Optical rotatory dispersion studies of poly-O-acetyl-gamma-hydroxy-L-proline forms I and II

Poly-O-acetyl-hydroxy-L -proline, forms I and II have been studied by optical rotatory dispersion (ORD) and ultraviolet spectrophotometry in solution and in the solid state. Cotton effects of opposite sign, but not mirror images, were observed in the 250 mμ region for the two forms (Form I, peak 278 mμ; crossover, 254 mμ; trough, 244 mμ: Form II, trough 270 mμ; crossover, 248 mμ; peak, 238 mμ). Thus, the Cotton effects for a right-handed and left-handed helix have been shown to be opposite for the proline type helices I and II. The ORD of films of form I was found to have a positive Cotton effect further into the ultraviolet region with peak at 218 mμ. Absorption spectra showed a shift of 8 mμ in the absorption peak in the 200 mμ region for the two forms (form. I, 211 mμ; form II, 203 mμ). A shoulder was demonstrated in the film absorption spectra in the 250 mμ region where the Cotton effects are found. The mixing of the n, π* and π, π* states of the amide chromophore and n, π* state of the ester chromophore was suggested as being responsible for the Cotton effects in the 250 mμ region.     

Studies on Respiratory Enzymes in Rice Kernel

Glutathione reductase as an acidic flavoprotein, has been isolated from the acidic protein fraction of rice embryos and purified by procedures involving ammonium sulfate fractionation, gel filration on Sephadex G–75 and G–100, ion exchange chromatography on CM-and DEAE-Sephadex and finally hydroxylapatite column chromatography. The preparation was homogeneous when examined by ultracentrifugation and almost pure on polyacrylamide gel electrophoresis. The flavoprotein exhibited an absorption spectrum characteristic of glutathione reductase having absorption maxima at 275, 370, 379, and 463 mμ with a clear double peak between 370 and 380 mμ and shoulders at around 430 and 490 mμ. The absorption ratio of A₂₇₅/A₄₆₃ and A₄₆₃/A₃₇₉ were 8.15 and 1.06, respectively. The purified enzyme was highly specific for NADPH and oxidized glutathione. The preparation had the average catalytic activity of 150 μmoles of NADPH oxidized per min per mg of protein.     

Cryptomonad biliprotein: phycocyanin-645 from a Chroomonas species.

The properties of phycocyanin-645 from the fresh water cryptomonad Chroomonas spec. were investigated after the pigment was isolated and purified by a combination of differential ammonium sulphate fractionation, gel filtration chromatography and ammonium sulphate gradient elution. Phycocyanin-645 is characterized by absorption maxima at 645 nm, 584 nm, 369 nm, 275 nm and shoulders at 340 nm and 620 nm. The CD spectrum has a negative maximum at 645 nm and a positive maximum at 584 nm with a shoulder at 610 nm. The fluorescence emission spectrum is asymmetrical and shows a maximum at 660 nm and a shoulder at approximately 715 nm. The molecular weight of the native phycocyanin-645, estimated by gel filtration, is 45000 for all multiple pigment forms below. Phycocyanin-645 is heterogeneous as revealed by isoelectric focusing with pIs at 7.03, 6.17, 5.75, 5.25 and 4.88, respectively, the main bands lying at pI 7.03 and pI 6.17. This was confirmed by polyacrylamide gel electrophoresis; five pigment compoents differing in mobility were found. We propose the term "multiple pigment forms" for these five phycocyanin-645 modifications. Calibrated SDS gel electrophoresis shows phycocyanin-645 to consist of three subunits, two light chains (alpha1, alpha2), having molecular weights of 9200 and 10400, respectively, and one heavy chain (beta), having a molecular weight of 15 500. Suggesting a 1:1:2 ratio between the subunits, the quaternary structure of the pigment molecule is alpha1beta--alpha2beta1.     

