Interpretation of multiple Q(0,0) bands in the absorption spectrum of Mg-mesoporphyrin embedded in horseradish peroxidase.

Mg-mesoporphyrin horseradish peroxidase (MgMP-HRP) and MgMP-HRP complexed with naphtohydroxamic acid (NHA) have been studied by fluorescence line narrowing (FLN) and pressure tuning spectral hole burning (SHB) techniques. In each sample, the low temperature absorption spectra show more than one transition in the origin range of the Q band. Comparisons with broad-band fluorescence spectra and FLN studies suggest that the multiple band feature originates from the presence of different configurations of the metal-porphyrin that are subject to Qx-Qy splitting within the protein cavity. This suggestion is supported by pressure tuning SHB studies. In the uncomplexed as well as in the NHA-complexed form of MgMP-HRP, irradiation in the Q band produces photoproduct bands, which has been attributed to a species with smaller Qx-Qy splitting. In an amorphous matrix, on the other hand, only one form of MgMP could be found, and no splitting could be observed. The binding of NHA does not significantly alter the bulk parameters of the protein matrix, but it reduces the structural variety in the configuration of MgMP to a single form with a more distorted structure and thus with an enlarged Qx-Qy splitting.     

Protochlorophyllide forms and energy transfer in dark-grown wheat leaves. Studies by conventional and laser excited fluorescence spectroscopy between 10 K–100 K

The fluorescence properties and role in energy transfer of protochlorophyllide (Pchlide) forms were studied in dark-grown wheat leaves by conventional and laser excited high resolution methods in the 10 K–100 K temperature range. The three major spectral bands, with emission maxima at 633, 657 (of highest intensity) and 670 nm as Bands I, II, and III were analyzed and interpreted as the contributions of six different structural forms. Band I is the envelope of three (0,0) emission bands with maxima at 628, 632 and 642 nm. Laser excitation studies in the range of Band II at 10 K reveal the presence of a spectrally close donor band besides the acceptor, Band II. The intensity in Band III originates mostly from being the vibronic satellite of Band II, but contains also a small (0,0) band with absorption maximum at 674 nm. Excitation spectra show that besides the Pchlides with absorption around 650 nm within Band II, another significant population of Band I with absorption around 640 nm is also coupled by energy transfer to the acceptor of Band II. The spectral difference between the two donor forms indicate different dipolar environments. Upon increasing the temperature, the intensity of Band II and its satellite, Band III decrease, while Band I remains unaffected. Band II shows also a broadening towards the blue side at higher temperatures. Both the quenching of fluorescence and the spectral change was explained by a thermally activated formation of a non-fluorescent intermediate state in the excited state of Pchlide acceptors.     

Hsp90 inhibition accelerates cell lysis. Anti-Hsp90 ribozyme reveals a complex mechanism of Hsp90 inhibitors involving both superoxide- and Hsp90-dependent events

The 90 kDa heat shock protein, Hsp90, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells. Here we analyzed the involvement of Hsp90 in the maintenance of cellular integrity using partial cell lysis as a measure. Inhibition of Hsp90 by geldanamycin, radicicol, cisplatin, and novobiocin induced a significant acceleration of detergent- and hypotonic shock-induced cell lysis. The concentration and time dependence of cell lysis acceleration was in agreement with the Hsp90 inhibition characteristics of the N-terminal inhibitors, geldanamycin and radicicol. Glutathione and other reducing agents partially blocked geldanamycin-induced acceleration of cell lysis but were largely ineffective with other inhibitors. Indeed, geldanamycin treatment led to superoxide production and a change in membrane fluidity. When Hsp90 content was diminished using anti-Hsp90 hammerhead ribozymes, an accelerated cell lysis was also observed. Hsp90 inhibition-induced cell lysis was more pronounced in eukaryotic (yeast, mouse red blood, and human T-lymphoma) cells than in bacteria. Our results indicate that besides the geldanamycin-induced superoxide production, and a consequent increase in cell lysis, inhibition or lack of Hsp90 alone can also compromise cellular integrity. Moreover, cell lysis after hypoxia and complement attack was also enhanced by any type of Hsp90 inhibition used, which shows that the maintenance of cellular integrity by Hsp90 is important in physiologically relevant lytic conditions of tumor cells.