Cross-talk between Cys34 and lysine residues in human serum albumin revealed by N-homocysteinylation

Protein N-homocysteinylation involves a post-translational modification by homocysteine (Hcy)-thiolactone. In humans, about 70% of circulating Hcy is N-linked to blood proteins, mostly to hemoglobin and albumin. It was unclear what protein site(s) were prone to Hcy attachment and how N-linked Hcy affected protein function. Here we show that Lys(525) is a predominant site of N-homocysteinylation in human serum albumin in vitro and in vivo. We also show that the reactivity of albumin lysine residues, including Lys(525), is affected by the status of Cys(34). The disulfide forms of circulating albumin, albumin-Cys(34)-S-S-Cys and albumin-Cys(34)-S-S-Hcy, are N-homocysteinylated faster than albumin-Cys(34)-SH. Although N-homocysteinylations of albumin-Cys(34)-SH and albumin-Cys(34)-S-S-Cys yield different primary products, subsequent thiol-disulfide exchange reactions result in the formation of a single product, N-(Hcy-S-S-Cys)-albumin-Cys(34)-SH. We also show that N-homocysteinylation affects the susceptibility of albumin to oxidation and proteolysis. The data suggest that a disulfide at Cys(34) of albumin promotes conversion of N-(Hcy-SH)-albumin-Cys(34)-SH to a proteolytically sensitive form N-(Hcy-S-S-Cys)-albumin-Cys(34)-SH, which would facilitate clearance of the N-homocysteinylated form of mercaptoalbumin.     

Circular dichroism and magnetic circular dichroism spectra of chlorophylls in nematic liquid crystals. I. Electric and weak magnetic field effects on the dichroism spectra

Dichroism spectra of chlorophyll a, chlorophyll b and bacteriochlorophyll a in various nematic liquid crystals are reported. The initial orientation of chlorophylls in such a sample is determined by the interaction of the aggregate formed from the pigment and the liquid crystal molecules with the electrode surface on the cell windows. Reorientation is carried out by either an electric or magnetic field. The analysis of the circular dichroism spectra obtained from these samples on the basis of the Mueller matrix shows that the intensity is predominantly related to the texture of the sample. Chlorophyll molecules can be aggregated with liquid crystals in two ways: (1) through the chlorin magnesium atom, which results in the liquid crystal chain being almost perpendicular to the porphyrin ring, or (2) attached parallel to the line connecting the first and third pyrrole rings of the chlorin, the chlorin now lying in the plane of the liquid crystal chains. By comparing the dichroism spectra of various chlorophylls in the same liquid crystal we can draw conclusions concerning the preferred type of aggregation, not only with liquid crystals, but also with biological molecules. These liquid crystal systems are models of the orientation effects found for chlorophyll in lamellae. The model studied in this work is much simpler than the lamellar system but it does exhibit several common properties with the latter. Both systems are anisotropic and show much more intense dichroism signals, often of opposite sign, compared with those observed for photosynthetic pigments in isotropic solutions. Dichroism signals of organism fragments are much more complex than those of our model, which can either be related to the occurrence in the organism of several types of pigments or, for a given type of pigment, could be the result of exciton splitting. On the basis of our model it is shown that small changes in the anisotropy of the pigment in the surroundings have a strong influence on the sign and amplitude of the observed circular dichroism signal. Such effects may be responsible for the structure of the dichroism spectra observed for biological samples. Such structures can be partially related to the superposition of the dichroism signal from various ‘domains’ of chromophore which are different in both pigment arrangement and in the anisotropy of the surroundings of the pigment molecules themselves.     

In Vivo Binding to Peripheral Benzodiazepine Binding Sites in Lesioned Rat Brain: Comparison Between [3H]PK11195 and [18F]PK14105 as Markers for Neuronal Damage

Peripheral-type benzodiazepine binding sites are not normally present in most cerebral tissues, but following neuronal damage, the cells involved in the ensuing gliosis show a marked expression of these sites. In a unilateral excitotoxic striatal lesion in the rat, we sought to determine whether the isoquinoline derivatives PK11195 and PK14105 bind to these sites in vivo and whether demonstration of these sites offers the potential of indirectly localising areas of neuronal damage. Binding was studied at several intervals after coinjection of [3H]PK11195 and [18F]PK14105 to determine the time courses of specific binding. Both compounds were rapidly extracted into all cerebral tissues, but in the absence of binding sites in nonlesioned tissues, this was followed by a rapid clearance of radioactivity. In lesioned areas, both [3H]PK11195 and [18F]PK14105 accumulated over the first 5 min followed by a much slower clearance of radioactivity, resulting in a "specific signal." [3H]PK11195 binding peaked at 20-30 min postinjection, with radioactivity in the lesioned striatum being three times greater than in its contralateral homologue. The specific signal was present for at least 60 min. The maximal [18 F]PK14105-specific signal was of similar magnitude but peaked earlier and was retained for only 45 min. Specific signals with both ligands were also detected in regions remote from the primary lesion site, e.g., in the hippocampus and substantia nigra. Predosing animals with a large dose of PK11195 (3 mg/kg), sufficient to saturate peripheral-type benzodiazepine binding sites, abolished in vivo binding of both [3H]PK11195 and [18F]PK14105 to both primary- and remote-lesioned tissues. The specific signal with both ligands could be of sufficient magnitude and duration to make tomographic studies in humans feasible.     

