Photothermal confocal spectromicroscopy of multiple cellular chromophores and fluorophores

Confocal fluorescence microscopy is a powerful biological tool providing high-resolution, three-dimensional (3D) imaging of fluorescent molecules. Many cellular components are weakly fluorescent, however, and thus their imaging requires additional labeling. As an alternative, label-free imaging can be performed by photothermal (PT) microscopy (PTM), based on nonradiative relaxation of absorbed energy into heat. Previously, little progress has been made in PT spectral identification of cellular chromophores at the 3D microscopic scale. Here, we introduce PTM integrating confocal thermal-lens scanning schematic, time-resolved detection, PT spectral identification, and nonlinear nanobubble-induced signal amplification with a tunable pulsed nanosecond laser. The capabilities of this confocal PTM were demonstrated for high-resolution 3D imaging and spectral identification of up to four chromophores and fluorophores in live cells and Caenorhabditis elegans. Examples include cytochrome c, green fluorescent protein, Mito-Tracker Red, Alexa-488, and natural drug-enhanced or genetically engineered melanin as a PT contrast agent. PTM was able to guide spectral burning of strong absorption background, which masked weakly absorbing chromophores (e.g., cytochromes in the melanin background). PTM provided label-free monitoring of stress-related changes to cytochrome c distribution, in C. elegans at the single-cell level. In nonlinear mode ultrasharp PT spectra from cyt c and the lateral resolution of 120 nm during calibration with 10-nm gold film were observed, suggesting a potential of PTM to break through the spectral and diffraction limits, respectively. Confocal PT spectromicroscopy could provide a valuable alternative or supplement to fluorescence microscopy for imaging of nonfluorescent chromophores and certain fluorophores.     

Synthesis and photoluminescent properties of a series of pnictogen-centered chromophores

A complete series of triarylpnictines of the form PnTbs₃ (Pn = a pnictogen, P, As, Sb, or Bi, Tbs = (E)-4-(4-t-butylstyryl)phenyl) has been prepared, and their absorption and photoluminescence spectral properties examined. Although it was anticipated that fluoresence would be significantly quenched for the heavier atom-centered pnictines in this series, unexpectedly high fluorescence was observed for AsTbs₃ and SbTbs₃. Phosphorescence was also observed to increase as the series was descended. This set of materials includes the first examples of stilbene-substituted As, Sb, and Bi materials.     

Separation and determination of cross-linking amino acids of elastin by high-voltage paper electrophoresis

A method is described for the separation and quantitative determination of the weakly basic (cross-linking) amino acids of elastin. A 6 N HCl hydrolyzate is submitted to high-voltage electrophoresis at pH 3.8. At least eight spots can be identified in the weakly basic region and quantitated by the ninhydrin-photodensitometric method. Some of these are spot 3 for desmosine + isodesmosine, and 7 for lysinonorleucine. Quantitative data given for two typical elastin preparations are in agreement with direct amino acid analysis.     

Characterization of insoluble elastin from copper-deficient pigs. Its usefulness in elastin sequence studies.

Insoluble elastin from copper-deficient animals has an amino acid composition intermediate between mature elastin and salt-soluble elastin (a higher lysine content and correspondingly low number of cross-links relative to the normal protein) and is solubilized by successive treatment with trypsin and chymotrypsin at 4 and 37 degrees C. Small amounts of B3H4 (11 mg--2 g of elastin) reduced allysine, allysine aldol, dehydronorleucine, and dehydromerodesmosine in insoluble elastin from copper-deficient pig aorta. In contrast, desmosine and isodesmosine were reduced only when a large excess of reductant (400 mg borohydride) was included in the reaction mixture. Reduction studies indicated that lysinonorleucine and merodesmosine were present in their dehydro forms to a greater extent in copper-deficient pig elastin than in normal elastin. After reduction with borohydride approximately 35% of the reduced form of the insoluble elastin remained insoluble after digestion with trypsin and chymotrypsin. A peptide containing the aldehyde oxidation product of lysine (allysine) and demonstrating an enrichment in glutamic acid was purified from the reduced form of copper-deficient pig elastin and partially sequenced. Its sequence (Gly-Ala-Glu-allysine-(Glu)...) and amino acid composition suggest: (1) clustering of glutamic acid residues in the elastin molecule, and (2) that allysine residues are not restricted to the alanine-enriched sites described for other elastin cross-links. Insoluble elastin from copper-deficient animals promises to be a useful tool for elastin sequence studies.     

Geometric and energy parameters in lysine-retinal chromophores.

The parameters used in the computer program ECEPP (Empirical Conformational Energy Program for Peptides) have been expanded to cover some key elements in retinal-containing proteins. These elements are 'all-trans retinal lysine with unprotonated imine', 'all-trans retinal lysine with protonated imine', '13-cis retinal lysine with unprotonated imine' and '13-cis retinal lysine with protonated imine' respectively. The geometric parameters of these four new 'amino acid residues' were derived by optimizing their molecular structures with the AM1 Hamiltonian included in MOPAC (Molecular Orbital PACkage), and their partial atomic charges were determined with a CNDO/2 (Complete Neglect of Differential Overlap) calculation. The parameters for nonbonded interactions and torsional potentials were obtained from the existing ECEPP parameters through a logical extension. The augmented ECEPP system thus obtained can be employed to investigate the conformation of bacteriorhodopsin and its proton-pumping mechanism from an energetic point of view. The computer modeling study on bacteriorhodopsin and other seven-helix membrane proteins, e.g. serotonin receptor and dopamine receptor, is under way in the Upjohn Laboratories.     

