Capillary circular dichroism

Circular dichroism (CD) has become an increasingly important tool in the study of biological molecules as it enables structural information to be obtained nondestructively on solution-phase samples. However, sample requirements for CD are often seen as being too high with protein backbone measurements in standard cuvettes typically requiring ～100-300 μL of 0.1 mg/ml protein. To address this issue, we have designed a new form of CD sample holder, which reduces the sample requirements of the technique by two orders of magnitude, with a sample requirement of less than 3 μl. This sample saving has been achieved through the use of extruded quartz capillaries, the sample being held within the internal diameter of the quartz capillary through capillary action. The extruded quartz capillaries exhibit remarkably little birefringence, although still transmitting high energy UV circularly polarized light. The optics associated with capillaries were investigated. A configuration has been adopted with the light beam of the spectrophotometer being focused in front of the front face of the capillary using a biconvex lens and advantage being taken of the additional focusing effect of the capillary itself. The focusing is vital to the low wavelength performance of the cell, where we have acquired reliable data down to 180 nm using a Jasco J-815 spectrophotometer. The system performance was validated with Na[Co(EDDS)].H(2)O (EDDS = N,N-ethylenediaminedisuccinic acid), concanavalin A, lysozyme, and progesterone.     

Linear dichroism and circular dichroism in photosynthesis research

The efficiency of photosynthetic light energy conversion depends largely on the molecular architecture of the photosynthetic membranes. Linear- and circular-dichroism (LD and CD) studies have contributed significantly to our knowledge of the molecular organization of pigment systems at different levels of complexity, in pigment–protein complexes, supercomplexes, and their macroassemblies, as well as in entire membranes and membrane systems. Many examples show that LD and CD data are in good agreement with structural data; hence, these spectroscopic tools serve as the basis for linking the structure of photosynthetic pigment–protein complexes to steady-state and time-resolved spectroscopy. They are also indispensable for identifying conformations and interactions in native environments, and for monitoring reorganizations during photosynthetic functions, and are important in characterizing reconstituted and artificially constructed systems. This educational review explains, in simple terms, the basic physical principles, and theory and practice of LD and CD spectroscopies and of some related quantities in the areas of differential polarization spectroscopy and microscopy.     

Magnetic circular dichroism of netropsin and natural circular dichroism of the netropsin-DNA complex

We report the first measurement of the magnetic circular dichroism (MCD) of the basic polypeptide antibiotic netropsin (Nt). The MCD shows that the longest wavelength absorption band of Nt is the sum of more than one component and permits a radically new interpretation of the circular dichroism of the complex which Nt forms with DNA. We conclude that Nt has no major effect on the CD and thus the helical structure of the bases of the DNA to which it is bound. Thus the ability of Nt to inhibit the function of DNA polymerase, RNA polymerase, and the photoreactivating enzyme must be mediated by factors other than a distortion of the helical structure of the bases.     

A circular dichroism microspectrophotometer

A microscope capable of measuring the CD of intact single eukaryotic cells, DNA microcrystals, and other microscopic structures has been constructed and tested. It can measure the CD spectra in the 200- and 800-nm wavelength range and consists of a modification to a standard Cary 60 CD machine in combination with a Zeiss uv microspectrometer. Preliminary CD spectra of red blood cells and lymphocytes are presented.     

Heparin-induced circular dichroism of chloroquine

Circular dichroism (CD) and UV/Visible absorption (UV/Vis) spectroscopy techniques were used to investigate the interaction between heparin and chloroquine, an antimalarial drug that has shown potential as an anti-prion agent. CD spectra of rac-chloroquine upon addition of heparin provide evidence of glycosaminoglycan (GAG) binding, support recent findings suggesting that interactions between heparin and antimalarial drugs are largely due to electrostatic interactions, and represent the first reported GAG-induced CD signal of a bicyclic, aromatic compound. The association constant (∼10³ M⁻¹) between chloroquine and heparin was calculated from a UV titration curve and provided additional insight into the nature of the association between these two compounds.     

Circular dichroism and magnetic circular dichroism of bismuth-induced,metallothionein-like proteins

Optical studies have been carried out on bismuth-containing proteins which were isolated from the livers and kidneys of rats following injections of BiCl₃. Absorption, circular dichroism and magnetic circular dichroism spectra of hepatic Bi,Zn-metallothionein 1 and 2 indicate that the spectra are dominated by transitions from the zinc thiolate chromophore. The data from the renal Bi,Cu-metallothionein 2 are quite different and it is suggested that these spectra involve a mixture of transitions from the bismuth and copper thiolate binding sites.     