Analysis of time-resolved emission spectra of oriented phycobilisomes

The polarized time-resolved (in ps range) fluorescence spectra of phycobilisomes obtained from cyanobacteria Tolypothrix tenuis embedded in poly(vinyl alcohol) films and oriented by film stretching have been analysed. Fluorescence spectra were deconvoluted on Gaussian components supposing the same positions of components maxima in three sets of time-resolved spectra taken in natural and polarized light. A good fit of the experimental and calculated spectra was obtained when using the following maxima: 580 and 595 nm in the phycoerythrin region, 634 and 650 nm in the phycocyanin region, 660 and 680 nm in the allophycocyanin region. The area under curve of the Gaussian component vs. time gives the shape of rise and decay of emission of chromophores contributing to the given component. These kinetics were analysed using several model functions. The experimental excitation profile was convoluted with a multiexponential model individually or "globally" e.g. assuming the same lifetime values for the given species in all sets of spectra. The Foerster-Hauser types of two- and three-dimensional models we also convoluted with excitation profile and fitted to the decay of primarily excited species. The first acceptor decay can be described well by the Foerster-Hauser models or by a monoexponential function. The accuracy of fit in either case of three- and two-dimensional Foerster-Hauser function is similar. The fluorescence rise and decay of the next species in a donor-acceptor chain can be analysed in terms of two or three exponential functions. Obtained lifetimes of fluorescence are similar to those reported in literature. The results suggest that there are more than one chain of excitation donors and acceptors in the phycobilisomes of cyanobacteria Tolypothrix tenuis.     

Photothermal spectroscopy of bacteriochlorophyll-lipoprotein complexes.

Photoacoustic spectra at room and 85 K temperatures as well as photothermal beam deflection spectra of bacteriochlorophyll-lipoprotein complexes from purple bacterium Chromatium minutissimum were measured. Spectra were compared and obtained differences were tentatively explained by various inertion of these two methods. Photothermal beam deflection method measure the heat which is generated in close surroundings of absorbing pigment molecule, whereas usage of more inert photoacoustic signal is averaged over contributions from various pigments located in a larger sample volume and therefore is similar to absorption spectra.     

Preliminary studies of phthalocyanine sensitizers incorporated into human leukemia cells from two cell-lines

Three phthalocyanine dyes-sensitizers were incorporated into two types of human T leukemia cells from two cell-lines: CCRF and MOLT 4. The efficiency of the dye incorporation into cells and photochemical properties of stained cells were investigated using fluorescence spectroscopy. The dyes exhibited different properties in each of the two cell-lines. Small differences in cell membrane properties have a strong influence on the efficiency of dye incorporation and on the course of photodynamic reaction. It is suggested that, for a given patient, an optimal dye-sensitizer should be established before photodynamic treatment.     

Identification of N-homocysteinylation sites in plasma proteins

A protocol for the identification of N-homocysteinylation sites in plasma proteins is described. Human plasma or purified fibrinogen is subjected to trypsin digestion and analysis of N-Hcy-peptides by liquid chromatography/mass spectroscopy (LC/MS). Human fibrinogen is isolated from the plasma by the glycine precipitation method. Identification of N-Hcy-Lys-peptides in tryptic digests of in vivo-derived samples is facilitated by the use of N-Hcy-albumin and N-Hcy-fibrinogen synthesized in vitro from commercially available human proteins. This protocol allows identification of N-homocysteinylation sites at Lys4, Lys12, Lys137, and Lys525 in albumin directly in trypsin-digested human serum samples. N-Hcy-Lys562, N-Hcy-Lys344, and N-Hcy-Lys385 were identified in human fibrinogen from patients with cystathionine β-synthase deficiency. The protocol can be completed in 4 days.     

Direct monitoring of albumin lysine-525 N-homocysteinylation in human serum by liquid chromatography/mass spectrometry

A posttranslational protein modification by homocysteine-thiolactone (N-homocysteinylation) is linked to human vascular and neurodegenerative diseases. Although chemical and immunological methods are available to detect and quantify the extent of protein N-homocysteinylation, the determination of site-specific N-homocysteinylation in vivo remains challenging. Here we describe a liquid chromatography/mass spectrometry method that monitors the extent of N-homocysteinylation at albumin lysine-525 in vivo directly in human serum. Using this method, we found that the extent of lysine-525 N-homocysteinylation was significantly increased in patients with cystathionine β-synthase deficiency.