用液相等电聚焦电泳纯化藻蓝蛋白亚基

以纯藻蓝蛋白(C-phycocyanin, C-PC)为材料, 采用Rotofor系统进行液相等电聚焦(Liquid-phase isoeletric focusing, LP-IEF)电泳纯化C-PC的α, β亚基, 探讨蛋白质亚基纯化的制备电泳(Preparative eletrophoresis)技术. 结果显示, 样品经2次等电聚焦电泳后, C-PC 的α, β亚基分别浓集在pH=4.9和pH=4.1附近, 平板超薄等电聚焦(Slab ultra thin IEF)和SDS-PAGE电泳鉴定表明分别为高纯度的C-PC α, β亚基. 提示LP-IEF是分离纯化等电点差异蛋白质活性亚基的简便有效的方法.     

PICOSECOND FLUORESCENCE FROM PHYCOCYANIN 612

The biliprotein, phycocyanin 612, was purified from a cryptomonad, Hemiselmis virescens. The protein, which is an α₂β₂ dimer having four spectrally different tetrapyrrole chromophores, was studied using picosecond fluorescence by exciting the various chromophores at three wavelengths, 565, 585 and 615 nm. These wavelengths were chosen to excite selectively the three highest energy chromophores. Decay times were measured as the excitation energy migrated from each of the three excited chromophores to the lowest-energy chromophore. The ps decay times were found to be 9, 13, and 12 ps for excitations at 565, 585, and 615 nm, respectively. A comparison is made between phycocyanin 612 and phycocyanin 645 with regard to the causes of their differing absorption maxima.     

ENERGY TRANSFER STUDIES ON CRYPTOMONAD BILIPROTEINS

Abstract. Fluorescence techniques of various types have been used to study the light-gathering and energy transfer modes for various cryptomonad biliproteins (phycocyanin or phycoerythrins). Analysis of fluorescence polarization and absorption data demonstrates that each cryptomonad biliprotein is composed of at least two distinct types of absorbing chromophore, each attached to the protein through covalent linkages to different polypeptide chains. Examination of the fluorescence emission spectra as a function of excitation at several wavelengths demonstrates that only one of these absorbing chromo-phores is responsible for the fluorescence. This behavior is consistent with a known phenomenon whereby photons are gathered by more than one chromophore and then after radiationless energy transfer are emitted by only one chromophore. Application of Förster dipole-dipole energy transfer theory is made to the study of the mode by which energy absorbed by biliproteins migrates to Chl a. The spectral overlap integral between phycocyanin (Chroomonas sp.) and Chl a is 7.13 ± 10^-10cm⁶mol^-1and between phycocyanin and Chl c₂ 0.25 ± 10^-10cm⁶mol^-1. This large difference in overlap suggests, although does not prove, that phycocyanin might transfer energy directly to Chl a without a Chl c₂ intermediary. The cryptomonad phycoerythrins may also use this method but a Chl c₂ intermediate could not be ruled out for them. Radiationless energy transfer among homogeneous biliproteins is shown to be feasible. All these calculations are based on in vitro spectra and the interpretations extrapolated to the cellular situation, and these tentative conclusions are reached without knowledge of other factors, such as chromophore-chro-mophore orientation and distance, which could greatly influence the energy transfer scheme.     

POLARIZED PHOTO ACOUSTIC SPECTRA OF PHYCOBILISOMES IN POLYVINYL ALCOHOL FILMS

Abstract— Phycobilisomes from Porphyridium cruentum , suspended in polyvinyl alcohol were found to be highly stable, and had normal absorption and fluorescence spectra. Intact phycobilisomes had a major emission peak at 680 nm, whereas upon partial dissociating the major emission was at 580 nm. Incorporation of phycobilisomes in thin polyvinyl alcohol films facilitated examination by photoacoustic spectroscopy. The photoacoustic spectra had a broad absorption maximum at 545–565 nm (phycoerythrin), which resolved as two peaks (545 and 563 nm) in absorption spectra. Stretching of films resulted in apparent chromophore reorientation in partially dissociated, but not in intact phycobilisomes. Only in dissociated phycobilisomes was observed a differential chromophore orientation at 685 nm by polarized fluorescence, which is attributed to a change in orientation of the terminal phycobilisome pigment relative to phycoerythrin.     

