in the significance of the "apparent" value of pKa = 13.3 observed for the Schiff base titration are discussed in light of possible alterations in the structure of bR resulting from the parallel titration of other protein groups. It is suggested that a light-induced pKa change of at least nine units takes place during the photocycle of light-adapted bR.

Original languageEnglish (US)
Pages (from-to)4953-4959
Number of pages7
JournalBiochemistry
Volume21
Issue number20
StatePublished - 1982
Externally publishedYes

Fingerprint

Bacteriorhodopsins
Acid-Base Equilibrium
Schiff Bases
Titration
Chromophores
Bleaching
Rate constants
Suspensions
Association reactions
Ions
Membranes
Purple Membrane
Halobacterium salinarum
Light
Proteins
hydroxide ion

ASJC Scopus subject areas

  • Biochemistry

Cite this

Druckmann, S., Ottolenghi, M., Pande, A., Pande, J., & Callender, R. (1982). Acid-base equilibrium of the Schiff base in bacteriorhodopsin. Biochemistry, 21(20), 4953-4959.

Acid-base equilibrium of the Schiff base in bacteriorhodopsin. / Druckmann, S.; Ottolenghi, M.; Pande, A.; Pande, J.; Callender, Robert.

In: Biochemistry, Vol. 21, No. 20, 1982, p. 4953-4959.

Research output: Contribution to journalArticle

Druckmann, S, Ottolenghi, M, Pande, A, Pande, J & Callender, R 1982, 'Acid-base equilibrium of the Schiff base in bacteriorhodopsin', Biochemistry, vol. 21, no. 20, pp. 4953-4959.
Druckmann S, Ottolenghi M, Pande A, Pande J, Callender R. Acid-base equilibrium of the Schiff base in bacteriorhodopsin. Biochemistry. 1982;21(20):4953-4959.
Druckmann, S. ; Ottolenghi, M. ; Pande, A. ; Pande, J. ; Callender, Robert. / Acid-base equilibrium of the Schiff base in bacteriorhodopsin. In: Biochemistry. 1982 ; Vol. 21, No. 20. pp. 4953-4959.
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N2 - Aqueous suspensions of dark-adapted bacteriorhodopsin (bR560) in the purple membrane of Halobacterium halobium are exposed to rapid jumps to high pH. Optical and resonance Raman measurements are carried out by using flow and stationary methods. Above pH ≃ 11.5 bR560 starts to be reversibly converted to a species absorbing at 460 nm (bR460) characterized by an unprotonated Schiff base chromophore. Above pH ≃ 13.0 bleaching takes place, first reversibly and subsequently irreversibly, to a species absorbing around 365 nm (bR365). This process competes with the formation of bR460. The pKa corresponding to the equilibrium bR560 + H2O ⇌ ka kd bR460 + H3O+ is determined as 13.3 ± 0.3. The value of the corresponding association rate constant determined from the reverse jumps (from pH 12.67 to pH 10 and 9.2) is ka = (3.5 ± 0.5) × 1011 M-1 s-1. Thus, starting with bR at pH 12.67 the reprotonation process is diffusion controlled as observed for homogeneous acid-base equilibria. The observed rate of dissociation when jumping from pH 6.5 to 12-13 is slower than that predicted by including the equilibrium bR560 + OH- ⇌ ka kd bR460 + H2O The results imply that the Schiff base is titratable in the dark, but its accessibility to external OH- ions is limited. The limitations in the significance of the "apparent" value of pKa = 13.3 observed for the Schiff base titration are discussed in light of possible alterations in the structure of bR resulting from the parallel titration of other protein groups. It is suggested that a light-induced pKa change of at least nine units takes place during the photocycle of light-adapted bR.

AB - Aqueous suspensions of dark-adapted bacteriorhodopsin (bR560) in the purple membrane of Halobacterium halobium are exposed to rapid jumps to high pH. Optical and resonance Raman measurements are carried out by using flow and stationary methods. Above pH ≃ 11.5 bR560 starts to be reversibly converted to a species absorbing at 460 nm (bR460) characterized by an unprotonated Schiff base chromophore. Above pH ≃ 13.0 bleaching takes place, first reversibly and subsequently irreversibly, to a species absorbing around 365 nm (bR365). This process competes with the formation of bR460. The pKa corresponding to the equilibrium bR560 + H2O ⇌ ka kd bR460 + H3O+ is determined as 13.3 ± 0.3. The value of the corresponding association rate constant determined from the reverse jumps (from pH 12.67 to pH 10 and 9.2) is ka = (3.5 ± 0.5) × 1011 M-1 s-1. Thus, starting with bR at pH 12.67 the reprotonation process is diffusion controlled as observed for homogeneous acid-base equilibria. The observed rate of dissociation when jumping from pH 6.5 to 12-13 is slower than that predicted by including the equilibrium bR560 + OH- ⇌ ka kd bR460 + H2O The results imply that the Schiff base is titratable in the dark, but its accessibility to external OH- ions is limited. The limitations in the significance of the "apparent" value of pKa = 13.3 observed for the Schiff base titration are discussed in light of possible alterations in the structure of bR resulting from the parallel titration of other protein groups. It is suggested that a light-induced pKa change of at least nine units takes place during the photocycle of light-adapted bR.

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