D-pantolactone as a circular dichromism (CD) calibration.

A critical study of circular dichromism (CD) measurement at the wavelength region around 220 nm was made. Test sample methods gave deviations of abou 20%, at most, between instruments calibrated at 290 nm with D-10-camphorsulfonic acid. A calculation of the CD from optical rotatory dispersion (ORD) data was made for d-pantolactone which gave a CD maximum at 219 nm, [θ]₂₁₉²⁵ = ?17.3 × 10³ in water. A proposal is made to use this value for calibration of CD in the 220-nm region.     

Conformational changes in erythrocyte membranes by prostaglandins as measured by circular dichroism

Membranes were prepared from fresh, washed human erythrocytes by hemolysis and washing with 5 mm sodium phosphate buffer (pH 7.4). The mean residue ellipticity, [θ], of erythrocyte membrane circular dichroism was altered by prostaglandin E₁ or prostaglandin F_2α at 37 °C when observed from 250 nm to 190 nm. The decrease in negativity of [θ] with 10^?6m prostaglandin E₁ was 12.7% at 222 nm and 17.7% at 208 nm, and with 10^?6m prostaglandin F_2α 22.5% and 34.2%, respectively (P < 0.01). Similar changes in [θ] were observed at lower concentrations of prostaglandins. No strict relationship between amount of change of [θ] and prostaglandin concentrations of 3 × 10^?5m to 3 × 10^?12m was evident. A persistent alteration of [θ] with prostaglandin was observed at 37 °C. Transient change of [θ] occurred at 25 °C with prostaglandin. No change of [θ] was observed at 15 or 20 °C. Buffer or palmitic acid were without effect on membrane [θ]. Phosphatidyl inositol or methyl arachidonate caused an increase in negativity of membrane spectra. The observed alterations of membrane [θ] did not arise from changes in light scattering as the OD₇₀₀–OD₂₀₀ of membranes was not changed by prostaglandin. Effects of prostaglandin were not dependent on light path length. The prostaglandin E₁ antagonist, 7-oxa-13-prostynoic acid, at 10^?7m produced no change of [θ] of membrane spectra and prevented the otherwise demonstrable effects of 10^?10m prostaglandin E₁ on [θ]. The decrease in negativity of [θ] at 222 nm is indicative of a decrease in ellipticity of membrane protein. These studies suggest that prostaglandins may act by inducing a conformational change in membrane protein.     

The influence of salt concentration on CD spectra of tRNA and aminoacyl-tRNA synthetase

The CD spectra of serine tRNA or seryl-tRNA synthetase were measured. The [theta] values at 210 nm were minimum at 50mM - 0.2M NaCl, at that concentration the velocity of aminoacylation was maximum. This results suggest that A . U and G . C base pairs loosened. The [theta] values at 200 nm decreased according to the decreasing of salt concentration, suggesting the decomposition of A . U base pairs. The CD spectra of seryl-tRNA synthetase at 210-240 nm were not changed in the range of 10mM-0.3M NaCl but the spectra at 260-290 nm showed minimum in the range between 50mM-0.2M NaCl. These results suggest that the influence of salt concentration on the velocity of aminoacylation depends on both the conformational changes of tRNA and seryl-tRNA synthetase.     

Photochemical properties of Yt base in aqueous solution.

Photoreactivity of Yt base [I] has been studied in aqueous solution [pH approximately 6] saturated with oxygen. Two photoproducts (II,III], resulting from irradiation at lambda = 253.7 nm and lambda greater than or equal to 290 nm, were isolated and their structures determined. The quantum yield for Yt base disappearance [zeta dis] is 0.002 [lambda = 313 nm]. It was shown that dye-sensitized photooxidation of Yt base in aqueous solution occurs according to a Type I mechanism, as well as with participation of singlet state oxygen. Quantum yields, fluorescence decay times and phosphorescence of Yt base have been also determined.     

