Circular dichroism of lipoxygenase-1 from soybeans.

The circular dichroism spectra of the three forms of lipoxygenase-1 from soybeans show characteristic differences in the region between 300 and 600 nm. Native lipoxygenase-1 only shows a negative dichroic band around 330 nm. Yellow lipoxygenase-1, obtained by addition of an equimolar amount of 13-F-hydroperoxylinoleic acid to the native enzyme, shows a positive Cotton effect at 425 nm, while the negative band band at 330 nm has increased in intensity. The blue enzyme, representing a complex of yellow enzyme with 13-L-hydroperoxylinoleic acid exhibits a negative dichroic band at 580 nm and positive bands at 410 and 391 nm. The near-ultraviolet CD spectra of the three forms of lipoxygenase are very similar, showing several well resolved positive dichroic bands at 0 degrees C. Using the method of Chen et al. (Chen, Y.-H., Yang, J.T. and Martinez, H.M. (1972) Biochemistry 11, 4120--4131) the contents of alpha-helix, beta- and unordered form of native lipoxygenase-1 were estimated to be 34, 27 and 39% respectively.     

Linear dichroism studies of tryptophanase and its quasisubstrate complexes oriented in polyacrylamide gel

Tryptophanase from Escherichia coli was oriented in a compressed slab of polyacrylamide gel and its linear dichroism (LD) and absorption spectra have been measured. The free enzyme displays four LD bands at 305, 340, 425 and 490 nm. Two bands at 340 and 425 nm belong to the internal coenzyme-lysine aldimine. The 305-nm band apparently belongs to an aromatic amino acid residue. The 490-nm band disappears after treatment with NaBH4 or after incubation with L-alanine and subsequent dialysis. It is suggested that the 490-nm band belongs to a quinonoid enzyme subform. The reaction of tryptophanase with threo-3-phenyl-DL-serine, L-threonine and D-alanine leads to formation of an external aldimine with an intense absorption band at 420-425 nm. The values of reduced LD (delta A/A) in this band strongly differ from that in the 420-nm band of the free enzyme. The LD value of the complex with D-alanine is intermediate between those of the free enzyme and the complex with 3-phenylserine. In the presence of indole the complex with D-alanine displays the same LD as that observed with 3-phenylserine. The reaction of tryptophanase with L-alanine or oxindolyl-L-alanine leads to formation of a quinonoid intermediate with an absorption band near 500 nm. The LD value in this band is close to that of an external aldimine with L-threonine. It is concluded that reorientations of the coenzyme occur in the course of the tryptophanase reaction.     

Study of coenzyme reorientation in the active center of tryptophanase by linear dichroism method

Tryptophanase from E.coli was oriented in a compressed slab of polyacrylamide gel and its linear dichroism (LD) and absorption spectra were measured. The free enzyme displays four LD bands at 305, 340, 425 and 490 nm. Two bands at 340 and 425 nm belong to the internal coenzyme-lysine aldimine. The 305 nm band apparently belongs to an aromatic amino acid residue; the sign and form of this band are changed upon the enzyme reaction with substrate analogs. The 490 nm band is present in the LD spectra of holo- and apoenzyme and disappears after treatment with NaBH4. It is suggested that the 490 nm band belongs to a quinoid enzyme subform. The reaction of tryptophanase with threo-beta-phenyl-DL-serine and L-threonine leads to formation of the external aldimine with a strong absorption band at 420-425 nm. The reduced LD (delta A/A) in this band is one order of magnitude greater than that in the 420 nm of the free enzyme. The complex with D-alanine is characterized by an intermediate LD value in the 425 nm band. In the presence of indole this complex displays the same LD as that observed with beta-phenylserine. The reaction of tryptophanase with L-alanine and oxindolyl-L-alanine leads to formation of the quinoid intermediate with a 500 nm absorption band. The LD value in this band differs from those in the absorption bands of the free enzyme. It is concluded that reorientations of the coenzyme occur in the course of the tryptophanase reaction.     