Capillary electrophoresis for analysis of microheterogeneous glutelin subunits in rice (Oryza sativa L.)

Glutelin, the major storage protein of rice seed, consists of microheterogenous subunits and partially exists in a macromolecular form that is polymerized by intersubunit disulfide bonds. In order to analyze the glutelin subunits using high-throughput CE, we first identified a sample preparation procedure suitable for CE. The polymerized glutelin treated with a reductant could not dissociate into its constituent monomer subunits when it was dissolved in an acidic solution. However, the glutelin dissociated into its subunits and component α and β polypeptides when it was denatured and reduced by an appropriate amount of urea and 2-mercaptoethanol at a specific incubation time and temperature. The molecular species of the completely dissociated α and β polypeptides were identified and quantitatively analyzed by CE using glutelin mutants. The CE analysis also demonstrated that the actual subunit variation in terms of the charge and/or size of the β polypeptides is much smaller than predicted when compared with that of α polypeptides, even under denaturing and reducing condition. Thus, the combined analytical system described here will be useful for basic and applied research, such as the kinetic characterization of higher-order structure and the quantitative evaluation of glutelin in a large number of diverse rice varieties.     

Isolation and characterization of a new subunit of phycocyanin from Chroomonas placoidea

A new phycocyanin（PC） fluorescent subunit namedβ₂（18kDa） was isolated and characterized by both SDS-PAGE and isoelectric focusing（IEF） from a species of cryptophytic alga Chroomonas placoidea.PC was separated and purified by ammonium sulfate sedimentation followed by two steps of Sephadex G-100 chromatography.After denatured in 4 mol/L urea for 48 h,PC was divided into two fractions by passing through a Sephacryl S-100 chromatography column twice.The blue fraction（S-1） containedβsubunits with a maximal absorbance at 595 nm in visible light region.While the green fraction（S-2） enriched inαsubunits showed a characteristic long wavelength absorbance at 680-700 nm region and exhibited a relatively low molecular weight of 9.4（α₁） and 8.5 kDa（α₂）.Fraction S-1 also consisted of two different fluorescent subunits with molecular weight of 20.1 kDa（β₁） and 18 kDa （β₂） and differed from each other on isoelectric points of pH 5.7（ft） and 6.0（ft）,respectively.Further investigation of peptide sequence will help a lot in elucidating the new subunit ft that was smaller in size and more neutral than the known ft subunit,and may provide an alternative explanation in structure of cryptophytic phycobiliproteins.     

THE SUBUNIT STRUCTURE OF YEAST DNA PHOTOLYASE AND THE PURIFICATION OF A FLUORESCENT ACTIVATOR OF THE ENZYME

Abstract— Yeast DNA photolyase purified twice by affinity chromatography was analyzed by electrophoresis on polyacrylamide gradient gels or by sedimentation velocity through 5–200/, sucrose gradients containing 0.4MKC1. Its molecular weight estimated by both these methods was 130 ,000 and 136 ,000, respectively. However, the enzyme dissociated into two bands having molecular weights of 60 ,000 and 85 ,000 when it was examined by electrophoresis on SDS polyacrylamide gradient gels. The subunit structure of the enzyme was confirmed when two absorption maxima corresponding to polypeptides of 54 ,000 and 82 ,500 daltons were observed in sucrose gradients run in 1.0 M KCI. Upon mixing these two fractions, a time-dependent increase in activity occurred, demonstrating that active enzyme could be reconstituted from these subunits.
The activity of photolyase purified by affinity chromatography is enhanced by a compound (activator III) obtained from yeast by acidification, neutralization, ion exchange chromatography and gel filtration. Activator III emits at 350 and 440 nm when excited at 290 nm, and emits at 440 nm when excited at 358 nm. After acid hydrolysis, emission at 440 nm is produced only by excitation at 358 nm, indicating that it contains two separate chromophoric moieties. The chromophore excited by 358 nm light has a pK of 9–11, while the other has a pK of 4–5. Enhancement of photolyase activity by activator III at a concentration equimolar with that of the enzyme and the similarity of the fluorescent spectra of the activator and heat-denatured photolyase suggest that the activator may be the chromophore associated with the enzyme.     