Synthesis and conformational study of poly(N -benzyl-L-lysine) and its benzyloxycarbonyl derivative

The solvent-and pH-induced conformational changes are examined in order to investigate the influence of benzyl group. Polymer was prepared via N^?-benzyloxycarbonyl, N^?-benzyl-N^α-carboxy-L -lysine anhydride. The resulting poly (N^?-benzyloxycarbonyl, N^?-benzyl-L -lysine) was obtained in high yield and had a high molecular weight. The protected polymer was removed into poly (N^?-benzyl-L -lysine) by treating it with hydrogen bromide. From the results of the ORD and CD, the protected polymer has a righthanded α-helix, showing [m′]₂₃₃ = –10,300, [θ]₂₂₀ = –27,600 and [θ]₂₀₇ = –25,100 in dioxane. The breakdown of the helical conformation is found to occur at 8% dichloroacetic acid in chloroform-dichloroacetic acid mixture. In the pH range 3.35–6.90, poly (N^?-benzyl-L -lysine) is in a random coil structure. In the pH range 7.50–13.0, the polypeptide has a right-handed α-helix structure; [m′]₂₃₃ = –12,000, [0]₂₂₀ = –27,200, and [0]₂₀₇ = –27,000. In comparison with poly-L -lysine, the coil-to-helix transition is observed at lower pH range in 50% n-propanol. Above pH 8 by heating, the α ? β transition of poly (N^?-benzyl-L -lysine) is not observed in an aqueous media.     

The metabolic pathway catalyzed by the tyrosinase of Agaricus bisporus

N-t-Butyloxycarbonyl-gamma-L-glutaminyl-2-bromo-4-hydroxybenzene alpha-benzyl ester was synthesized as a precursor to gamma-L-glutaminyl-4-hydroxy[2-3H]benzene. With this labeled compound and the previously synthesized gamma-L-glutaminyl-4-hydroxy[3,5-3H]benzene, the stoichiometry of ring substitution was determined for the tyrosinase-catalyzed metabolic pathway of Agaricus bisporus. In this pathway, gamma-L-glutaminyl-4-hydroxybenzene is hydroxylated to gamma-L-glutaminyl-3,4-dihydroxybenzene which is oxidized to gamma-L-glutaminyl-3,4-benzoquinone and a compound of previously unknown structure, "490." The results indicated that the "490" quinone was derived from gamma-L-glutaminyl-3,4-benzoquinone without further ring substitution. A base-catalyzed, nonenzymatic reaction of gamma-L-glutaminyl-3,4-benzoquinone was observed which yielded a compound with a 490 nm chromophore. gamma-Glutamyl transpeptidase cleavage of gamma-L-glutaminyl-3,4-dihydroxybenzene led to the release of 4-aminocatechol which air-oxidized to a compound with identical spectral properties to "490." The structure of "490" was thus determined to be 2-hydroxy-4-imino-2,5-cyclohexadiene-1-one(2-hydroxy-4-iminoquinone). The tyrosinase-catalyzed hydroxylation of gamma-L-glutaminyl-4-hydroxybenzene was found to be optimal at pH 8.0, while the enzymatic oxidation of gamma-L-glutaminyl-3,4-dihydroxybenzene was optimal at pH 6.0.     

Secondary Structure of Nuclease P1

The molecular conformation of nuclease P₁ in aqueous solution was investigated by measuring the optical rotatory dispersion (ORD) and circular dichroism (CD). The optical rotatory dispersion constant, λ was 281 nm. The Moffit-Yang parameters, a₀ and b₀, were ?2 and ?195, respectively. The ORD spectrum showed a minimum at 234 nm and the reduced mean residue rotation at 233 nm, [m]233, was ?5880. The CD spectrum showed a double minimum at 213 and 226 nm and the molecular ellipticity at 222 nm, [θ]22, was -11,900. From these data, the α-helix content was calculated to be 29 to 31 %. The computer fit of CD suggests that the α-structure is about 6% and the random coil is about 63%. The helical structure was found to be quite stable to denaturing reagents such as urea and guanidine hydrochloride. However, removal of zinc atoms from the enzyme resulted in disruption of the helical structure with inactivation.     

Site-directed mutagenesis of the active site serine290 in flavanone 3beta-hydroxylase from Petunia hybrida.

