Quantitative analysis of protein mixtures by second derivative absorption spectroscopy

A new method for the quantitative analysis of protein mixtures based on multicomponent analysis of the second derivative near uv spectra is described. Using bovine eye lens crystallins, we demonstrate that the technique can provide precise concentrations of closely related proteins within mixtures, under both native and denaturing conditions. We have also successfully used the method to analyze the subunit composition of a heteromultimeric protein aggregate. The method is more rapid and precise than alternative approaches and offers the advantage of substantially reduced interference from many extraneous solution components and light scattering. It is also nondestructive and extremely sensitive, requiring only small volumes of sample at low total protein concentrations. Prospective applications are proposed for the study of eye lens crystallins, as well as for other protein/protein and protein/nonprotein mixtures.     

Differences between Fragaria clones by fourth derivative room temperature spectroscopy of intact leaves

Clones of Fragaria were characterized by the attenuance (absorption) spectra of their photosynthetic apparatus. This method uses fourth derivative analysis of spectra of intact lamina of detached leaves at room temperature (20–25 C). Twenty-one different species and hybrids of Fragaria were examined. F. chiloensis accessions frequently have characteristic fourth derivative spectra distinct from that of other Fragaria species. These spectra are characterized by a strong band that appears to be photosystem I component Ca 693; and is correlated with a higher chlorophyll b content per unit area of leaf, and a lower chlorophyll a/b ratio.Abbreviations Chl chlorophyll - chiloensis Fragaria chiloensis - 4th deriv fourth derivative - PSI photosystem I - PSH photosystem II     

Low spin quantitation of NiFeC EPR signal from carbon monoxide dehydrogenase is not due to damage incurred during protein purification

Evidence is presented that the O₂-sensitive, nickel- and iron-containing enzyme carbon monoxide dehydrogenase from Clostridium thermoaceticum was purified without significantly inactivating either its CO oxidation or CO/acetyl-CoA exchange activities. All CO oxidation activity from the crude extract was recovered in the purified enzyme (and side fractions). The exchange activity could not be quantified similarly, because the crude extract and early purification step fractions exhibited little or no exchange activity. Later purification fractions exhibited much more exchange activity, suggesting that an inhibitor was present in the impure fractions. The NiFeC EPR signal intensity was used as an indicator of the enzyme's capacity to catalyze exchange. This signal was extremely sensitive to oxygen; exposure to as little as 0.5 equiv/mol enzyme dimer resulted in substantial loss of intensity. The NiFeC intensities at each step in the purification were virtually invariant, indicating that the enzyme had not been exposed to oxygen and had not been inactivated towards catalyzing exchange. The ability to purify carbon monoxide dehydrogenase (CODH) without inactivating nearly any of the molecules suggests that it is quite stable under anaerobic conditions. The purified enzyme, which could not have lost functional metal ions during purification, contained 1.9 Ni and 11.3 Fe, similar to previous reports. The NiFeC EPR signal intensity from each purification fraction (0.2 spins/mol enzyme dimer) was as low as from previous preparations, indicating that its low spin quantitation is not the result of damage incurred during purification. If the low intensity arises from heterogeneity as proposed earlier, the heterogeneity must originate prior to purification.     

Immunoaffinity purification and quantitation of a rat protein similar to the human decidua-associated protein (hDP) 71

The role of human decidua-associated protein (hDP) 71, copurified consistently with hDP 200, identified as rheumatoid factor, remains undetermined. The possibility of using a rat as an experimental model for the further research of hDp 71, was examined. A rat protein, similar to the hDp 71, was immunoaffinity purified using the same monoclonal antibody recognizing hDp 71. The protein was named rat decidua- associated protein (rDP) 71. The level of hDp 71 in extracts of endometrial epithelium and stroma, as well as in uterine washings, was measured throughout the oestrous cycle and on 5 consecutive days, starting the day after the rats mated. Moreover, the effect of oestrogen and progesterone on the level of hDp 71 was examined. The results demonstrate the oestrogen-dependent accumulation of rDP 71 in uterine lumen, and support the use of a rat as an experimental animal model to investigate the possible physiological role of this protein in the reproductive process.     

