Absorption Spectral Shifts Research Articles

Site-specific methylation of a strategic lysyl residue in aspartate aminotransferase.

Conditions for reductive methylation of amine groups in proteins using formaldehyde and cyanoborohydride can be chosen to modify selectively the active site lysyl residue of aspartate aminotransferase among the 19 lysyl residues in each subunit of this protein. Apoenzyme must be treated, under mildly acidic conditions (pH = 6), at a relatively low molar ratio of formaldehyde to protein (40:1); and, upon reduction with sodium cyanoborohydride, 85% of the formaldehyde is incorporated at Lysine 258 and 15% at the amino-terminal alanyl residue. The modified protein, characterized after tryptic hydrolysis, separation of the peptides by high performance liquid chromatography procedures and subsequent amino acid analysis, shows that lysine 258 is preferentially modified as a dimethylated derivative. Modified apoenzyme can accept and tightly bind added coenzyme pyridoxal phosphate, as measured by circular dichroism procedures. The methylated enzyme is essentially catalytically inactive when measured by standard enzymatic assays. On the other hand, addition of the substrate, glutamate, produces the characteristic absorption spectral shifts for conversion of the active site-bound pyridoxal form of the coenzyme (absorbance at 400 nm) to its pyridoxamine form (absorbance at 330 nm). Such a half-transamination-like process occurs as in native enzyme, albeit at several orders of magnitude lower rate. This event takes place even though the characteristic internal holoenzyme Schiff's base between Lys-258 and aldehyde of bound pyridoxal phosphate does not exist in methylated, reconstituted holoenzyme. It is concluded that this chemically transformed enzyme can undergo a half-transamination reaction with conversion of active site-bound coenzyme from a pyridoxal to a pyridoxamine form, even when overall catalytic turnover transamination cannot be detected.

Ultraviolet Studies and Fluorescence Quenching of Some Aromatic Primary Amines

Abstract The fluorescence quenching of o-aminobenzoic acid, 1-naphthylamine, aniline, p-toluidine, m-toluidine, and N,N-dimethylaniline by 2-hydroxy-5-methyl-1,3-benzenedicarbaldehyde (VII) was studied. The equilibrium constant of hydrogen bonding in the ground state was evaluated from the absorption spectral change for various donor–acceptor (D–A) pairs. The rate constants of hydrogen-bond formation as well as decomposition in the excited state were evaluated in the case of several donor–acceptor pairs from fluorescence intensity measurements. In addition, the mechanism of fluorescence quenching and absorption spectral shift when VII acted as a quencher/proton donor (D) was discussed on the basis of charge-transfer and hydrogen-bond interaction.

Chloroplast biogenesis—46. Calculation of net spectral shifts induced by axial ligand coordination in metalated tetrapyrroles

Three sets of equations are described, for the Soret and visible regions of the electromagnetic spectrum, which allow the calculation of net absorption and fluorescence spectral shifts, directly induced by coordination of metalated tetrapyrroles to axial ligands. This was made possible by separating mathematically the axial ligand-induced spectral shifts from other spectral shifts induced by (a) non-coordination solvent effects; (b) peripheral group substitution; (c) temperature and (d) central metal substitution. In calculating the net spectral shifts, we made use of vapor spectral data published by others, and when such data were not available, solvated free-base spectral data were used. The importance of being able to calculate axial ligand-induced net spectral shifts lies in their quantitative relationship to the positive charge density on the central metal atom of metalated tetrapyrroles, which in turn plays an important role in orienting metallotetrapyrroles within biological membranes.

Chloroplast biogenesis—47. Spectroscopic study of net spectral shifts induced by axial ligand coordination in metalated tetrapyrroles