Circular dichroism and magnetic circular dichroism of Azotobacter vinelandii ferredoxin I

Room temperature circular dichroism (CD) and low temperature magnetic circular dichroism (MCD) spectra of air-oxidized and dithionite-reduced Azotobacter vinelandii ferredoxin I (FdI), a [( 4Fe-4S]2+/1+, [3Fe-4S]1+/0) protein, are reported. Unlike the CD of oxidized FdI, the CD of dithionite-reduced FdI exhibits significant pH dependence, consistent with protonation-deprotonation at or near the cluster reduced: the [3Fe-4S] cluster. The MCD of reduced FdI, which originates in the paramagnetic reduced [3Fe-4S]0 cluster, is also pH-dependent. Detailed studies of the field dependence and temperature dependence of the MCD of oxidized and reduced FdI, in the latter case at pH 6.0 and 8.3, are reported. The low-field temperature dependence of the MCD of oxidized FdI, which originates in the paramagnetic oxidized [3Fe-4S]1+ cluster, establishes the absence of a significant population of excited electronic states of this cluster up to 60 K. The low-field temperature dependence of the MCD of reduced FdI establishes that the ground-state manifold of the reduced [3Fe-4S]0 cluster possesses S greater than or equal to 2 at both pH 6.0 and 8.3. Analysis, assuming S = 2 and an axial zero-field splitting Hamiltonian, leads to D = -2.0 and -3.5 cm-1 at pH 6.0 and 8.3, respectively. The site of the (de)protonation affecting the spectroscopic properties of the [3Fe-4S] cluster remains unknown.     

Absorption, circular dichroism, magnetic circular dichroism and emission study of rat kidney Cd,Cu-metallothionein

Absorption, circular dichroism (CD), magnetic circular dichroism (MCD) and emission spectra of rat liver and rat kidney cadmium-, zinc- and copper-containing metallothioneins (MT) are reported. The absorption, CD and MCD data of native rat kidney Cd,Cu-MT protein closely resemble data recorded for the rat liver Cd,Zn-MT. This suggests that the major features in all three spectra of the native Cd,Cu-MT are dominated by cadmium-related bands. The CD spectrum of the Cd,Cu-MT recorded at pH 2.7 has the same band envelope that is observed for a Cd,Cu-MT formed in vitro by titration of Cd,Zn-MT with Cu(I), suggesting that the copper occupies the zinc sites in Cd,Cu-MT formed both in vivo and, at low molar ratios, in vitro. Remetallalion of the metallothionein from low pH in the presence of both copper and cadmium results in considerably less cadmium bound to the protein than was present in the native sample. It is suggested that this is due to the effect of the distribution of the copper amongst all available binding sites, thus inhibiting cluster formation by the cadmium. Emission spectra are reported for the first time for a cadmium- and copper-containing metallothionein. An emission band at 610 nm is shown to be a sensitive indicator of Cu(I) binding to metallothionein. Both the native Cd,Cu-MT and a Cd,Cu-MT formed in vitro exhibit an excitation spectrum with a band in the copper-thiolate charge-transfer region.     

Conformation and circular dichroism of DNA.

CD spectra of calf thymus, C. perfringens, E. coli, and M. luteus DNA have been measured in the vacuum-uv region to about 168 nm for the A-, B-, and C-forms. The positive band at about 187 nm and the negative band at about 170 nm found for each type and form of DNA are sensitive to the source of the DNA and the base–base interactions of the double-stranded helix. The A-form spectra confirm that these bands are indeed sensitive to secondary structure. In the near-uv, the CD of B-form DNA is well analyzed as a linear combination of 27% A-form and 78% C-form. However, an analysis of the extended spectrum demonstrates that the near-uv analysis is not correct. The extended analysis shows that the base–base interactions are similar for B- and C-forms in solution, which implies that these two forms have nearly the same number of base pairs per turn. Various types of CD difference spectra are also discussed.     