Preparative high-performance liquid chromatography purification of polyunsaturated phospholipids and characterization using ultraviolet derivative spectroscopy

A preparative reversed-phase HPLC system utilizing an isocratic mobile phase to purify up to 10-mg quantities of phospholipids is described. The method was developed to separate oxidation products of polyunsaturated phospholipids from intact, parent lipids. The method is useful for phosphatidylcholine and phosphatidylethanolamine on a preparative scale and for phosphatidylserine and phosphatidic acid on an analytical scale. Both intact phospholipids and oxidized phospholipids were monitored by absorbance at 206 nm. The oxidation products were simultaneously monitored at 234 nm where the intact phospholipids have only a very slight end absorption. Second-derivative uv spectroscopy proved to be extremely useful to identify the presence or to verify the absence of oxidation products in phospholipid samples. For autoxidized docosahexaenoic acid containing phospholipids, the absorbance maximum of diene oxidation products is 237 nm for the trans,trans (t,t) isomer and 246 nm for the cis,trans (c,t) isomers. Similarly, five classes of triene oxidation product stereoisomers have distinct absorbance maxima detected by second-derivative spectroscopy ranging from 269 to 292 nm.     

Temperature derivative fluorescence spectroscopy as a tool to study dynamical changes in protein crystals

Motions through the energy landscape of proteins lead to biological function. At temperatures below a dynamical transition (150-250 K), some of these motions are arrested and the activity of some proteins ceases. Here, we introduce the technique of temperature-derivative fluorescence microspectrophotometry to investigate the dynamical behavior of single protein crystals. The observation of glass transitions in thin films of water/glycerol mixtures allowed us to demonstrate the potential of the technique. Then, protein crystals were investigated, after soaking the samples in a small amount of fluorescein. If the fluorophore resides within the crystal channels, temperature-dependent changes in solvent dynamics can be monitored. Alternatively, if the fluorophore binds to the protein, local dynamical transitions within the biomolecule can be probed directly. A clear dynamical transition was observed at 175 K in the active site of crystalline human butyrylcholinesterase. The results suggest that the dynamics of crystalline proteins is strongly dependent on solvent composition and confinement in the crystal channels. Beyond applications in the field of kinetic crystallography, the highly sensitive temperature-derivative fluorescence microspectrophotometry technique opens the way to many studies on the dynamics of biological nanosamples.     

High-order derivative spectroscopy of infrared absorption spectra of the reaction centers from Rhodobacter sphaeroides

The infrared absorption spectra of reduced and chemically oxidized reaction center preparations from the purple bacterium Rhodobacter sphaeroides were investigated by means of high-order derivative spectroscopy. The model Gaussian band with a maximum at 810 nm and a half-band of 15 nm found in the absorption spectrum of the reduced reaction center preparation is eliminated after the oxidation of photoactive bacteriochlorophyll dimer (P). This band was related to the absorption of the P(+)y excitonic band of P. On the basis of experimental results, it was concluded that the bleaching of the P(+)y absorption band at 810 nm in the oxidized reaction center preparations gives the main contribution to the blue shift of the 800 nm absorption band of Rb. sphaeroides reaction centers.     

Time-resolved evanescent wave absorption spectroscopy for real-time monitoring of heme protein adsorption to glass

Evanescent wave has been recognized as a highly sensitive optical probe for surface monitoring. By use of slab optical waveguides, time-resolved evanescent wave absorption spectroscopy was developed for the investigation of the interfacial behavior of biomolecules with a chromophore. In this study, 30-microm thick glass sheets served as freestanding multimode waveguides that were combined with a simple fiber-coupling method to lead to a broadband evanescent wave absorption spectrometer. With such a homemade instrument, adsorption of heme proteins onto glass slides from static aqueous solution was monitored in situ. The experimental results reveal that the interfacial behavior of myoglobin (Mb) is different from that of hemoglobin (Hb) and cytochrome c (Cc). Formation of dynamic equilibrium for Mb adsorption at both hydrophilic and hydrophobic surfaces always is behind the occurrence of the maximum coverage. However, simultaneous formation of the dynamic equilibrium and the maximum coverage was observed for Hb and Cc adsorptions.     