Flavanone 3beta-hydroxylase (FHT) catalyzes a pivotal reaction in the formation of flavonoids, catechins, proanthocyanidins and anthocyanidins. In the presence of oxygen and ferrous ions the enzyme couples the oxidative decarboxylation of 2-oxoglutarate, releasing carbon dioxide and succinate, with the oxidation of flavanones to produce dihydroflavonols. The hydroxylase had been cloned from Petunia hybrida and expressed in Escherichia coli, and a rapid isolation method for the highly active, recombinant enzyme had been developed. Sequence alignments of the Petunia hydroxylase with various hydroxylating 2-oxoglutarate-dependent dioxygenases revealed few conserved amino acids, including a strictly conserved serine residue (Ser290). This serine was mutated to threonine, alanine or valine, which represent amino acids found at the corresponding sequence position in other 2-oxoglutarate-dependent enzymes. The mutant enzymes were expressed in E. coli and purified to homogeneity. The catalytic activities of [Thr290]FHT and [Ala290]FHT were still significant, albeit greatly reduced to 20 and 8%, respectively, in comparison to the wild-type enzyme, whereas the activity of [Val290]FHT was negligible (about 1%). Kinetic analyses of purified wild-type and mutant enzymes revealed the functional significance of Ser290 for 2-oxoglutarate-binding. The spatial configurations of the related Fe(II)-dependent isopenicillin N and deacetoxycephalosporin C synthases have been reported recently and provide the lead structures for the conformation of other dioxygenases. Circular dichroism spectroscopy was employed to compare the conformation of pure flavanone 3beta-hydroxylase with that of isopenicillin N synthase. A double minimum in the far ultraviolet region at 222 nm and 208-210 nm and a maximum at 191-193 nm which are characteristic for alpha-helical regions were observed, and the spectra of the two dioxygenases fully matched revealing their close structural relationship. Furthermore, the spectrum remained unchanged after addition of either ferrous ions, 2-oxoglutarate or both of these cofactors, ruling out a significant conformational change of the enzyme on cofactor-binding.     

Vacuum ultraviolet circular dichroism spectrum of beta-turn in solution.

The vacuum ultraviolet circular dichroism spectrum of an isolated 4 → 1 hydrogen bonded β-turn is reported. The observed spectrum of N-acetyl-Pro-Gly-Leu-OH at ? 40°C in trifluoroethanol is in good agreement with the theoretically calculated CD spectrum of the β-turn conformation. This spectrum, particularly the presence of a strong negative band around 180 nm and a large ratio $\text{[θ]}{}_{201}\text{[θ]}{}_{225}$, can be taken as a characteristic feature of the isolated β-turn conformation. These CD spectral features can thus be used to distinguish the β-turn conformation from the β-structure in solution.     

Effect of metal ions and EGTA on the optical properties of concanavalin A at alkaline pH

In our earlier communications, we reported the effect of salts and alcohols on alpha-chymotrypsinogen [1] and the existence of stable intermediates at low pH in bromelain [2] and glucose oxidase [3]. In the present study, the role of metal ions and EGTA on the conformation of concanavalin A at alkaline pH was studied by near- and far-UV circular dichroism, fluorescence emission spectroscopy and binding of a hydrophobic dye, 1-anilino-8-naphthalene sulfonate (ANS). Far-UV CD spectra showed the transition from an ordered secondary structure at pH 7 with a trough at 223 nm to a relatively unordered state at pH 12. Near-UV CD spectra showed the loss of signal at 290 nm, thereby indicating the disruption of native three dimensional structure. Maximum ANS binding occurred at pH 12 suggesting the presence of an intermediate or molten globule-like state at alkaline pH.     