Circular dichroism and magnetic circular dichroism spectra of chlorophylls a and b in nematic liquid crystals. II. Magnetic circular dichroism spectra

Absorption and magnetic circular dichroism (MCD) spectra are reported for chlorophyll (Chl) a and Chl b dissolved in nematic liquid crystal solvents. The spectra were measured with the dye molecules oriented uniaxially along the direction of. the magnetic field and measuring light beam. It is significant that under such conditions the MCD spectra recorded in the wavelength region of the Q and Soret bands of the chlorophyll are essentially unchanged with respect to rotation of the sample cell around this axis, even though there is almost complete orientation of the chlorophyll molecules by the liquid crystals. The MCD spectra of Chl a and b in the nematic liquid crystal solvents used in this study are surprisingly similar to the spectra obtained under isotropic conditions. These results illustrate an important technique with which to examine the optical spectra of dyes oriented in liquid crystal matrices in which the anisotropic effects can be reduced the negligible proportions by the application of a strong magnetic field parallel to the direction of the measuring light beam. The first deconvolution calculations are reported that describe the deconvolution of pairs of absorption and MCD spectra, in the Q and B band regions, for both Chl a and b. The spectral analysis to obtain quantitative estimates of transition energies was accomplished by carrying out detailed deconvolution calculations in which the both the absorption and MCD spectral envelopes were fitted with the same number of components; each pair of components had the same hand centres and bandwidth values. This procedure resulted in an assignment of each of the main transitions in the absorption spectra of both Chl a and b. Chl a is clearly monomeric, with Qy, Qx, By and Bx located at 671, 582, 439 and 431 nm, respectively. Analysis of the spectral data for Chl b located Qy, By and Bx, at 662, 476 and 464 nm, respectively.     

Induced circular dichroism of DNA-dye complexes

The binding of methylene blue, proflavine, and ethidium bromide with DNA has been studied by spectrophotometric titration. Methylene blue and proflavine or methylene blue and ethidium bromide were simultaneously titrated by DNA. The results indicate that all of these dyes compete for the same bindine sites. The binding properties are discussed in terms of symmetry. The optical properties of the dye–DNA complexes have been studied as a function of DNA/dye ratio. The induced circular dichriosm due to dye–dye interaction was measured at low dye/DNA ratios for cases involving both the same dye and different dyes. A positive Cotton effect for DNA–proflavine complex may be induced at 465 mμ by eithr proflavine or ethidium bromide, whereas a netgative Cotton effect at 465 mμ may be induced by methylene blue. The limiting circular dichroism, with no dye–dye interaction, and the induced circular dichroism spectra are discussed in terms of symmetry rules.     

The circular dichroism spectrum of poly-L-acetoxyproline

We report the circular dichroism (CD) spectrum of poly-L acetoxyproline in trifluoroethanol, a solvent in which the form I to form II conformational isomerization occurs slowly enough to permit observation of the spectral changes. A comparison is made to poly-L proline. As judged by the similarity of the CD spectra, the conformations of the corresponding forms of the two polymers must be nearly the same. Transition assignments are proposed; these are shown to agree with the theoretical calculations of Pysh. There is a serious unexplained discrepancy between our solution data and those of Fasman for poly-L acetoxyproline.     

Vacuum-ultraviolet circular dichroism analysis of biomolecules

The vacuum-ultraviolet circular dichroism (VUVCD) spectra of various amino acids, saccharides, and proteins were measured using a synchrotron-radiation CD spectrophotometer at HiSOR/HSRC that is capable of measuring the CD spectra down to 140 nm in aqueous solution. L-Isomers of amino acids show two successive positive peaks at around 200 and 180 nm depending on the side chain. The ab initio assignment by time-dependent density functional theory predicts that these peaks are attributed to n-pi* and pi-pi* transitions of the carboxyl group, respectively. Most mono- and disaccharides exhibit characteristic peaks at around 170 nm, sensitively depending on the anomeric and axial/equatorial configurations of hydroxyl groups, trans-gauche conformations of the hydroxymethyl group, and the type of glycosidic linkage. The VUVCD spectra of 31 globular proteins allow us to estimate more accurately the content and number of alpha-helix and beta-strand segments by extending the short-wavelength limit of the analytical program SELCON3 down to 160 nm. These results demonstrate that synchrotron-radiation VUVCD spectroscopy is a useful tool for structure analyses of biomolecules in solution based on the higher energy transitions of chromophores.