Application of vesicle chromatography in protein purification

A recently described vesicular packing material (VP) representing clusters of microcapsules (derived from plant cells) was tested with respect to its application for protein purification. Protein elution behaviour was investigated with 28 defined proteins and several protein containing preparations and biological fluids. All proteins were eluted with a neutral buffer without retardation as peaks in the permeable or excluded fraction. Due to its sharp separation limits VP can be used for the separation of proteins with small differences in size. In special cases, proteins of nearly equal molecular weight (e.g. carboxypeptidase A and pepsin) may be separated due to differences in the electrical charge of the protein molecules and resulting differences in the electric in the electrical charge of the protein molecules and resulting differnces in the electric interaction with the negatively charged polygalacturonan matrix of the vesicle membrane (cell wall). Vesicle chromatography is a biocompatible process. The VP may be applied on a large scale. Complete separation between excluded and permeable proteins may be reached if the columns are loaded with concentrated protein samples (e.g., blood plasma). Size fractionation by the VP seems to be applicable in the following fields:

• 1 Preparative separation of an excluded protein from an excess of permeable macromolecules, especially if the difference in STOKES ' diameter is too small for an effective separation by gel chromatography or conventional membrane techniques.
• 2 Preparative separation of permeable proteins from an excess of excluded proteins.
• 3 Chromatography of proteins in the presence of alcohol, polyethylene glycol or detergents.

     

Standardization and validation of a new atomic absorption spectroscopy technique for determination and quantitation of Aluminium adjuvant in immunobiologicals

In the present study, Aluminium quantification in immunobiologicals has been described using atomic absorption spectroscopy (AAS) technique. The assay was found to be linear in 25-125 microg/ml Aluminium range. The procedure was found to be accurate for different vaccines with recoveries of external additions ranging between 93.26 and 103.41%. The mean Limit of Variation (L.V.) for both intra- and inter-assay precision was calculated to be 1.62 and 2.22%, respectively. Further the procedure was found to be robust in relation to digestion temperature, alteration in acid (HNO(3) and H(2)SO(4)) ratio used for sample digestion and storage of digested vaccine samples up to a period of 15 days. After validation, AAS method was compared for its equivalency with routinely used complexometric titration method. On simultaneously applying on seven different groups of both bacterial and viral vaccines, viz., DPT, DT, TT, Hepatitis-A and B, Antirabies vaccine (cell culture) and tetravalent DPT-Hib, a high degree of positive correlation (+0.85-0.998) among AAS and titration methods was observed. Further AAS method was found to have an edge over complexometric titration method that a group of vaccines, viz., ARV (cell culture, adsorbed) and Hepatitis-A, in which Aluminium estimation is not feasible by pharmacopoeial approved complexometric titration method (possibly due to some interference in the sample matrix), this newly described and validated AAS assay procedure delivered accurate and reproducible results.     

Toward the fourth dimension of membrane protein structure: insight into dynamics from spin-labeling EPR spectroscopy

Trapping membrane proteins in the confines of a crystal lattice obscures dynamic modes essential for interconversion between multiple conformations in the functional cycle. Moreover, lattice forces could conspire with detergent solubilization to stabilize a minor conformer in an ensemble thus confounding mechanistic interpretation. Spin labeling in conjunction with electron paramagnetic resonance (EPR) spectroscopy offers an exquisite window into membrane protein dynamics in the native-like environment of a lipid bilayer. Systematic application of spin labeling and EPR identifies sequence-specific secondary structures, defines their topology and their packing in the tertiary fold. Long range distance measurements (60 ?-80 ?) between pairs of spin labels enable quantitative analysis of equilibrium dynamics and triggered conformational changes. This review highlights the contribution of spin labeling to bridging structure and mechanism. Efforts to develop methods for determining structures from EPR restraints and to increase sensitivity and throughput promise to expand spin labeling applications in membrane protein structural biology.     