Synthesis, mechanism and fluorescence properties of 8-(aryl)-3-beta-D-ribofuranosylimidazo[2,1-i]purine 5'-phosphate derivatives

The synthesis of new fluorescent nucleotides is described. This synthesis comprises two parallel reactions, the Kornblum oxidation and imidazole formation, which lead to 8-(aryl)-3-beta-D-ribofuranosylimidazo[2,1-i]purine 5'-phosphates 2 from AMP or ATP. A detailed mechanism is proposed based on monitoring the reaction by 1H- and 13C-NMR spectroscopy, MS, FAB, HPLC, and pH meter. The spectral and fluorescent properties of the new derivatives at various pH values are described. Excitation and emission maxima for 3 were observed at 290 and 420 nm, respectively, in both basic and neutral media. In acidic media, the emission maximum shifted to 410 nm, however, the fluorescence intensity increased 1.5-fold. ATP analogues 2b and 3b exhibited relative stability regarding hydrolysis by type II ATPDase. Compound 3b is relatively chemically stable at pH 10.4 and 7.4.     

Circular dichroism and redox properties of high redox potential ferredoxins

The circular dichroism (CD) spectra of 13 examples of high-potential iron-sulfur proteins (HiPIPs), a class of [4Fe-4S] ferredoxins, have been determined. In contrast to the proposal of Carter [Carter, C. W., Jr. (1977) J. Biol. Chem. 252, 7802-7811], no strict correlation between visible CD features and utilization of the [4Fe-4S]2+/[4Fe-4S]3+ oxidation levels was found. Although most HiPIPs have these features, the model requires their presence in all species. There is also no simple relationship between CD spectral features and the presence of conserved tyrosine-19. In addition, no apparent correlation between CD properties and oxidation-reduction potential could be detected. However, amino acid side chains in close contact to the iron-sulfur cluster appear to be important in modulating spectral and oxidation-reduction properties. In particular, the negative shoulder at 290 nm and negative maximum at 230 nm correlate with the presence of Trp-80 (Chromatium vinosum numbering). Two HiPIPs that do not have Trp at this position have positive bands at 290 and 230 nm. These bands in the Ectothiorhodospira halophila HiPIPs are apparently associated with Trp-49, which is located on the opposite side of the effective mirror plane of the cluster from Trp-80. The effect of pH on circular dichroism and redox potential in Thiocapsa roseopersicina HiPIP, which has a histidine at position 49, is consistent with the interaction of the side chain with the cluster. Despite specific differences in their CD spectra, the various HiPIPs studied show general similarity consistent with structural homology within this class of iron-sulfur proteins.     

Inhibition of RNA chain initiation in E. coli core RNA polymerase with 2-hydroxy-5-nitrobenzyl bromide

The modification of $\text{E. coli}$ core RNA polymerase with 2-hydroxy-5-nitrobenzyl bromide (Koshland's Reagent) resulted in the benzylation of 6 out of 13 cysteines, and 10 out of 20 tryptophans in the polymerase, and occurred with an 8% decrease in its [θ]₂₂₀. The modification resulted in a maximal inhibition of 60% of the RNA chains on both calf thymus and micrococcal DNA templates. γ-³²P-ATP studies showed the inhibition occurred at RNA chain initiation. This study raises the possibility that the modified core polymerase may synthesize specific RNA(s).     

Benzoxazinone kanamycin A conjugate. A new fluorescent probe suitable to detect mycoplasmas in cell culture

The synthesis of a new benzoxazinone derivative suitable to detect early infection of cultured cells with mycoplasmas is described. p-[beta-(7-dimethylamino 1,4-benzoxazin 2-one 3yl)-vinyl]- phenylpropenoic acid was coupled to kanamycin A, an aminoglycoside leading to a cationic fluorescent probe which fluoresces at 600 nm upon excitation at 490 nm. This fluorescent probe is shown to heavily label the glycocallix of all the mycoplasma strains tested which are found to be associated with contaminated cultured cells and to allow an easy and rapid detection of contamination by fluorescence microscopy and flow cytometry.     