Optical activity of simple cyclic amides in solution

The cyclic dipeptide, L -alanylglycyl anhydride, has been studied by optical rotatory dispersion; both L -alanylglycyl anhydride and the lactam, L -3-aminopyrrolidin-2-one, have been studied by circular dichroism. In hydroxylic solvents the circular dichroism spectra of 3-aminopyrrolidin-2-one can be attributed to an n–π* transition near 220 mμ and a π–π* transition near 190 mμ. In these solvents the optical activity of L -alanylglycyl anhydride can be explained as being due to contributions of n–π* transitions and a split π–π* transition. In acetonitrile, however, the circular dichroism spectrum of 3-aminopyrrolidin-2-one shows an additional apparent minimum near 200 mμ. The CD spectrum of the dipeptide is also quite distinctive in this solvent. The possible nature of the band at 200 mμ and the implications of these findings are discussed.     

Optical activity of single-stranded polydeoxyadenylic and polyriboadenylic acids; dependence of adenine chromophore cotton effects on polymer conformation

Circular dichroism (CD) curves are reported for poly dA, (pdA)₆, (pdA)₂, poly A, ApAp, ApA, AMP, dApA, pdApA, A-2′-O-methyl pA, and A-2′-O-methyl pAp. Analysis of these curves indicated the presence of single CD bands at 228–230 mμ and at 278–280 mμ in oligomers longer than dinucleotides. In the case of dinucleotides and mononucleotides (from the literature, in addition to those studied here), the 230 mμ CD of band appears but the 280 mμ CD band does not. We assign the 230 mμ band to a very weak π–π* transition at this wavelength. From theoretical considerations, we show that the 280 mμ band is not an exciton component of the strong π–π* transition at 260 mμ in adenine. We conclude that the 280 mμ CD band must be assigned to a distinct absorption, not previously reported, which we suggest arises from an n–π* transition. The fact that the n–π* CD band at 280 mμ is not seen in mononucleotides or dinucleotides is ascribed to solvation of the adenine ring by water, which shifts the band to shorter wavelengths. Therefore, only interior residues of oligomers have the 280 mμ band, and the optical activity of a polymer cannot be computed from that of a dinucleotide, by using a nearest-neighbor approximation. The existence of this end effect hag been tested, by taking it into account in computing the rotational strengths of the 278 mμ n–π* transition for several oligomers; it is pointed out that a more sensitive test of this end effect would require CD data for the oligo dA series of 3 to 5 residues. We speculate about the structural and optical differences between poly dA and poly A, and point out the need for a theoretical treatment of n–π* Cotton effects in polynucleotides.     

Theoretical optical properties of poly-L-proline

A non-perturbational technique is used to calculate the circular dichroism and absorption spectra of polypeptide chains having conformations similar to that of poly-L -proline II. The method employs a Bogoliubov exciton formalism, from which the various optical terms associated with parallel and perpendicular components of the exciton band are obtained. A simple model for the peptide unit, consisting of three Gaussian absorption bands, leads to reasonable results for the polymer spectra, provided the lowest energy peptide π → π* transition is taken at 207 mμ and the value of the Ramachandran angle Ψ is taken to be 390°. The calculations suggest that the polymer circular dichroism spectrum is the resultant of strong interference among the two Gaussian exciton terms and the non-Gaussian helix term. Consequently, the CD spectrum is very sensitive to the value of Ψ. It is found that the small positive CD band in the vicinity of 230 mμ arises partly from the effect of the static (crystal) field interactions on the n → π* CD band.     

Bactericidal substances in cytoplasm of leucocytes. An attempt to separate lysozyme by gel filtration

A substance with a bactericidal effect onEscherichia coli was isolated from the cytoplasm of polymorphonuclear leucocytes. It consists of a mixture of different active substances and therefore a gel filtration using s Sephadex G-100 column was used to separate lysozyme from other active substances. The extract was separated by this procedure into five peaks, out of which peak I. possesed a significant bactericidal activity, peak II. and III. had a weak one, whereas the lysozyme activity was present in peak III., IV. and V.     

Characterization of the Malaria Pigment (Hemozoin) from the Avian Malaria Parasite Plasmodium lophurae*

SYNOPSIS. Plasmodium lophurae hemozoin (malaria pigment) is a heme-containing protein which is distinctly different from hemoglobin and hematin by immunologic, spectrophotometric, fingerprint, heme-iron, gel filtration, and starch gel electrophoretic analyses. The calculated average molecular weight of P. lophurae hemozoin is ca. 40,000. Hemozoin contains at least 3 antigenic components and shows some indication of cross-reaction with hemoglobin.     