Complexes of aspartate aminotransferase with hydroxylamine derivatives: spectral studies in solution and in the crystalline state

Hydroxylamine and its derivatives of general formula H2NOR react with aldehydes and aldimines to produce oximes. If R corresponds to the side chain of a natural amino acid, such compounds can be thought of as analogs of the corresponding amino acids, lacking the alpha-carboxylate group. Oximes formed between such compounds and pyridoxal phosphate in the active site of aspartate amino-transferase mimic external aldimine intermediates that occur during catalysis by this enzyme. The properties of oxime derivatives of mitochondrial aspartate aminotransferase with hydroxylamine and 6 compounds H2NOR were studied by absorption spectroscopy and circular dichroism in solution and by linear dichroism in crystals. Stable oximes, absorbing at lambda max congruent to 380 nm and exhibiting a negative Cotton effect, were obtained with the carboxylate-containing compounds. The oximes formed with carboxylate-free compounds showed somewhat different properties and stability. With H-Tyr a stable complex absorbing at lambda max congruent to 370 nm rather than at 380 nm, was obtained, H-Ala and H-Phe produced unstable oximes with the initial absorption band at lambda max congruent to 380 nm that was gradually replaced by a band at lambda max congruent to 340 nm. The species absorbing at 340 nm were shown to be coenzyme-inhibitor complexes which were gradually released from the enzyme. A similar 330-340 nm absorption band was observed upon reaction of the free coenzyme with all hydroxylamine inhibitors at neutral pH-values. The results of the circular dichroism experiments in solution and the linear dichroism studies in microcrystals of mAspAT indicate that the coenzyme conformation in these inhibitor/enzyme complexes is similar to that occurring in an external aldimine analogue, the 2-MeAsp/mAspAT complex. Co-crystallizations of the enzyme with the H2NOR compounds were also carried out. Triclinic crystals were obtained in all cases, suggesting that the "closed" structure cannot be stabilized by a single carboxylate group.     

Circular dichroism and conformational properties of modeccin,a toxic protein

According to its circular dichroism (CD) spectrum, modeccin, a toxic lectin from the roots of the South African plantModecca digitata, is structurally similar to the ricins and abrins. In nearly neutral and weakly alkaline solutions (pH 7.6–9.0) the CD spectra of modeccin displayed a positive CD band at 190–195 nm and a negative band at 210–220 nm, indicating the presence of some α-helix and β-sheet structures. In the near-ultraviolet zone, we observed positive CD bands at 232 and 245 nm and weak negative bands at 285 and 293 nm. In more strongly alkaline solutions of pH 9.5–10.2 the CD bands in the farultraviolet zone were not affected, but the CD band at 232 nm diminished and the CD band at 245 nm was enhanced. These transitions were reversible. At pH 11.2–11.5 the CD band at 232 nm disappeared completely, and the CD bands in the far-ultraviolet diminished. The CD bands at 285 and 293 nm were affected very little by the alkali, and these bands were assigned to buried tryptophan side chains. Sodium dodecyl sulfate and 2,2,2-trifluoroethanol disorganized the tertiary structure of modeccin and reconstructed the secondary structure into a new form with a higher helix content than in the native protein.     

Ceruloplasmin-anion interaction. A circular dichroism spectroscopic study.

The effect of anion binding to ceruloplasmin has been studied using absorption and cirbular dichroism spectral data. At anion to ceruloplasmin molar ratios approaching infinite, OCN-, N3- and SCN- bind to ceruloplasmin giving rise to similar alterations in circular dichroism and absorption spectra. The positive bands at 610 and 520 nm in circular dichroism spectra disappear, a negative one apperars at 600 nm and the peak at 450 nm is only slightly modified. There is a new negative band at 410 nm well-defined in OCN- ceruloplasmin spectra. The decrease in absorption at 610 nm is ascribed to the disruption of one type I Cu-S(cysteine) bond owing presumably to the changes induced by anions in the protein secondary structure. The new band at 410 nm is assigned to a charge transfer transition from the ligand replacing cysteine at its binding site. Both absorption and circular dichroism spectra show isobestic points indicating that anion binding to the enzyme, disruption of one of the two type I Cu-S bonds and coordination of this Cu to another protein residue take place simultaneously.     