Light and dark adaptation of halorhodopsin

Dark incubation of envelope vesicles derived from a strain of Halobacterium halobium that lacks bacteriorhodopsin but contains halorhodopsin and a third rhodopsin-like pigment caused a decrease in the flash yield [the amplitude of a transient absorbance change of flash reactive component(s) by flash] of halorhodopsin but not the rhodopsin-like pigment. The flash yield decreased to reach a low steady level after incubation for about 4 days in the dark. The flash yield of halorhodopsin at any stage of dark incubation was increased by actinic illumination of the vesicles. The flash yield at 490 nm (absorbance increase) was found to be approximately proportional to that at 590 nm (absorbance decrease). These results indicate that halorhodopsin in the envelope vesicles has two forms, dark and light adapted, and that the halorhodopsin phototransient absorbing at 490 nm is originated from the light-adapted form. A difference spectrum between these two forms of halorhodopsin shows that the light-adapted halorhodopsin was red-shifted from the dark-adapted form. The light-induced membrane potential was measured by tetraphenylphosphonium uptake. The uptake by the dark-adapted vesicles was slower than that by the light-adapted vesicles, suggesting that only the light-adapted halorhodopsin has ion-transporting activity.     

Dihydrofolate reductase from soybean seedlings: a fluorescence and circular dichroism study.

The fluorescence emission spectrum of soybean dihydrofolate reductase suggests that the emitting tryptophan residues are situated in a hydrophobic microenvironment. The dissociation constants determined from fluorescence and circular dichroism data reveal that the soybean enzyme has a lower affinity for substrates and substrate analogs than that determined for dihydrofolate reductases isolated from other sources. The binding of methotrexate to the soybean enzyme does not affect the binding of NADPH. Similarly, the binding of NADPH has no effect on subsequent methotrexate binding. Polarimetric study indicates that the enzyme has a low (ca. 5%) α-helical content. Addition of dihydrofolate to the soybean enzyme results in the generation of a positive ellipticity band at 298 nm with a molar ellipticity, [θ], of 186,000, whereas the binding of folate induces a negative ellipticity band at 280 nm with [θ] of ?181,000. The qualitative and quantitative differences in the circular dichroism of the enzyme-dihydrofolate and enzyme-folate complexes indicate that the mode of binding of these ligands may be different. The formation of an enzyme-NADPH complex is accompanied by a negative Cotton effect at 270 nm. These studies indicate that the binding of substrates or inhibitors causes significant conformational changes in the enzyme and also leads to the formation of a number of spectroscopically identifiable complexes.     

Circular dichroism of cattle rhodopsin and bathorhodopsin at liquid nitrogen temperatures

The photoevent in vision has been considered to be the conversion of rhodopsin to bathorhodopsin, which is caused by photoisomerization of the chromophoric retinal. Recently some objections were raised to this hypothesis. The reliability of the hypothesis was verified by measurement of circular dichroism of bathorhodopsin.The measurement of circular dichroism of rhodopsin extract (containing 66% or 75% of glycerol) at liquid nitrogen temperatures (?195°C) by a conventional spectropolarimeter induced an extraordinary large signal, owing to linear dichroism originated from conversion of rhodopsin to bathorhodopsin by the measuring light. The similar linear dichroism can be induced by irradiation of rhodopsin extract at ?195°C with polarized light or natural light. At photosteady state the linear dichroism disappeared.Circular dichroism spectrum of cattle rhodopsin displayed two positive peaks ([θ]_max = 80 800 degrees at 335 nm, and [θ]_max = 42 600 degrees at 500 nm) at ?195°C, whereas, bathorhodopsin displayed a positive peak ([θ]_max = 43 100 degrees at 334 nm) and a negative peak ([θ]_max = 163 000 degrees at 540 nm).The change of the positive sign to negative one at α-band of circular dichroism spectrum supports the hypothesis that the conversion of rhodopsin is due to rotation of the chromophoric retinal about C-11—12 double bond (‘photoisomerization model’).     

Dissolved organic matter in Cumbrian lakes and streams

SUMMARY. 1. A survey of dissolved organic carbon concentration [DOC] and optical absorbance at 340 nm (A340) was carried out for thirteen lakes and twenty-three streams in Cumbria over the period May 1986 to April 1987.
2. Values of [DOC] were in the range 0-6.2 mg 1-1-1, no water having a mean [DOC] greater than 3.6mgI-1. Streamwaters showed no gross seasonal dependence in [DOC], but there was a peak in [DOC] associated with the first major rain event of autumn 1986. Summer values of [DOC] in lakes were greater than winter values, probably because of the production of DOC by phytoplankton.
3. In streams, [DOC] was weakly positively correlated (r2 <0.3) with discharge (as estimated from rainfall); A340 was more strongly correlated (0.4 < r2 < 0.6). Linear regression of mean A340 against mean [DOC] for streams gave a slope consistent with the absorptivities of aquatic humic substances, and a positive intercept on the abscissa that suggests the presence of, on average 0.6 mg 1-1 of DOC that does not absorb at 340 nm. Lake samples taken in winter gave a similar A.340/[DOC] relationship to that for streams, but in summer A340/[DOC] ratios were lower, suggesting that phytoplankton-derived [DOC] absorbs weakly or not at all at 340nm.     