过渡金属离子(Cu2+、Cd2+)对藻红蛋白单体的漂白机制

藻胆蛋白是一组同源捕光色素蛋白,由多肽链及相连的开链四吡咯环色团构成。多肽链对色团的调控形成不同藻胆蛋白的特征吸收及荧光光谱。这些色团是藻蓝胆素(吸收带590～670 nm)、藻红胆素(吸收带530～570 nm)和藻尿胆素(吸收带490～500nm)。藻尿胆素由于在共轭体系中含有较少的双键,所以与前二者比,在较短波长有吸收。它们总是与藻红胆素伴生,在藻胆蛋白中作为敏化基团存在。     

SPECTRAL PROPERTIES OF PHYCOBILISOMES AND PHYCOBILIPROTEINS FROM THE BLUE-GREEN ALGA-NOSTOC SP.*

Abstract— An improved method for phycobilisome isolation from a blue-green alga Nostoc sp. was developed using 1% Triton X-100. The phycobilisome preparations showed little fragmentation and had structures similar in size to those observed in thin sections of the organism. Phycobiliproteins isolated from phycobilisomes and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, had subunits with the following molecular weights: phycoerythrin (PE), 20,000 and 16,900; phycocyanin (PC), 14,700 and 16,300; and allophycocyanin (APC), 14,000. Isoelectric focusing of each phycobiliprotein resulted in major bands isoelectric at the following pH values: PE, 4.43, 4.45; PC 4.32; APC, 4.38. Absorption spectra at -196°c showed maxima at 551 and 566 nm for PE; 598 and 631 nm for PC; and 590, 600, 629 and 650 nm for APC. Concentrated vs dilute difference spectra of phycobiliproteins showed increased absorption at 574 nm (PE), 630 nm (PC) and 651 nm (APC) suggesting that spectral changes resulted from aggregation. Fluorescence analysis of each phycobiliprotein and of intact phycobilisome preparations showed that energy absorbed by phycoerythrin is transferred to allophycocyanin, possibly by a resonance transfer mechanism. These observations support a model where allophycocyanin forms the base of the phycobilisome which is attached to the photosynthetic membrane. The next layer is assumed to be phycocyanin, which in turn is followed by a phycoerythrin layer that is the outermost layer (on the stroma side) of the phycobilisome.     

Effects of PAR and UV Radiation on the Structural and Functional Integrity of Phycocyanin,Phycoerythrin and Allophycocyanin Isolated from the Marine Cyanobacterium Lyngbya sp. A09DM

An in vitro analysis of the effects of photosynthetically active and ultraviolet radiations was executed to assess the photostability of biologically relevant pigments phycocyanin (PC), phycoerythrin (PE) and allophycocyanin (APC) isolated from Lyngbya sp. A09DM. Ultraviolet (UV) irradiances significantly affected the integrity of PC, PE and APC; however, PAR showed least effect. UV radiation affected the bilin chromophores covalently attached to phycobiliproteins (PBPs). Almost complete elimination of the chromophore bands associated with α‐ and β‐subunit of PE and APC occurred after 4 h of UV‐B exposure. After 5 h of UV‐B exposure, the content of PC, PE and APC decreased by 51.65%, 96.8% and 96.53%, respectively. Contrary to PAR and UV‐A radiation, a severe decrease in fluorescence of all PBPs was observed under UV‐B irradiation. The fluorescence activity of extracted PBP was gradually inhibited immediately after 15–30 min of UV‐B exposure. In comparison to the PC, the fluorescence properties of PE and APC were severely lost under UV‐B radiation. Moreover, the present study indicates that UV‐B radiation can damage the structural and functional integrity of phycobiliproteins leading to the loss of their ecological and biological functions.     