Resonance Raman spectral isolation of the a and a3 chromophores in cytochrome oxidase.

Resonance Raman spectra of reduced CO-bound cytochrome oxidase obtained at two different excitation frequencies (441.6 and 413.1 nm) are compared with the spectra of the fully reduced enzyme. In the spectra of the CO-bound complex only the cytochrome a modes are strongly enhanced with 441.6 nm excitation and only the modes of the CO-bound cytochrome a3 heme are strongly enhanced with 413.1-nm excitation. In the fully reduced complex with both excitation frequencies, modes of both cytochrome a and a3 are enhanced. By subtraction we are able to uncover the complete spectrum of the fully reduced ligand-free cytochrome a3 heme. Thus, we report the discrete resonance Raman spectra of cytochromes a2+, a2+3, and a2+3 (CO). The spectra of fully reduced cytochrome a and ligand-free cytochrome a3 are very different especially in the low frequency region. Binding CO to ferrous cytochrome a3 results in electronic structure changes in the heme analogous to those in hemoglobin and myoglobin, from which we conclude that there is nothing electronically unique in the ferrous cytochrome a3 heme to account for its catalytic properties.     

Crosslinkage of salt-soluble elastin in vitro.

Radioactively labeled soluble elastin, synthesized $\text{in vitro}$ by viable copper-deficient pig aorta in a culture medium containing L-[4,5-³H] lysine, was incubated with normal newborn pig aorta. The insoluble residue, after extraction of the aorta with cold 0.5M NaCl at pH 7.4, was reduced with NaBH₄. Insoluble elastin, prepared from this by autoclaving after extraction with guanidine, was hydrolyzed with HCl and the hydrolysate was chromatographed on Aminex A-5. Among the radioactive residues eluted in the basic region, four elastin crosslinks (isodesmosine, desmosine, lysinonorleucine and merodesmosine) were identified by comparison with known standards on the Beckman amino acid analyzer. This provides the first direct evidence that soluble elastin is a precursor of insoluble elastin.     

Photoreduction of copper chromophores in blue oxidases.

The low temperature (77 K) irradiation of oxidized ceruloplasmin and Rhus vernicifera laccase at the 330 nm absorption which arises from type 3 copper leads to the reduction of type 1 copper as demonstrated by bleaching of the 610 nm chromophore and the decrease of the EPR signal associated with this species. Type 2 copper remains unaffected. Concomitant with the type 1 copper reduction, a new EPR signal which is possibly that of a biradical appears. Upon thawing, type 1 copper is reversibly oxidized and the radical signal disappears. Irradiation of oxidized protein at the absorption band of type 1 copper produces no spectral change. An EPR study at room temperature confirms the wave-length specificity and reversibility of the photoreduction of type 1 copper and radical formation. Radical appearance and disappearance at room temperature are extremely slow (tau1/2 approximately 30 min). Optical studies at room temperature show that upon anaerobic irradiation of laccase in the 330 nm absorption band, both type 3 and type 1 chromophores are slowly reduced. Upon return to the dark and in the presence of O2, both type 3 and type 1 centers are reoxidized. Oxidizing equivalents either from O2 or K3Fe(CN)6 are required for the reoxidation reaction. These studies demonstrate that there is a direct energy transfer between type 3 and type 1 copper sites in blue copper oxidases.     

Two new elastin cross-links having pyridine skeleton. Implication of ammonia in elastin cross-linking in vivo

Isolation and structure analysis of two amino acids from bovine ligamentum nuchae elastin hydrolysates revealed the presence of pyridine cross-links in elastin. The structures of these amino acids were determined to have 3,4,5- and 2,3,5-trisubstituted pyridine skeletons both with three carboxylic acids and a mass of 396 (C(18)H(28)N(4)0(6)) identified as 4-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3-carboxypropyl)-pyridine and 2-(4-amino-4-carboxybutyl)-3,5-di-(3-amino-3-carboxypropyl)-pyridine. We have named these pyridine cross-links desmopyridine (DESP) and isodesmopyridine (IDP), respectively. Structure analysis of these pyridine cross-links implied that the formation of these cross-links involved the condensation reaction between ammonia and allysine. The elastin incubated with ammonium chloride showed that DESP and IDP levels increased as the allysine content decreased. DESP and IDP were measured by high pressure liquid chromatography (HPLC) with UV detection and were found in a variety of bovine tissues. The DESP/desmosine (DES) and IDP/isodesmosine (IDE) ratios in aorta elastin were higher than in other tissues. DESP and IDP contents in human aorta elastin were found to be gradually increased with age. The concentration of IDP was significantly elevated in aorta elastin of rat with chronic liver cirrhosis induced by carbon tetrachloride (mean +/- S.D.; 11.1 +/- 0.9 nmol/mg elastin) when compared with normal rats (5.9 +/- 1.5 nmol/mg elastin). Although DESP and IDP are present at only trace concentrations in the tissue elastin, these pyridine cross-links may be useful biomarkers for the aortic elastin damaged by ammonia.     