An internally quenched fluorescent substrate for collagenase

A synthetic collagenase substrate containing the internal peptide sequence--Gly-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Pro--has been synthesized, with an N-terminus 4-((4-(dimethylamino)phenyl)azo)-benzoyl (DABCYL) group and C-terminus 5-[2-(acetamido)ethylamino] naphthalene-1-sulfonic acid (AEDANS) moiety resulting in internal quenching of AEDANS fluorescence. Peptide bond hydrolysis results in a large increase in fluorescence at 490 nm upon excitation at 336 nm. The substrate is cleaved exclusively by Clostridium histolyticum collagenase and is completely resistant to attack by proteases like thermolysin, proteinase K, and trypsin. K(m) and V(max) values for substrate hydrolysis by collagenase have been determined, establishing the peptide as one of the best binding substrates for the enzyme. MALDI mass spectrometry using a derivative of the substrate establishes that the sites of cleavage lie within the collagen like domain. The CD spectrum of an analog peptide lacking the donor and acceptor groups reveals spectral features that are reminiscent of weak polyproline structures.     

A polynucleotide CD probe: interactions with oligomers of a polycationic amine that exhibit positive extrinsic bands at greater than 300 nm

A set of large positive extrinsic CD bands ([θ]₃₃₃ = 2.6 X 10⁴ deg-cm²/decimole phosphate) in the > 300 nm region as well as diminution of the intrinsic signals (θ₂₇₅) have been observed in the CD spectra of various nucleic acids complexed with the achiral compound, N-poly{α-[N-(4-pyridylethylene-4-pyridyl-N′-)α′-p-xylyl]dibromide}-4-pyridylethylene-4-pyridinium bromide, (polymer X).^1,2,5 The signal changes are attributed to the binding of polymer X chromophores isogeometrically to the DNA helix in an ordered chiral arrangement. Fractionation of polymer X gives 10 well-separated oligomers. The oligomers were characterized by nmr. Their interactions with DNA have been investigated with respect to r(r = ratio of equivalents of polymer X charge/g-atoms DNA phosphorus) and n (oligomer chain length). In all cases where n ≥ 1, [θ]₃₃₃ increases linearly with increasing r between 0 and 0.32, and is accompanied by a corresponding decrease in [θ]₂₇₅, which becomes negative as r approaches .32. Extrinsic band intensities reveal a dependence on n up to n = 5, above which increases in nonspecific binding result in a reduction in normalized band intensities. Polymer X shows a strong preference for B-form nucleic acids and induces maximum extrinsic CD signal intensities with A-T homopolymers. Alterations in helix hydration are believed to accompany complex formation. Inversions in [θ]₂₇₅ of the octamer X-poly(dA-dT) complex have been attributed to the “alternating B” conformation of poly(dA-dT).³ Similar inversions are not observed in other nucleic acid-octamer X complexes. Visible and CD spectrometry data from competition studies in the presence of the antibiotics actinomycin D (AMD), daunomycin (DM), and distamycin A (DST) are consistent with “nonclassical” intercalation as the mode of binding, and these data place the potential binding site in or near the hydrophobic region of the minor groove. Reductions in [θ]₃₃₃ with increasing urea further implicate the involvement of hydrophobic interactions in the formation of an asymmetric complex. Stabilization of the helix results in all cases as evidenced by alterations in T_m; corresponding changes, however, in cooperativity are not clearly discernable. Viscosity and light-scattering data indicate no changes in molecular weight due to aggregation, and as such are not consistent with a transition to the ψ-DNA upon complex formation.