Effects of D-serine on bacterial D-amino acid transaminase: accumulation of an intermediate and inactivation of the enzyme

Incubation of pure bacterial D-amino acid transaminase with D-serine or erythro-beta-hydroxy-DL-aspartic acid, which are relatively poor substrates, leads to generation of a new absorbance band at 493 nm that is probably the quinonoid intermediate. The 420-nm absorbance band (due to the pyridoxal phosphate coenzyme) decreases, and the 338-nm absorbance band (due to the pyridoxamine phosphate or some other form of the coenzyme) increases. A negative Cotton effect at 493 nm in the circular dichroism spectra is also generated. Closely related D amino acids do not lead to generation of this new absorption band, which has a half-life of the order of several hours. Treatment of the enzyme with the good substrate D-alanine leads to a small but detectable amount of the same absorbance band. D-Serine but not erythro-beta-hydroxyaspartate leads to inactivation of D-amino acid transaminase, and D-alanine affords partial protection. The results indicate that D-serine is a unique type of inhibitor in which the initial steps of the half-reaction of transamination are so slow that a quinonoid intermediate with a 493-nm absorption band accumulates. A derivative formed from this intermediate inactivates the enzyme.     

Circular dichroism and proton magnetic resonance studies of random chain poly-L-lysine

The 218-nm peak, characteristic of the circular dichroism of randomly coiled poly-α-amino acids can be demonstrated in solutions of penta-L -lysine, α-glycyl-L -lysine, as well as poly-L -lysine. The thermal stability of the particular state that gives rise to this 218-nm band in the CD is similar for all three peptides. These results eliminate the possibility that poly-L -lysine forms a structure with long-range order in acidic aqueous solution since the stability of such a structure would be expected to be greater for a higher molecular weight polymer than for a pentamer. The intrinsic viscosity of poly-L -lysine of molecular weight 180,000 varies only slightly between 25 and 60°C. The proton magnetic resonance spectra of poly-L -lysine and penta-L -lysine are indistinguishable on the basis of the chemical shift of all resonances, their line widths, and the exchange rates of the N? H protons. This demonstrates that poly-L -lysine does not possess a cooperatively formed ordered structure in acidic solutions. A weak band at 238 nm is observed in the circular dichroism of poly-L -lysine and other peptides. It is suggested that the effects of change in temperature, salt concentration, or polymer on both the magnitude and position of the 238-nm band may be explained if it is assumed that it is a shoulder of a lower wavelength peak.     

Reconstitution of the B873 light-harvesting complex of Rhodospirillum rubrum from the separately isolated alpha- and beta-polypeptides and bacteriochlorophyll a

The light-harvesting complex of Rhodospirillum rubrum was reversibly dissociated into its component parts: bacteriochlorophyll and two 6-kilodalton polypeptides. The dissociation of the complex by n-octyl beta-D-glucopyranoside was accompanied by a shift of the absorbance maximum from 873 to 820 nm (a stable intermediate form) and finally to 777 nm. In the latter state, bacteriochlorophyll was shown to be free from the protein. Complexes absorbing at 820 and 873 nm could be re-formed from the fully dissociated state with over 80% yield by dilution of the detergent. Absorbance and circular dichroism properties of the re-formed B820 complex were essentially identical with those of B820 formed from chromatophores. Phospholipids and higher concentrations of complex were required to obtain the in vivo circular dichroism spectrum for reassociated B873. Reconstitution of the light-harvesting complexes from separately isolated alpha- and beta-polypeptides and bacteriochlorophyll was also demonstrated. Absorbance and circular dichroism spectra of these complexes were identical with those of complexes formed by the reassociation of the dissociated complex. Bacteriochlorophyll and the beta-polypeptide alone formed a complex that had an absorbance at 820 nm, but an 873-nm complex could not be formed without addition of the alpha-polypeptide. The alpha-polypeptide alone with bacteriochlorophyll did not form any red-shifted complex. In preliminary structure-function studies, some analogues of bacteriochlorophyll were also tested for reconstitution.     