Synthesis of elastin in aortas from chick embryos. Conversion of newly secreted elastin to cross-linked elastin without apparent proteolysis of the molecule

The biosynthesis of elastin was examined in matrix-free cells isolated by enzymic digestion of aortas from 17-day old chick embryos. After the cells were incubated with [14C]proline and then were rapidly boiled in buffer containing high concentrations of protease inhibitors and sodiumdodecyl sulfate, about one-quarter of the intracellular 14C-labeled protein was recovered as an elastin component with an apparent molecular weight of about 72 000. Examination of the medium from the cell suspension indicated that the largest elastin component secreted by the cells also had an apparent molecular weight of about 72 000. Pulse-chase experiments with intact aortas demonstrated that about two-thirds of the 72 000-dalton component disappeared in 2 h, apparently because it was converted to cross-linked fibers. When cross-linking was inhibited with penicillamine, the 72 000-dalton component persisted in the tissue 5 h. When cross-linking was inhibited with beta-aminopropionitrile, the elastin component of 72 000 daltons persisted for about 2 h, but thereafter it was gradually degraded to small peptides which were recovered in the incubation medium. The results suggest that elastin is secreted by cells in chick aorta as a polypeptide of about 72 000 daltons and that the secreted protein is incorporated into elastin fibers without cleavage to a protein of considerably smaller size.     

Dynamic mechanical properties of elastin.

The dynamic mechanical properties of water-swollen elastin under physiological conditions have been investigated. When elastin is tested as a colsed, fixed-volume system, mechanical data could be temperature shifted to produce master curves. Master curves for elastin hydrated at 36°C (water content, 0.46 g water/g protein) and 55°C (water content, 0.41 g/g) were constructed, and in both cases elastin goes through a glass transition, with the glass transition temperatures of -46 and -21°C, respectively. Temperature shift data used to construct the master curves follow the WLF equation, and the glass transition appears to be characteristic of an amorphous, random-polymer network. For elastin tested as an open, variable-volume system free to change its swollen volume as temperature is changed, dynamic mechanical properties appear to be virtually independent of temperature. No glass transition is observed because elastin swelling increases with decreased temperature, and the increase in water content shifts elastin away from its glass transition. It is suggested that the hydrophobic character of elastin, which gives rise to the unusual swelling properties of elastin, evolved to provide a temperature-independent elastomer for the cold-blooded, lower vertebrates.     

The polyelectrolyte properties of elastin.

The charge structure and ionic interactions of elastin prepared from the pig thoracic aorta by acid, alkali, or CNBr extraction have been investigated by potentiometric titration and radiotracer techniques. The number of charged groups was consistent with the amino acid composition, comparable to elastin from other sources and insensitive to the method of preparation. The enthalpies of ionization of the basic groups were comparable for those previously found for proteins but those of the acidic groups were higher. Ionic interactions were predominantly electrostatic although a strong affinity for chloride ions was noted. Changes in ionic interactions as the elastin was stretched had a similar effect to an increase in the apparent fixed charge density of the tissue. Mechanical strain altered the protonation of the elastin and the pK of the carboxyl groups. Conversely, the conformation of the elastin network varied with ionic strength and pH, being particularly sensitive to the degree of ionization of the more basic groups and with the ionic strength and anion composition of the medium. We speculate that strain induced changes in the conformation of elastin altering its reactivity towards lipids, ions or matrix macromolecules or changes in its mechanical properties resulting from changes in its ionic environment may be of physiological or pathological importance.     

A comparative analysis of the amino acid and cDNA sequences of bovine elastin a and chick elastin

A comparative analysis of the amino acid and cDNA sequences of bovine elastin a and chick elastin shows that there are considerable differences between these proteins. There is evidence that duplication of segments of DNA and gene rearrangement have occurred in the gene for chick elastin as compared with the gene for bovine elastin. The length of the polypeptide chain of elastin in both species is similar. Therefore, the duplications are compensated for by deletions in the gene for chick elastin.     

Separation of damage specific DNA endonuclease activities present in calf thymus

A DNA endonuclease activity present in calf thymus specific for incision on DNA damaged by ultraviolet light, osmium tetroxide, potassium permanganate, hydrogen peroxide and acid has been purified from whole cell extracts. The enzymatic activity was heterogeneous both with regard to molecular mass and charge. The molecular mass of the enzyme varied from 25 to 35 kDa, but the different enzymatic species appeared to possess similar activities. The enzymes acted equally well on damage in supercoiled and relaxed forms of DNA. It further had a narrow optimum with regard to salt concentrations, the optimum activity being observed at a concentration of KCl from 40 to 65 mM.