EXCITATION ENERGY MIGRATION IN PHYCOBILISOMES: COMPARISON OF EXPERIMENTAL RESULTS AND THEORETICAL PREDICTIONS

Abstract— Quantum yield and fluorescence polarization determinations on phycobilisomes and their constituent phycobiliproteins show that phycobilisomes are energetically effective macromolecular structures. Energy migration within the phycobilisome to allophycocyanin, the longest wavelength absorbing and emitting phycobiliprotein, was indicated by the predominant allophycocyanin fluorescence emission which was independent of the phycobiliprotein being excited. The high efficiency of the energy migration inside the phycobilisome was reflected by the low polarized fluorescence. Excitation of phycobilisomes in the region of major absorption (500–650 nm) resulted in degrees of fluorescence polarization between +0.02 and –0.02, whereas in isolated phycobiliproteins the values were 2 to 12 times greater. Furthermore, 94–98° of the excitation energy of phycoerythrin was transferred to phycocyanin and allophycocyanin as determined from comparisons of fluorescence spectra of intact and dissociated phycobilisomes. The fluorescence quantum yields of phycobilisomes were about 0.60–0.68, very similar to that of pure allophycocyanin in solution (0.68). Phycobilisomes isolated from Fremyella diplosiphon and Nostoc sp. (blue-gree algae) have respective quantum yields of 0.68 and 0. 65, and those isolated from Porphyridium cruentum (red alga), about 0.60. In Fremyella diplosiphon and Nostoc sp., which showed a striking adaptation to different wavelengths, the phycobilisome quantum yields only varied from 0.68 to 0.67 and from 0.65 to 0. 60, respectively. The mean transfer time, calculated on the basis of experimental results, was about 280 ± 40 ps for transfer of excitation from the phycoerythrin to the phycocyanin layer in phycobilisomes. This time corresponds to the mean number of jumps, about 28, of the excitation in the phycoerythrin layer before it is captured by phycocyanin. These values are in reasonable agreement with the values of 250 ± 30 ps and 25 jumps, calculated on the basis of a phycobilisome model (of Porphyridium cruentum) and Pearlstein's theory of energy migration devised for a three-dimensional photosynthetic unit. It was also shown that Paillotin's theory of energy migration predicts similar values for mean transfer time and mean number of jumps, if one assumes that phycocyanin is a perfect sink for phycoerythrin excitation.     

The study for isolation and purification of R-phycoerythrin from a red alga

An effective procedure for the rapid extraction and purification of the biliprotein R-phycoerythrin from a red alga,Ceramium isogonum, was developed. The purified R-phycoerythrin ofC. isogonum consisted of three components with mol wt 180,000 (6β subunits), 70,000 (6α subunits), and 30,400 (γ subunit), respectively. The phycoerythrin is suitable for use as a natural food coloring and can also be used as a fluorescent label.     

A new way to understand quaternary structure changes of hemoglobin upon ligand binding on the basis of UV-resonance Raman evaluation of intersubunit interactions

The single residue vibrational spectra of tryptophan (Trp) and tyrosine (Tyr) residues in human adult hemoglobin (HbA), which play important roles in cooperative oxygen binding, were determined for the deoxy and CO-bound forms by applying UV resonance Raman spectroscopy to various variant Hbs. It was found that Trpβ37, Tyrα42, Tyrα140, and Tyrβ145 at the α(1)-β(2) subunit interface underwent transitions between two contact states (named as T and R) upon ligand binding, while Trpα14, Trpβ15, and Tyrβ35 displayed little changes. The corresponding spectral changes were identified only for the α(2)β(2) tetramer, but not the isolated α and β chains in the oligomeric forms, and therefore were exclusively attributed to a quaternary structure change. Ligand binding as well as allosteric effectors and pH altered only the number of the T-contacted Tyr and Trp residues without varying the two contact states themselves. A new method to semiquantitatively evaluate the amount of T-contacted Tyr and Trp residues in a given liganded form is here proposed, and with it a quaternary structure was determined for various symmetrically half-liganded forms obtained with ligand-hybrid, metal-hybrid, and valency-hybrid Hbs. It was found that ligand binding to the α or β subunits yielded different subunit contacts and that the contact changes of the Trp and Tyr residues were not always concerted. The contact changes at the α(1)-β(2) (α(2)-β(1)) interface are correlated with the proximal strain exerted on the Fe-His(F8) bond, which is noted to be much larger in the α than β subunits in the α(2)β(2) tetramer.     