PHOTOINDUCTION AND PHOTOREVERSAL OF THE NOSTOCACEAN DEVELOPMENTAL CYCLE1

The developmental cycle of Nostoc muscorum, a nitrogen-fixing blue-green alga, is controlled by the spectral quality of illumination. Red light with peak activity at 650 mμ induces development of filaments from a nonfilamentous (aseriate) stage of the life cycle. Red-light photoinduction is reversed by simultaneous or subsequent exposure to light from a broad band in the green region of the spectrum. Photoreversibility of the red-light induction, by green light, decays very slowly, remaining at an appreciable level for over 24 hr after the primary stimulus. Allophycocyanin is indicated to be the photoreceptor for red-light induction. One or more phycoeythrins may operate as photoreceptors for reversal of induction. The dosage response and wavelength dependence of developmental photocontrol in Nostoc muscorum A indicate that a nonphotosynthetic mechanism is involved in both developmental photoinduction and its photoreversal.     

A novel pigment protein in the ovary of echinothurid sea urchins,Asthenosoma ijimai and Araeosoma owstoni

• 1.1. A protein conjugated with a pigment, which showed a peak of absorbance at 385 nm, was identified and partially purified from the ovary of Asthenosoma ijimai and Araeosoma owstoni by butanol extraction, gel filtration, ion-exchange chromatography and adsorption chromatography. This protein was observed only in ovaries, but not in testes.
• 2.2. This protein of A. ijimai showed a molecular weight of 600 kDa on gel filtration. The isoelectric point of the protein was 4.7.
• 3.3. The possible presence of this protein was examined by gel filtration chromatography in the ovaries of 11 other species of sea urchins, Glyptocidaris crenularis, Diadema setosum, Temnopleurus hardwicki, Toxopneustes pileolus, Pseudocentrotus depressus, Hemicentrotus pulcherrimus, Strongylocentrotus intermedius, S. nudus, Echinostrephus aciculatus, Anthocidaris crassispina, Echinometra mathaei and Echinocardium cordatum. However, it was not detected.

     

Vacuum ultraviolet absorption spectra of sublimed films of nucleic acid bases

Measurements of the optical absorption spectra of the sublimed films of adenine, guanine, cytosine, thymine, and uracil were extended down to 120 mμ at room temperature. Several remarkable absorption peaks were found to exist below 190 mμ in addition to the already known ones near 260 mμ and 200 mμ. The intensities of these peaks were comparable to or, in some cases, even larger than those near 260 mμ and 200 mμ. It was found that the relative intensities of the absorption peaks differed considerably from sample to sample for all five bases, probably due to variation in the arrangement of the bases in the sublimed films. It was found also that the absorption spectra of the sublimed films change with aging on standing in vacuo at room temperature. On comparing the spectra of several fresh samples it was found that the intensities of the transitions with wavelength longer than 160 mμ of adenine, guanine, and uracil films are inversely correlated with those of the transitions shorter than 160mμ.     

Isolation and Characterization of the Putative Photoreceptor for Phototaxis in Amoebae of the Cellular Slime Mold,Dictyostelium discoideum

Amoebae of the cellular slime mold Dictyostelium discoideum (strain AX2) produce a pigment with an absorption spectrum that closely resembles the action spectrum for phototaxis. The protein-pigment complex was isolated and purified by sucrose gradient centrifugation, fast protein liquid chromatography (FPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). It is tightly membrane-bound and the bulk of it is located in the mitochondrial membrane fraction, while a small part is located in the cytoplasmic membrane fraction, as indicated by marker enzyme tests (succinate dehydrogenase for mitochondria and alkaline phosphatase for the cytoplasmic membrane). It is speculated that the pigment bound to the cytoplasmic membrane acts as photoreceptor and that bound to the mitochondria operates as a shading pigment in the light direction perception mechanism of Dictyostelium amoebae.     

