Optical and IR absorption as probe of dynamics of heme proteins

The spectroscopy of horseradish peroxidase with and without the substrate analogue benzohydroxamic acid (BHA) was monitored in different solvents as a function of the temperature in the interval from 10 to 300 K. Thermal broadening of the Q(0,0) optical absorption band arises mainly from interaction of the electronic pi --> pi transition with the heme vibrations. In contrast, the width of the IR absorption band of CO bound to heme is controlled by the coupling of the CO transition moment to the electric field of the protein matrix. The IR bandwidth of the substrate free enzyme in the glycerol/H2O solvent hardly changes in the glassy matrix and strongly increases upon heating above the glass transition. Heating of the same enzyme in the trehalose/H2O glass considerably broadens the band. The binding of the substrate strongly diminishes the temperature broadening of the CO band. This result is consistent with the view that the BHA strongly reduces the amplitude of vibrations of the heme pocket environment. Unusually strong thermal broadening of the CO band above the glass transition is interpreted to be caused by thermal population of a very flexible excited conformational substate. The thermal broadening of the same band in the trehalose glass is caused by an increase of the protein vibrational amplitude in each of the conformational substates, their population being independent of the temperature in the glassy matrix.     

Carbonmonoxy horseradish peroxidase as a function of pH and substrate: influence of local electric fields on the optical and infrared spectra

Infrared and optical spectra of carbonmonoxy horseradish peroxidase were monitored as a function of pH and substrate binding. The analyses of experimental results together with semiempirical calculations show that the CO-porphyrin complex is sensitive to environmental changes. The electronic Q(0,0) band of the porphyrin and the CO stretching mode respond to external perturbations with different symmetry dependencies. In this way, the complex is nonisotropic, and the combined spectral analyses constitute a valuable tool for the investigation of structure. In the absence of substrate and at pH 6.0, the low-spin heme optical Q(0,0) absorption band is a single peak that narrows as the temperature decreases. Under these conditions, the CO vibrational stretch frequency is at 1903 cm(-1). Addition of the substrates benzohydroxamic acid or naphthohydroxamic acid produces a split of approximately 320 cm(-1) in the Q(0,0) absorption band that is clearly evident at < 100 K and shifts the CO absorption to 1916 cm(-1). Increasing the pH to 9.3 also causes a split in the Q(0,0) optical band and elicits a shift in nu(CO) to a higher frequency (1936 cm(-1)). The splitting of the Q(0,0) band and the shifts in the IR spectra are both consistent with changes in the local electric field produced by the proximity of the electronegative carbonyl of the substrate near the heme or the protonation and/or deprotonation of the distal histidine, although other effects are also considered. The larger effect on the Q(0,0) band with substrate at low pH and the shift of nu(CO) at high pH can be rationalized by the directionality of the field and the orientation dependence of dipolar interactions.     

Hepatitis viruses and hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is among the most frequent malignancies worldwide. Hepatitis viruses, such as the hepatitis B virus (HBV) and hepatitis C virus (HCV) are undoubtedly listed in the etiology of HCC. Studies show that, in the near future, viral hepatitis will carry increasing weight in the etiology of HCC. This review briefly discusses the known carcinogenic effects of HBV and HCV in the light of experimental and human studies. The data show that viral proteins may directly interfere with gene products responsible for cell proliferation and cell growth. Many other signal transduction cascades may be affected as well. Direct integration of HBV viral sequences into the host genome increases the genomic instability. The genomic imbalance allows the development and survival of malignant clones bearing defected genomic information. HBV and HCV infection induces indirect and direct mechanisms through cellular damage, increased regeneration and cell proliferation, therefore enhancing the development of HCC.     

Influence of static and dynamic disorder on the visible and infrared absorption spectra of carbonmonoxy horseradish peroxidase.

Spectroscopy of horseradish peroxidase with and without the substrate analog, benzohydroxamic acid, was monitored in a glycerol/water solvent as a function of temperature. It was determined from the water infrared (IR) absorption that the solvent has a glass transition at 170-180 K. In the absence of substrate, both the heme optical Q(0,0) absorption band and the IR absorption band of CO bound to heme broaden markedly upon heating from 10-300 K. The Q(0,0) band broadens smoothly in the whole temperature interval, whereas the IR bandwidth is constant in the glassy matrix and increases from 7 to 16 cm(-1) upon heating above the glass transition. Binding of substrate strongly diminishes temperature broadening of both the bands. The results are consistent with the view that the substrate strongly reduces the amplitude of motions of amino acids forming the heme pocket. The main contribution to the Q(0,0) bandwidth arises from the heme vibrations that are not affected by the phase transition. The CO band thermal broadening stems from the anharmonic coupling with motions of the heme environment, which, in the glassy state, are frozen in. Unusually strong temperature broadening of the CO band is interpreted to be caused by thermal population of a very flexible excited conformational substrate. Analysis of literature data on the thermal broadening of the A(0) band of Mb(CO) (Ansari et al., 1987. Biophys. Chem. 26:337-355) shows that such a state presents itself also in myoglobin.     

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.     