Properties of a water-soluble, yellow protein isolated from a halophilic phototrophic bacterium that has photochemical activity analogous to sensory rhodopsin

A water-soluble yellow protein, previously discovered in the purple photosynthetic bacterium Ectothiorhodospira halophila, contains a chromophore which has an absorbance maximum at 446 nm. The protein is now shown to be photoactive. A pulse of 445-nm laser light caused the 446-nm peak to be partially bleached and red-shifted in a time less than 1 microsecond. The intermediate thus formed was subsequently further bleached in the dark in a biphasic process occurring in approximately 20 ms. Finally, the absorbance of native protein was restored in a first-order process occurring over several seconds. These kinetic processes are remarkably similar to those of sensory rhodopsin from Halobacterium, and to a lesser extent bacteriorhodopsin and halorhodopsin; although these proteins are membrane-bound, they have absorbance maxima at about 570 nm, and they cycle more rapidly. In attempts to remove the chromophore for identification, it was found that a variety of methods of denaturation of the protein caused transient or permanent conversion to a form which has an absorbance maximum near 340 nm. Thus, by analogy to the rhodopsins, the absorption at 446 nm in the native protein appears to result from a 106-nm red shift of the chromophore induced by the protein. Acid denaturation followed by extraction with organic solvents established that the chromophore could be removed from the protein. It is not identical with all-trans-retinal and remains to be identified, although it could still be a related pigment. The E. halophila yellow protein has a circular dichroism spectrum which indicates little alpha-helical secondary structure (19%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical and physical properties of the disulfides of bovine neurophysin-II.

Bovine neurophysin-II is shown to be very susceptible to partial reduction in the absence of urea. Reduction of an average of one disulfide leads to major changes in conformation and disulfide optical activity, manifest in part by pronounced far-uv ellipticity changes, complete loss of the 248-nm ellipticity band, and a shift of the 278-nm ellipticity band to shorter wavelengths with loss of half its intensity; the reduction process generates a mixture of products and appears to be accompanied by disulfide interchange. The circular dichroism data indicate that the disulfide(s) most susceptible to reduction or interchange are either the principal contributors to the 248- and 278-nm ellipticity bands or that the optical activity of other disulfides is dependent on their integrity. Peptides that bind to the hormone-binding site of neurophysin-II protect against reduction. On reoxidation of partially reduced neurophysin-II there is only a partial return of the native circular dichroism spectrum and electrophoretic behavior. The percentage of native protein in samples reoxidized following different degrees of reduction was estimated by comparison of the circular dichroism spectra of these samples with those of the fractionated native and denatured components of monoreduced-reoxidized neurophysin. Under our reoxidation conditions, less than 50% native protein was found in monoreduced-reoxidized neurophysin and less than 10% native protein was found in completely reduced-reoxidized neurophysin. The results are interpreted with qualified reference to a model in which one or more disulfides are "strained" in the native state and in which the native protein is unstable relative to species in which the disulfides are differently paired.     

Allosteric transitions in cobalt hemoglobins.

Circular dichroism and difference ultraviolet visible spectra were obtained for cobalt hemoglobin derivatives. At 287 nm the ellipticity difference between the oxy- and deoxycobaltohemoglobin is about one-half as great as that for the native proteins indicating smaller quaternary conformational changes for the former. Deoxygenation increases the Soret rotational strengths of both iron and cobalt hemoglobins to comparable degrees suggesting similar conformational changes for their aromatic residues near the "heme." Deoxygenation causes a much larger decrease of L band ellipticity for iron than cobalt hemoglobin. Circular dichroism spectra of nitrosylcobaltohemoglobin indicate the molecule to have a T quaternary structure. The circular dichroism spectra of cobaltihemoglobin do not seem to fit the patterns of the other cobalt derivatives and its 287 nm ellipticity is pH-dependent. From the shape of the Soret circular dichroism spectra, it is estimated that the transition dipole makes an angle with the line joining the two opposing pyrrole nitrogens of about 60 degrees for oxy- and deoxycobaltohemoglobin, 80 degrees for cobaltihemoglobin, as compared to 70 degrees for the native oxy- and deoxyhemoglobins. Inositol hexaphosphate has little or no effect on the circular dichroism spectra of cobalt hemoglobins in the 287 nm region, but it significantly increases the Soret rotational strength and decreases the L band ellipticity. The results are interpreted to mean that polyphosphates modify primarily the protein structure of hemoglobins at the tertiary level, and that the intersubunit interactions are weak in cobalt hemoglobins.     