pH值及金属离子对樟树果实红色素吸收光谱的影响

以樟树果实为材料,运用800～400 nm光谱扫描分析了pH值、金属离子、加热时间及温度、光照等稳定性因子对樟树果实红色素吸收光谱的影响。结果表明:最大吸收波长是517 nm,酸性条件对色素吸收光谱无明显影响,当pH为8.6的强碱性环境,红色素结构变化,最大吸收峰漂移至402 nm;100℃内,色素性质稳定,当加热时间延长至120 min,最大吸收峰在512.5 nm;红色素耐光性较好,但避光更利于保存;金属离子对红色素吸收光谱的影响强弱为:Fe3+>Al3+>Mg2+>Ca2+>Na+>K+,Fe3+短时内引起色素变色、发生沉淀,最大吸收峰漂移,浓度越高影响越大,Na+、K+对红色素吸收光谱无明显影响。     

Ultraviolet dichroic ratio of DNA from T2 and T5 bacteriophages

The dichroic ratios of T5st-O and T2H bacteriophage DNA molecules were measured throughout the ultraviolet region of the spectrum. Two methods of DNA orientation were studied: (1) orientation in solution in a Shimadzu flow dichroism instrument attached to a Beckman DU spectrophotometer, and (2) alcohol precipitation of the DNA from solution and orientation in a thin film on the quartz face of a humidity chamber. Spectra in the latter case were recorded using a Gary Model 14 spectrophotomcter fitted with Glan prisms. The lower wavelength limit was 215 mμ in both systems. The DNA preparations were carefully characterized as to spectral purity, sedimentation coefficient, hyperchromicity, protein content, and DNA content. In addition, the structure of the DNA oriented in films was inferred from x-ray diffraction patterns of fibers of the precipitated DXA. The A and B configurations of DNA in films could not be distinguished by the dichroic ratio measuiements. The dichroic ratio obtained for the film-oriented DNA at high relative humidity shows the same wavelength dependence as for the flow-oriented DNA. The same wavelength dependence for DNA in the fibrous state and in solution, when considered together with the x-ray diffract ion results, indicates that DNA in solution maintains an orientation of bases which is similar to that in fibers. I¹Or both solutions and films of DNA, the dichroic ratio is constant from 290mμ to 240 mμ and increases at wavelengths below 240 mμ. The increased parallel absorption below 240 mμ is consistent with the existence of an n→π* transition. The inherent molecular dichroic ratio is found to be the same for T5st-O DNA and T2H DNA in solution, and is a maximum of 0.09 ± 0.02 at 260 mμ.     

Isolation and characterization of a small molecular weight protein of linseed meal

A small M_r, protein from linseed meal has been isolated by CM-Sephadex chromatography. The protein was found to be homogeneous by the techniques of gel filtration, polyacrylamide gel electrophoresis and ultracentrifugation. It had S_20,w value of 1.6S. Amino acid composition of the protein revealed a high amount of glutamic acid, cystine, arginine and glycine. The absorption spectrum of the protein consisted of a peak at 280 nm with a shoulder at 290 nm. The fluorescence emission maximum was at 340 nm. The protein contained large amounts of α-helix and β-structure. SDS-PAGE showed the protein to consist of a single polypeptide chain. The M_r estimated by Archibald's method, sedimentation-diffusion method and gel filtration was 17 000,16 000 and 15 000 respectively. Difference spectra studies as a function of pH and temperature showed no variation in the conformation of the protein, probably due to disulphide bridges.     