Isolation and purification of food‐grade C‐phycocyanin from Arthrospira platensis and its determination in confectionery by HPLC with diode array detection

C‐Phycocyanin is the major phycobiliprotein in Arthrospira platensis, also known as Spirulina, which is a cyanobacterium used as a dietary supplement because of its powerful effects on body and brain. C‐phycocyanin is a blue‐colored accessory photosynthetic pigment with multiple applications in food industry as natural dye or additive, and in pharmaceuticals. This study presents a simple protocol for the extraction and purification of food‐grade C‐phycocyanin from Arthrospira platensis. The cell lysis of cyanobacterium was performed by sonication combined with repeated freezing and thawing cycles. The purification of the crude extract of C‐phycocyanin was carried out by ammonium sulfate precipitation followed by ion exchange chromatography resulting in 2.5 purity. The purity of phycocyanobilin chromophore has been tested by UV‐visible spectrophotometry by monitoring the absorption after each stage of purification. A high‐performance liquid chromatography method has been developed and validated for the determination of food‐grade C‐phycocyanin. Intra‐ and interday precision values less than 5.6% and recovery greater than 91.2% indicated high precision and accuracy of the method for analysis of C‐phycocyanin. The method has been applied to commercial confectionery of blue color and to the purified protein obtained in the first stage of the study.     

Ion-exchange high-performance liquid chromatographic purification of bovine cardiac and rabbit skeletal muscle troponin subunits

Bovine cardiac and rabbit skeletal troponin complexes were separated into their respective subunits employing high-performance liquid chromatographic (HPLC) techniques on CM-300 and Q-300 ion-exchangers. Bovine cardiac and rabbit skeletal subunits were separated on the strong anion-exchanger, Q-300, in 8 M urea, 50 mM Tris, 2 mM EGTA, 0.5 mM dithiothreitol, pH 7.5, employing a linear salt gradient and on the weak cation-exchanger, CM-300, in 8 M urea, 50 mM potassium dihydrogen phosphate, 2 mM EGTA, 0.5 mM dithiothreitol, pH 6.5, using a linear salt gradient. To obtain complete purification of all components of troponin both ion-exchangers were required. The initial separation of troponin was carried out on the strong anion-exchanger followed by weak cation-exchange chromatography of the troponin I collected from the strong anion-exchange column. The troponin T subunits obtained from Q-300 chromatography demonstrated heterogeneity (three components: T1, T2 and T3) while the troponin I collected from both sources on the Q-300 column were both resolved into major doublets (I1 and I2) when rechromatographed on the CM-300 column. The three troponin T fractions and two troponin I fractions isolated from ion-exchange HPLC were examined by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis to confirm that the heterogeneity was due to differences in charge and not molecular weight. These results were in agreement with the charge differences observed from retention times on ion-exchange HPLC. When comparing the same troponin subunit from different muscle sources, considerable differences in the content of charged amino acid residues were also observed.     

Polymorphic structure and physical properties of the polyester/ether poly(ethylene-1,2-diphenoxyethane-p,p′ -dicarboxylate)

X-ray diffraction studies of fibers of the polyester/ether poly(ethylene-1,2-diphenoxyethane-p,p′ -dicarboxylate) (PEET) produced by high-speed melt spinning show the existence of two polymorphic forms, designated α and β, in the solid state. The α form is obtained by annealing filaments melt spun at takeup speeds below 3000 m/min and is also found in samples crystallized from the melt and from dilute solutions. The α form has a monoclinic unit cell with dimensions a = 7.83, b = 10.33, c = 18.68 Å, and β = 83.1°. The equilibrium melting temperature and heat of fusion of the α form are 288.3°C and 19.1 cal/g, respectively. The β form predominates in highly oriented filaments obtained at takeup velocities above 6000 m/min. The unit cell is orthorhombic with dimensions a = 7.28, b = 5.65, and c = 18.64 Å. The β form does not transform to the α form on annealing.     

