TY  - JOUR
AU  - Seemann, Klaus M.
AU  - Kiefersauer, Reiner
AU  - Jacob, Uwe
AU  - Kuhn, Bernd
TI  - Optical pH Detection within a Protein Crystal
JO  - The journal of physical chemistry <Washington, DC> / B
VL  - 116
SN  - 1520-6106
CY  - Washington, DC
PB  - Soc.
M1  - PreJuSER-111840
SP  - 9873-9881
PY  - 2012
N1  - Record converted from VDB: 16.11.2012
AB  - The pH is one of the key parameters governing protein conformation and activity. In protein crystals, however, the pH is so far not accessible by experiment. Here, we report on the optical detection of the pH in a lysozyme crystal employing the pH-sensitive fluorescent dyes SNARF-1 and SNARF-4F. The molecular probes were loaded into the crystal by diffusion. Two-dimensional fluorescence spectra of the labeled protein crystal were recorded, and the average pH of the crystal at different bath pH's was determined by calibrating fluorescence peak ratios. In addition, we used two-photon microscopy to spatially resolve the pH inside a lysozyme crystal three-dimensionally and to follow pH changes in response to a pH change of the bath over time. At equilibrium at bath pH between 5.5 and 8.0, we found a pH in the water-filled crystal channels that was ΔpH = -0.3 to -1.0 lower than that of the bath. This corresponds to a 2- to 10-fold higher proton concentration in the crystal channels than in the bath. The lower pH at equilibrium in the crystal channels can be explained by slower proton diffusion in the channels than in the bath and a resulting proton accumulation in the crystal for conservation of mass and so an equilibrium of proton flux.
KW  - J (WoSType)
LB  - PUB:(DE-HGF)16
C6  - pmid:22834887
UR  - <Go to ISI:>//WOS:000307749100001
DO  - DOI:10.1021/jp2103512
UR  - https://juser.fz-juelich.de/record/111840
ER  -