内含肽在构建亲和纯化系统中的应用

内含肽是前体未成熟蛋白中的一段具有自我剪接功能的多肽链,在蛋白质纯化、蛋白质连接、环肽制备、蛋白标记以及生物传感器等方面广泛应用。本文综述了内含肽应用于蛋白质亲和纯化的发展历程,分别对层析型和非层析型内含肽纯化体系进行了分析和讨论,并总结了对控制内含肽断裂反应所进行的研究,为进一步改善内含肽介导蛋白质纯化提供依据和线索。     

Detection, quantitation, purification, and identification of cardiac proteins S-thiolated during ischemia and reperfusion.

We have developed methods that allow detection, quantitation, purification, and identification of cardiac proteins S-thiolated during ischemia and reperfusion. Cysteine was biotinylated and loaded into isolated rat hearts. During oxidative stress, biotin-cysteine forms a disulfide bond with reactive protein cysteines, and these can be detected by probing Western blots with streptavidin-horseradish peroxidase. S-Thiolated proteins were purified using streptavidin-agarose. Thus, we demonstrated that reperfusion and diamide treatment increased S-thiolation of a number of cardiac proteins by 3- and 10-fold, respectively. Dithiothreitol treatment of homogenates fully abolished the signals detected. Fractionation studies indicated that the modified proteins are located within the cytosol, membrane, and myofilament/cytoskeletal compartments of the cardiac cells. This shows that biotin-cysteine gains rapid and efficient intracellular access and acts as a probe for reactive protein cysteines in all cellular locations. Using Western blotting of affinity-purified proteins we identified actin, glyceraldehyde-3-phosphate dehydrogenase, HSP27, protein-tyrosine phosphatase 1B, protein kinase Calpha, and the small G-protein ras as substrates for S-thiolation during reperfusion of the ischemic rat heart. MALDI-TOF mass fingerprint analysis of tryptic peptides independently confirmed actin and glyceraldehyde-3-phosphate dehydrogenase S-thiolation during reperfusion. This approach has also shown that triosephosphate isomerase, aconitate hydratase, M-protein, nucleoside diphosphate kinase B, and myoglobin are S-thiolated during post-ischemic reperfusion.     

The purification and quantitation of myosin from cultured cells.

Myosin has been purified from the following cultured cell lines: normal rat kidney fibroblast (NRK), HeLa-Rhino (HeLa), human choriocarcinoma, human acute lymphoblastic leukemia, rat hepatoma (HTC), monkey kidney (VERO), pigmented mouse melanoma, Y-1 rat adrenal cortex, and growth hormone-secreting GH-1. Myosin constitutes 0.5-5.4% of the protein of these cells. It was not detected in washed human erythrocytes or in two types of mouse plasmacytoma cells. Two methods for the purification of myosin from cultured cells have been employed. With Method I highly purified myosin was prepared by Sepharose 4B and DEAE-cellulose chromatography from 10(10) L-929 cells as well as from mouse uterus. Those myosins have similar molecular and subunit weights as well as ATPase activity but are immunologically distinct. Method II involving ultracentrifugation and Sepharose 4B chromatography, is suitable for the production of moderately pure myosin in good yield from as few as 5-10(7) cells (five 100-mm Petrie dishes).     

Microwave absorption spectroscopy of DNA

By utilizing a novel approach to microwave spectrometry, we have measured the absolute absorption spectrum of plasmid DNA (pUC8.c2), in buffered aqueous solution, from 5 to 20 GHz. Our technique does not suffer from the same experimental difficulties that plague other methods. We observe no absorption resonances in this frequency range, but we do see broadband differences, between DNA and pure buffer, that are attributable to changes in the ionic conductivity of the solutions. These results constitute the first verification, by a totally different technique, of the absence of resonances in the microwave absorption spectrum of DNA, and the first data obtained by any technique in the 10–20-GHz band. © 1993 John Wiley & Sons, Inc.     

Application of Fourier transform infrared spectroscopy to studies of aqueous protein solutions.