Properties of rooster serum high density lipoproteins.

1. Holo-superoxide dismutase from bovine erythrocytes has been shown to undergo a reversible structural modification in the pH 3-5 range. 2. The spectral alterations observed on changing from neutrality to pH 2 were: a slight attenuation of the 680 nm absorbance; the loss of the 450 nm shoulder, apparent in the optical spectrum of the native protein; and a new band appeared at 330 nm. The circular dichroism at 600 nm was essentially lost while a weak negative band appeared at approx. 380 nm and a positive band at 310 nm. 3. The EPR spectrum was also modified on changing from the native to the low pH form: A parallel increased from approximately 130 to approximately 150 G, g parallel remained unchanged at approximately 2.27, and gm decreased from approximately 2.09 to approximately 2.08. The apparent linewidth remained essentially constant. 4. High resolution (220 MHz) PMR spectra of holo- and apoproteins revealed that the metals influence the three-dimensional structure of the protein. 5. PMR studies indicated that at pH 3 the apoprotein existed almost entirely in a random coil form and that it assumed a compact well-ordered structure on returning to neutral pH. The holoprotein maintained a compact, apparently dimeric, structure even at pH 3.     

Cholera toxin does not impair hormonal inhibition of adenylate cyclase and concomitant stimulation of a GTPase in adipocyte membranes

Factor H of the human complement system exhibits an unusual circular dichroism spectrum. The CD spectrum of Factor H exhibits a positive extreme at 230 nm and a negative extreme at 190 nm. No apparent alpha-helical or beta-sheet conformations were present in the native protein structure. However, when the disulfide bridges are reduced, followed either by reoxidation or alkylation, the structure of Factor H is modified so that it now exhibits conventional protein secondary structure as determined from its CD spectra in the far ultraviolet region. Factor H also fails to mediate its regulatory function of inhibiting the alternative pathway convertase once the disulfides have been ruptured and conformational rearrangement has occurred. CD studies indicate that minor conformational changes take place when Factor H and C3b associate in free solution.     

Circular dichroism of isolated ricin A- and B-chains

An analysis of the circular dichroism (CD) spectra of isolated ricin A- and B-chains revealed several bands not apparent in the spectrum of intact ricin. Arithmetic combination of the A- and B-chain spectra gave a composite spectrum resembling that of native ricin, indicating that the two chains did not undergo any major conformational change upon dissociation. The addition of lactose to the B-chain at pH 7.2 caused a slight perturbation of a tryptophan-derived negative CD band centred at 283 nm without change to the overall structure of the polypeptide.     

Induced optical activity in poly-L-lysine-methyl orange system

The absorption and cicular dichroism spectra of the complex of poly-L -lysine (PLL) in the random coil form with methyl orange (MO) have been measured in aqueous solution. A new absorption band is observed at the shorter wavelength compared with that of the free dye. Although MO does not show a formation of dimer or aggregation with an increase in concentration, circular dichroism bands are observed at the wavelength corresponding to the wavelength of the new absorption band. These induced circular dichroism bands may arise from the dimeric MO molecules bound to PLL in the random coil form. The main contribution to the interaction between MO molecules is shown to be the electro static interaction. The observed circular dichroism spectra and the configuration of dimeric MO molecules bound to PLL can be explained by the dipole couping mechanism.     