Fluorescent derivatives of bovine neurotensin 8–13 fragment

Fluorescent derivatives of bovine neurotensin 8–13 fragment were prepared. For N-terminal labelling, 4-[7-hydroxycoumaryl]acetic acid (Hca), 4-[7-methoxycoumaryl]acetic acid (Mca) and 2-amino-3-[4-[7-methoxycoumaryl]]propionic acid (Amp) were used while the C-terminus of the peptide chain was elongated with Amp. The fluorescence excitation and emission spectra of the peptide derivatives were studied. Hca- and Mca/Amp-derivatives were easily distinguishable because of the 60 nm shift of their emission maxima. Compared with the natural sequence, the presence of an N-terminal label did not influence the biological potency in a longitudinal muscle strip of guinea-pig ileum, while labelling at the C-terminus considerably reduced the activity of the peptide.     

Horseradish peroxidase monitored by infrared spectroscopy: effect of temperature, substrate and calcium

Horseradish peroxidase was examined as a function of Ca and substrate binding using infrared spectroscopy in the temperature range of 10-300 K. The Ca complex could be identified by the carboxylate stretches. The amide peak positions indicate that the protein remains stable from room temperature to 10 K. Shifts in these peaks are consistent with increased hydrogen bonding as temperature decreases, but the protein conformation is maintained at cryogenic temperatures. The substrate, benzohydroxamic acid, produced no detectable change in the infrared spectrum, consistent with X-ray crystallography results. With removal of Ca, the protein maintained its overall helicity.     

Solvent dependent and independent motions of CO-horseradish peroxidase examined by infrared spectroscopy and molecular dynamics calculations

The role of the solvent matrix in affecting CO bound to ferrous horseradish peroxidase was examined by comparing band-widths of nu(CO) for the protein in aqueous solutions and in trehalose/sucrose glasses. We have previously observed that the optical absorption band and the CO stretching mode respond to the glass transition of glycerol/water in ways that depend upon the presence of substrate (Biochemistry 40 (2001) 3483). It is now demonstrated that the CO group band-width for the protein with bound inhibitor benzhydroxamic acid is relatively insensitive to temperature or the glass transition of the solvent. In contrast, in the absence of inhibitor, the band-width varies with the temperature that the glass is formed. The results show that solvent dependent and independent motions can be distinguished, and that the presence of substrate changes the protein such that the Fe[bond]CO site is occluded from the solvent conditions. Molecular dynamic calculations, based upon X-ray structures, showed that the presence of benzhydroxamic acid decreases the distance between His42 and Arg38 and this leads for closer distances to the O of the CO from these residues. These results are invoked to account for the observed line width changes of the CO band.     

Variability of cnidae within a small clonal sea anemone (Isactinia sp.)

The cnidom and intraspecific variability of cnidae within the small sea anemone Isactinia sp. were verified. The specific cnidae within the cnidom of four discrete morphological structures (tentacle, actinopharynx, mesenterial filaments, and body column) within Isactinia sp. was investigated. Microbasic b-mastigophores, microbasic p-mastigophores, basitrichs, microbasic p-amastigophores, and spirocysts were found in this species. In addition, two morphologically distinct basitrich forms, distinguishable only in a discharged state, were also found, of which basitrichs with the distinctly shorter thread were found predominately only on the body column. The distribution and abundance of cnidae types differed significantly around the body in the sea anemones, as did the length of basitrichs and spirocysts among tissue types. Cnidae length also differed significantly among individuals. Correlations between cnidae length and sea anemone size were variable, whereby cnidae size was significantly negatively correlated to sea anemone size in seven cnidae–tissue combinations, positively correlated in one, and not correlated in two. Linear regression revealed that sea anemone size was able to explain 33%–68% of variation in size of b-mastigophores, p-amastigophores, and small basitrichs from within the mesenterial filaments. Correlations were negligible or not significant in remaining cnidae–tissue combinations, however. While providing key taxonomic cnidae information, this study also highlights the variability of cnidae that may occur within a species of Isactinia.     

In situ detection of ALA-stimulated porphyrin metabolic products in Escherichia coli B by fluorescence line narrowing spectroscopy.

In a recent work [Photochem. Photobiol. B: Biol. 50 (1999) 8] the successful photodynamic inactivation of Escherichia coli bacteria by visible light was reported based on delta-aminolevulinic acid (ALA)-induced endogenous porphyrin accumulation. In this work, the identification of these porphyrin derivatives in intact bacteria was performed by low-temperature conventional fluorescence and fluorescence line narrowing (FLN) techniques. Conventional fluorescence emission spectroscopy at cryogenic temperatures revealed the presence of the free-base porphyrins, identified earlier by high-performance liquid chromatography analysis of disintegrated bacterial cells after ALA induction; however, emission maxima characteristic for metal porphyrins were also observed. We demonstrated that the primary reason for this signal is that metal porphyrins are formed from free-base porphyrins by Mg2+ ions present in the culturing medium. Incorporation of Zn ions originating from the glassware could also be supposed. In the FLN experiment, the energy selection effect could be clearly demonstrated for (0,0) emissions of both the free-base and the metal porphyrins. The comparison of the conventional emission spectra and the bands revealed by the FLN experiment show that the dominant monomeric structural population is that of metal porphyrins. The intensity and the shape of the FLN lines indicate an aggregated population of the free-base porphyrins, beside a small monomeric population.