Modern protein Fourier transform infrared (FT-IR) spectroscopy has proven to be a versatile and sensitive technique, applicable to many aspects of protein characterization. The major practical drawback for the FT-IR spectroscopy of proteins is the large absorbance band of water, which overlaps the amide I resonances. D2O is often substituted for H2O in infrared experiments. Removal of water from protein samples can be complicated and tedious and potentially lead to denaturation, aggregation, or sample loss. Solvent removal by dialysis is difficult for suspensions and sols. A new method called the D2O dilution technique (Ddt) is described which simplifies the sample preparation step and improves the solvent subtraction. The effect of the D2O concentration on the IR spectrum of aqueous solutions of several model proteins was studied. Dilution of aqueous samples with D2O yields good quality spectra. The Ddt has been evaluated for quantitative analysis using standard proteins and its applicability to solutions and suspensions of a genetically engineered malaria antigen is demonstrated. Use of resolution-enhancement techniques with spectra in mixed solvents has also been investigated.     

Application of high-performance liquid chromatographic peptide purification to protein microsequencing by solid-phase Edman degradation

Peptide purification via high-performance liquid chromatography (HPLC) and solid-phase sequencing were integrated to form a system allowing the determination of complete sequence information on a microscale without the use of radiolabels or modified phenylisothiocyanate. Mixtures of peptides (500 pmol to 10 nmol) resulting from proteolytic digestion or chemical cleavage were applied directly to reverse-phase columns. The columns, equilibrated in either 10 mm KP_i or 0.05% trifluoroacetic acid, were then developed using acetonitrile gradients. Eluates were monitored nondestructively by direct ultraviolet detection at both 214 and 254 nm. Each peak was collected as a discrete fraction, and purity was assessed by amino acid analysis prior to covalent attachment to a solid support for sequence analysis. Activation of the peptide carboxyl terminus via a water soluble carbonyldiimide was the solid-phase coupling method used 90% of the time. Coupling yields averaged 52% of starting material. Sequence analysis was performed in the range 100 pmol to 4 nmol of coupled peptide. Phenylthiohydantoin-amino acids were identified by reverse-phase HPLC using ultraviolet detection.     

Use of surface enhanced infrared absorption spectroscopy (SEIRA) to probe the functionality of a protein monolayer

We present a novel infrared method to investigate the functionality of a protein monolayer tethered to a metal substrate. The approach employs Surface Enhanced Infrared Absorption Spectroscopy (SEIRAS), which renders high surface sensitivity by enhancing the signal of the adsorbed protein by up to approximately 2 orders of magnitude. We demonstrate that the electrochemically induced absorption changes of a cytochrome c monolayer can be observed with excellent signal-to-noise ratio when the protein is adhered to a modified gold surface. To probe membrane proteins, a concept is introduced for the oriented incorporation into solid supported lipid bilayers. Recombinant cytochrome c oxidase solubilized in detergent is immobilized on a chemically modified gold surface via the affinity of its histidine (His)-tag to a nickel-chelating nitro-triacetic acid (NTA) surface. The protein monolayer is reconstituted into the lipid environment by detergent removal. Changing the orientation of the protein with respect to the metal surface is achieved by inserting the His-tag on either side of the membrane protein surface. Orientational control is mandatory for experiments in which electrons are injected from the electrode into the protein. The presented methodology opens new avenues to study the mechanism of the biomedically relevant class of electron and voltage-gated proteins on the atomic level.     

Study of different types of chlorophyll a aggregates in solutions and films by absorption and luminescence derivative spectroscopy]

The second derivative of absorption, fluorescence and fluorescence excitation spectra of chlorophyll a in concentrated solutions and films was investigated. More than 14 forms of pigment aggregates, which can be divided into two types--with narrow 8-10nm) and wide (25-40nm) low temperature (-196 degrees C) spectra bands, were found. For the most part of the aggregated forms, the position and half width of the bands, as well as the Stokes shift and relative quantum yield were determined. The comparison of the spectral characteristics points to the indentity of the aggregates and corresponding native forms of Chl. a. It is shown that the universal relationship between absorption and fluorescence bands in applicable to the aggregates of the two types and the energy of resonance interaction between monomers in the aggregates is evaluated.