Steady-state and picosecond time-resolved fluorescence studies on native and apo seed coat soybean peroxidase.
Seed coat soybean peroxidase (SBP) belongs to class III of the plant peroxidase super family. The protein has a very similar 3-dimensional structure with that of horseradish peroxidase (HRP-C). The fluorescence characteristics of the single tryptophan (Trp117) present in SBP and apo-SBP have been studied by steady-state and pico-second time-resolved fluorescence spectroscopy. Fluorescence decay curve of SBP was best described by a four exponential model that gave the lifetimes, 0.035 ns (97.0%), 0.30 ns (2.0%), 2.0 ns (0.8%), and 6.3 ns (0.2%). These lifetime values agreed very well with the values obtained by the model independent maximum entropy method (MEM). The three longer lifetimes that constituted 3% of the fluorophore population in the SBP sample are attributed to the presence of trace quantities of apo-SBP. The pico-second lifetime value of SBP is indicative of efficient energy transfer from Trp117 to heme. From fluorescence resonance energy transfer (FRET) calculations, the energy-transfer efficiency in SBP is found to be relatively higher as compared to HRP-C and is attributed mainly to the higher value of orientation factor, kappa(2) for SBP. Decay-associated spectra of SBP indicated that the tryptophan of SBP is relatively more solvent exposed as compared to HRP-C and is attributed to the various structural features of SBP. Linear Stern-Volmer plots obtained from the quenching measurements using acrylamide gave k(q) = 5.4 x 10(8) M(-1) s(-1) for SBP and 7.2 x 10(8) M(-1) s(-1) for apo-SBP and indicated that on removal of heme in SBP, Trp117 is more solvent exposed.
Kamal
J K
JK
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, Mumbai 400 005, India.
Behere
D V
DV
D019277:Entropy; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D010544:Peroxidases / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D010940:Plant Proteins; D006418:Heme; D014364:Tryptophan; D010544:Peroxidases; C120494:anionic peroxidase