Structural fluctuation and concerted motions in F1‐ATPase: A molecular dynamics study

F₁‐ATPase is an adenosine tri‐phosphate (ATP)‐driven rotary motor enzyme. We investigated the structural fluctuations and concerted motions of subunits in F₁‐ATPase using molecular dynamics (MD) simulations. An MD simulation for the α₃β₃γ complex was carried out for 30 ns. Although large fluctuations of the N‐terminal domain observed in simulations of the isolated β_E subunit were suppressed in the complex simulation, the magnitude of fluctuations in the C‐terminal domain was clearly different among the three β subunits (β_E, β_TP, and β_DP). Despite fairly similar conformations of the β_TP and β_DP subunits, the β_DP subunit exhibits smaller fluctuations in the C‐terminal domain than the β_TP subunit due to their dissimilar interface configurations. Compared with the β_TP subunit, the β_DP subunit stably interacts with both the adjacent α_DP and α_E subunits. This sandwiched configuration in the β_DP subunit leads to strongly correlated motions between the β_DP and adjacent α subunits. The β_DP subunit exhibits an extensive network of highly correlated motions with bound ATP and the γ subunit, as well as with the adjacent α subunits, suggesting that the structural changes occurring in the catalytically active β_DP subunit can effectively induce movements of the γ subunit. © 2010 Wiley Periodicals, Inc. J Comput Chem 2010     

Comparative Analysis of Oxy-Hemoglobin and Aquomet-Hemoglobin by Hydrogen/Deuterium Exchange Mass Spectrometry

The function of hemoglobin (Hb) as oxygen transporter is mediated by reversible O₂ binding to Fe(2+) heme in each of the α and β subunits. X-ray crystallography revealed different subunit arrangements in oxy-Hb and deoxy-Hb. The deoxy state is stabilized by additional contacts, causing a rigidification that results in strong protection against hydrogen/deuterium exchange (HDX). Aquomet-Hb is a dysfunctional degradation product with four water-bound Fe(3+) centers. Heme release from aquomet-Hb is relatively facile, triggering oxidative damage of membrane lipids. Aquomet-Hb crystallizes in virtually the same conformation as oxy-Hb. Hence, it is commonly implied that the solution-phase properties of aquomet-Hb should resemble those of the oxy state. This work compares the structural dynamics of oxy-Hb and aquomet-Hb by HDX mass spectrometry (MS). It is found that the aquomet state exhibits a solution-phase structure that is significantly more dynamic, as manifested by elevated HDX levels. These enhanced dynamics affect the aquomet α and β subunits in a different fashion. The latter undergoes global destabilization, whereas the former shows elevated HDX levels only in the heme binding region. It is proposed that these enhanced dynamics play a role in facilitating heme release from aquomet-Hb. Our findings should be of particular interest to the MS community because oxy-Hb and aquomet-Hb serve as widely used test analytes for probing the relationship between biomolecular structure in solution and in the gas phase. We are not aware of any prior comparative HDX/MS experiments on oxy-Hb and aquomet-Hb.

BILIPROTEINS FROM THE BUTTERFLY Pieris brassicae STUDIED BY TIME-RESOLVED FLUORESCENCE AND COHERENT ANTI-STOKES RAMAN SPECTROSCOPY

Abstract— The fluorescence decay time of the biliverdin IX7 chromophore present in biliproteins isolated from Pieris brassicae is determined to be 44 ± 3 ps. This value suggests a cyclic helical chromophore structure. The vibrational frequencies determined by CARS-spectroscopy are compared with those of model compounds. The data confirm that the chromophore in the protein-bound state adopts a cyclic-helical, flexible conformation.     

Phycobiliproteins encapsulated in sol-gel glass

Light transducing phycobiliproteins are encapsulated in optically transparent sol-gel matrices. Absorption and fluorescence spectroscopies are used to characterize the effect of the sol-gel encapsulation on the conformation and aggregation states of the three major phycobiliproteins present in phycobilisomes: phycoerythrin, phycocyanin, and allophycocyanin. It is found that the effects of sol-gel entrapment on the spectroscopic properties are significantly different for the three phycobiliproteins. The results indicate that phycoerythrin undergoes only minor change in its native structure when entrapped in sol-gel. However, significant changes in conformation and aggregation state occur when phycocyanin and allophycocyanin are entrapped in sol-gel matrices. A thin film of sol-gel encapsulated phycoerythrin is also coated on an optical fiber surface and strong fluorescence from the evanescent wave excitation is detected. The potential applications of sol-gel encapsulated phycobiliproteins in biosensors are discussed.