Net absorption and fluorescence spectral shifts, directly induced by coordination of metalated tetrapyrroles to axial ligands, were calculated for the Soret and visible regions of the electromagnetic spectrum. An examination of the calculated net spectral shifts confirmed the conclusions of several other investigators and revealed that the axial coordination potential of a metalated tetrapyrrole is strongly influenced (a) by functional group distribution around the periphery of the tetrapyrrole macrocycle; (b) by the temperature of the sample; (c) by the availability of adventitious ligands in the immediate environment of the metalated tetrapyrroles and (d) by the nature of the central metal atom of the metallotetrapyrrole. In general, electron withdrawing peripheral groups, low temperatures and the availability of unhindered Lewis bases all enhanced the formation of hexacoordinated complexes in Mg-tetrapyrroles. For example, in ether at room temperature, all Mg-tetrapyrroles coordinated to one axial ligand thus forming pentacoordinated complexes. At 77 K, all Mg-porphyrins with electron withdrawing side chains occurred mainly in the pentacoordinated state and to a much lesser extent in the hexacoordinated state. On the other hand in ether at 77 K, Mg-chlorins, such as monovinyl and divinyl chl(ide) a, coordinated to two axial ligands and occured predominantly in the hexacoordinated state. The relevance of these observations to the positive charge density on the central metal atom of metallotetrapyrroles and to the orientation and organization of Mg-tetrapyrroles in biological membranes is discussed.

Absorption spectral shift of meso-tetraphenylporphinatocadmium on axial ligation.

The effects of axial ligation to meso-tetraphenylporphinatocadmium (CdTPP) were studied in dimethyl sulfoxide (DMSO) or dichloromethane (CH2Cl2). Addition of neutral (L) or anionic ligands (X-) to CdTPP caused red shifts in the absorption spectrum. The magnitude of the red shift depended on the charge but not on the strength of the Cdligand bond. Further, a relationship between the ratio εα/εβ and the wavenumber of the Soret band was observed.

Laser-induced excited state and ultrasonic wave gratings: Amplitude and phase grating contributions to diffraction

A detailed analysis of diffraction from laser-induced gratings is presented. The changes which occur in both the real and imaginary parts of the index of refraction are accounted for when excited states are created. These lead to phase and amplitude grating contributions, respectively, to the diffraction from a laser-induced excited state grating. Experimental confirmation of the predicted wavelength dependence of these contributions is presented. Diffraction from laser-induced excited state gratings, ultrasonic wave gratings, and mixed excited state and acoustic gratings is analyzed with the phase and amplitude contributions to each accounted for. The results permit the interpretation of mixed grating data and predict conditions under which density-dependent absorption spectral shifts and excited state-phonon interactions can be measured.

Studies on ground and excited state prototropic equilibria of 2-fluorenaldehyde 2-pyridylhydrazone

The ground and excited state pK values of 2-fluorenaldehyde 2-pyridylhydrazone obtained from variations in fluorescence intensity with pH are reported. The two acid-base equilibria are separately treated by instrumental and chemical means. Absorption and fluorescence spectral shifts are employed to show that solute-solvent interactions through hydrogen-bonding are responsible for the disagreement between the excited state pK values obtained from Förster cycle calculations and from fluorimetric titration measurements.

Phospholipid lateral phase separation and the partition of cis-parinaric acid and trans-parinaric acid among aqueous, solid lipid, and fluid lipid phases

The partition of cis-parinaric acid (9,11,13,15-cis, trans, trans,cis-octadecatetraenoic acid, cis-PnA) and trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid, trans-PnA) among aqueous, solid lipid, and fluid lipid phases has been measured by three spectroscopic parameters: absorption spectral shifts, fluorescence quantum yield, and fluorescence polarization. The solid lipid was dipalmitoylphosphatidylcholine (DPPC); the fluid lipid was palmitoyldocosahexaenoylphosphatidylcholine (PDPC). Mole fraction partition coefficients between lipid and water were determined by absorption spectroscopy to be for ci--PnA, 5.3 X 10(5) with a solid lipid and 9 X 10(5) with fluid lipid and, for trans-PnA, 5 X 10(6) with solid lipid and 1.7 X 10(6) with fluid lipid. Ratios of the solid to the fluid partition coefficients (Kps/f) are 0.6 +/- 0.2 for cis-PnA and 3 +/- 1 for trans-PnA. A phase diagram for codispersions of DPPC and PDPC has been constructed from the measurements of the temperature dependence of the fluorescence quantum yield and polarization of cis-PnA and trans-PnA and their methyl ester derivatives. A simple analysis based on the phase diagram and fluorescence data allows additional calculations of Kps/f's which are determined to be 0.7 +/- 0.2 for the cis probes and 4 +/- 1 for the trans probes. The relative preference of trans-PnA for solid phase lipids and its enhanced quantum yield in solid phase lipids make it sensitive to a few percent solid. The trans probes provide evidence that structural order may persist in dispersions of these phospholipids 10 degrees C or more above their transition temperature. It is concluded that measurements of PnA fluorescence polarization vs. temperature are better suited than measurements of quantum yield vs. temperature for determining phospholipid phase separation.