Bovine Epizootic Fever

It was found that the ruins of Bovine Epizootic Fever appeared to be RNA as its replication was not inhibited by 5-iodo-2′-deoxyuridine. It had a buoyant density of 1.196, was not filterable through membrane filter (Millipore Filter Corp. Mass.) of 100 mμ pore size, and was sensitive to ether, chloroform and deoxycholate, was inactivated by trypsin and ultraviolet irradiation, precipitated by protamine sulfate, readily inactivated at pH 3.0, fairly labile at 56 C but readily preserved at —80 C, not stabilized at 50 C by I M MgCl₂, and resisted repeated freeze-thawing. The virus appears not to require a DNA-depcndent RNA synthesis in the host cell for its replication, as chromomycin A3 did not inhibit its replication. Sucrose density gradient centrifugation was unsuitable for purification because of a very poor recovery of infectivity. CsCl equilibrium density gradient centrifugation was successfully used for this purpose, and electron microscopy of the resulting fractions by phosphotungstic negative staining technique revealed virus-like bullet-shape particles, about 140 mμ in length and 80 mμ across, in the fraction of peak infectivity titer. The particle is probably the virion of the virus. These findings suggest relation of the virus to Rhabdoviruses [6], but further studies are necessary. The physicochemical properties of the virus provide additional evidence for similarity of the virus to bovine ephemeral fever virus, and emphasizes the desirability of further detailed comparative studies to decide whether they are one and the same or merely very closely related.     

PHOTOINDUCTION AND PHOTOREVERSAL OF THE NOSTOCACEAN DEVELOPMENTAL CYCLE1

The developmental cycle of Nostoc muscorum, a nitrogen-fixing blue-green alga, is controlled by the spectral quality of illumination. Red light with peak activity at 650 mμ induces development of filaments from a nonfilamentous (aseriate) stage of the life cycle. Red-light photoinduction is reversed by simultaneous or subsequent exposure to light from a broad band in the green region of the spectrum. Photo-reversibility of the red-light induction, by green light, decays very slowly, remaining at an appreciable level for over 24 hr after the primary stimulus. Allophycocyanin is indicated to be the photoreceptor for red-light induction. One or more phyco-erythrins may operate as photoreceptors for reversal of induction. The dosage response and wavelength dependence of developmental photocontrol in Nostoc muscorum A indicate that a nonphotosynthetic mechanism is involved in both developmental photoinduction and its photor ever sal.     

A carotenoprotein from chromatophoreses of Rhodospirillum rubrum

A carotenoprotein has been obtained by SDS-solubilization of $\text{Rhodospirillum rubrum}$ chromatophores. It was then purified by (NH₄)₂SO₄ precipitation and Sephadex G-200 filtration. SDS-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of about 12,000. The absorption spectrum of the complex is entirely different from the usual three peaked carotenoid spectrum, it has only a major peak at 370 nm. However, after acetone extraction the spectrum of spirilloxanthin reappears. The fact that the carotenoid associates with a specific protein provides strong evidence that the complex originates from the chromatophores and is not a preparative artefact.     

Subcellular distribution of free and esterified forms of vitamin a in the pigment epithelium of the retina and in liver

• 1.1. The distribution of vitamin A was examined in various subcellular fractions of rat liver and bovine retinal pigment epithelium. In rat liver, the major portion of the vitamin is in the cytosol, whereas in pigment epithelium, it is concentrated mainly in the microsomes. The microsomal vitamin A of pigment epithelium is tightly bound to membranes, as shown by the inability to release it except by organic solvent extraction or incubation with Triton X-100.
• 2.2. In both tissues, two different forms of cytosol vitamin A could be distinguished by ultracentrifugation. The major portion in liver is in the flotating lipid phase and consists mainly of retinyl ester. The remainder (less than 10% of the total) is in the underlying infranatant; about 90% of the vitamin A in this fraction is esterified. By contrast, two-thirds of the vitamin A of pigment epithelial cell cytosol is in the infranatant; it consists of both esterified and unesterified retinol. The floating layer in the pigment epithelial cytosol consists entirely of retinyl ester.
• 3.3. These two forms of cytosol vitamin A in the pigment epithelium could also be separated by gel filtration on Sephadex G-100 which yielded two distinct fluorescent peaks. The first, which appeared in the void volume and corresponded in all probability to the floating layer obtained by ultracentrifugation, consisted only of retinyl ester. The second peak, which was eluted in approximately the same position as myoglobin, contained only unesterified retinol. It was abolished completely by preincubation with pronase. These findings support the view that the second peak represents the endogenous retinol-retinol binding protein complex of pigment epithelial cytosol. The fluorescent enhancement of the retinol bound to protein in this peak was about 4–5-fold compared to retinol in organic solvents.