Vacuum-ultraviolet circular dichroism of acetylated glucans

We have measured the solution and film vacuum ultraviolet circular dichroism of a series of acetylated glucans containing α- and β-(1→3), (1→4), and (1→6) linkages. In addition to the 210-nm band studied previously, we observe the entire π-π* band near 190 nm; these bands are negative for all triacetates regardless of configuration and conformation. A band near 170 nm shows configurational sensitivity for (1→3)- and (1→6)-linked polysaccharides. The band is positive for both (1→4)-linked triacetates, but when cellulose triacetate is partially deacetylated, the 170-nm band becomes negative, thus making the correlation complete. The positive 170-nm band in cellulose triacetate films is more than an order of magnitude more intense than in any other case and, further, is accompanied by an equally large negative band near 153 nm, raising the possibility that the dichroism in the triacetate arises from strong excitonic interactions which are disrupted upon partial deacetylation.     

Protochlorophyll forms with different molecular arrangements

Spectral properties of protochlorophyll (PChl) forms were investigated in solid-film model systems by absorption. fluorescence and circular dichroism (CD) spectroscopy. The solid films were prepared from diethyl ether solution of PChl on a cover glass surface by evaporation of the solvent. After preparation the films usually showed an absorption maximum at 635 nm or in some cases at 640 nm. The PChl form with 635 nm absorption maximum had no CD signal, whilst the films with absorption maximum at 640 nm gave an intense negative CD band at about 640 nm and a positive one at 668 nm. The treatment of the films with ammonia or acetone vapour resulted in a red shift of the absorption maximum from 635 nm or 640 nm to 650 nm. The study of the CD spectra of the films with different PChl forms showed that, depending on the treatment, forms of PChl with similar absorption and fluorescence spectra, but with opposite CD signals, can exist. It is suggested that the differences of the CD spectra are mainly due to different arrangements of the aggregates.     

6-Thioguanine in DNA as CD-spectroscopic probe to study local structural changes upon protein binding

A combination of experimental and theoretical circular dichroism (CD) spectroscopy was used to study local deformations of DNA caused by binding of the base flipping DNA methyltransferase M.TaqI. To selectively study the structural changes within the DNA, we replaced single guanine residues at six different positions in duplex DNA with 6-thioguanine (s(6)G), which absorbs at 342 nm where unmodified DNA and the enzyme are transparent. The shape and the transition wavelength of a CD signal around 340 nm in the spectra of the free DNA and the M.TaqI-bound DNA were found to depend on the position of the s(6)G probe. Theoretical rotational strengths were calculated employing the matrix method which is frequently used to model the CD of large biomolecules. The only chromophores in these calculations were the nucleic acid bases. Comparison of the measured and the calculated CD spectra showed that the applied computational method qualitatively reproduces the dominant band observed around 340 nm in all cases. From our results we conclude that the spectral changes observed upon binding of the enzyme to the DNA are indeed predominantly due to structural changes within the DNA and not to other effects caused by the presence of the enzyme.     

Circular dichroic spectra of elapid cardiotoxins

Cardiotoxins isolated from elapid snake venoms constitute a chemically homogeneous family of molecules. Within this group several biologically different subclasses exist. We report a comparative analysis of the structure of 20 cardiotoxins using circular dichroism, immunological methods and secondary-structure prediction. It is shown that cardiotoxins fall within two structural subclasses. Toxins of group I are characterized by (a) CD spectra having an intense positive band close to 192.5 nm and a negative trough at 225 nm with no positive band around 230 nm, (b) strong cross-reactivity with a polyclonal antiserum specific for Naja nigricollis toxin gamma and (c) a high tendency to form a reverse turn in the region of position 11. Toxins of group II are characterized by (a) CD spectra displaying a much weaker positive band at 192.5 nm, a negative band around 210 nm and a positive band at 230 nm, (b) little cross-reactivity with the aforementioned antiserum and (c) a high reverse-turn potential at position 31. It is suggested that the observed differences result from differing curvatures in the antiparallel beta sheet which constitutes the main secondary structure of cardiotoxins.