Mechanisms of spectral shifts in lobster carotenoproteins The resonance raman spectra of ovoverdin and the crustacyanins

Resonance Raman data have been used to elucidate the mechanisms of the absorption spectral shifts occurring for astaxanthin upon binding to the carotenoproteins, ovoverdin and α-, β- and γ-crustacyanins, from the lobster Homarus americanus. Although distinguishable on the basis of small differences in their resonance Raman spectra the binding sites of the crustacyanins, giving rise to λ max at 605 ± 25 nm, are essentially the same. The large red shift in λ max for the crustacyanins compared to free astaxanthin ( λ max 480 nm), is accounted for by a charge-polarisation mechanism in which charged groups and possibly hydrogen bonds in the binding site set up π electron polarisation in the ligand. Several alternate mechanisms can be eliminated. Ovoverdin is found to consist of three polypeptide chains of molecular weight 105 000, 95 000 and 78 000 which are not linked by disulfide bridges. The visible absorption peaks of ovoverdin at 460 and 640 nm are shown to arise from two astaxanthin molecules each bound at a different site. The spectral characteristics of the 460 nm site suggest a rigid hydrophobic environment for astaxanthin, in which no charge-ligand interactions occur. The mechanism of the spectral shift in the 640 nm site is the same as in the crustacyanins, i.e. a charge-polarisation effect. Resonance Raman spectra of ovoverdin and the crustacyanins could be obtained in situ; they were identical to the spectra of the purified proteins showing that the carotenoid sites were unperturbed by protein isolation.

Conjugated polyene fatty acids as fluorescent membrane probes: model system studies.

The use of conjugated polyene fatty acids as probes of membrane structure is examined, alpha- and beta-parinaric acid (cis, trans, trans, cis- and all trans-9, 11, 13, 15-octadecatetraenoic acid) and synthetic lecithins containing an alpha-parinaric acid chai in position 2 have been prepared, and their absorption and fluorescence properties have been determined. Their absorption spectra are at sufficiently long wavelength to be unobscured by cellular chromophores such as nucleotides and aromatic amin acids. Parinaric acid absorption does, however, overlap tryptophan emission which allows fluorescence energy transfer. Potential uses of these fluorescent probes are presented with studies on mode systems with known physical properties. Dipalmitoyl phosphatidylcholine exhibits a sharp phase transition 1 degree wide at 42 degrees C, as monitored by the fluorescence intensit of parinaric acid. The magnitude of the transition is independent of probe concentration, but the width of the transition and hysteresis are dependent upon such factors as the probe concentration and whether or not sonication is used in sample preparation. Using both fluorescence and absorption properties of the probe, we show that the addition of cholesterol to the dispersion broadens and decreases the magnitude of the transition. These results are interpreted in terms of a change in the polarizability of the acyl chains of a lipid bilayer undergoes a thermal transition. Lipid-protein interactions are studied by the binding of alpha-parinaric acid to bovine serum albumin. Fluorescence enhancement, absorption spectral shifts, and quenching of tryptophan fluorescnece are observed when alpha-parinaric acid binds to bovine serum albumin. Calculations based on these measurements are consistent with two binding sites of KB approximately 10(8) (M-1) and three to four binding sites of KB approximately 10(6)-10(7) (M-1), similar to known values for the binding of other long-chain fatty acids. Biosynthetic incorporation of beta-parinaric acid into the E. coli fatty acid auxotroph 30E betaox has been accomplished and phase transitions in cells and isolated phospholipids are shown.

Effects of ultrasonic radiation on Escherichia coli: Spectrophotometric studies

The heritably altered staining property of E. coli B with vital fluorochrome acridine orange after exposure to ultrasonic radiation is studied spectrophotometrically in the visible and ultraviolet regions. Sustained absorption spectral shifts are observed for bacterial suspensions in vivo immediately after ultrasonic treatment and subsequent staining in the visible and without staining in the ultraviolet regions. An increased uptake of the dye has also been observed after treatment. However, it is observed that the viability is substantially retained and there is no indication of any change in the growth rate or nutritional requirements. The possible role of the nucleic acids in these observed spectral shifts, the significance of these results and their genetic implications are discussed.