CHARACTERIZATION OF PROTEINS DURING AGGREGATION USING TURBIDIMETRY

The dissolution process for hydrophobic proteins as well as protein denaturation-renaturation reactions often lead to the formation of aggregates. The characterization of the aggregates in terms of sire and the characterization of the physico-chemical changes undergone by the proteins are key to the elucidation of the kinetics of both, the aggregation processes and the denaturation-renaturation reactions. The aggregation processes and the denaturation-renaturation reactions are amenable to characterization through spectroscopy techniques. This paper reports on a technique for the estimation of the particle size and the absorption coefficients of proteins in solution and in protein aggregates present during denaturation-renaluration reactions. The technique reported is based on the application of Mie theory to turbidity measurements of polydisperse particulate systems. Mie theory is applied to both, the region where the particles are non-absorbing and, to the spectral region where the protein intrinsic chromophores absorb. This technique yields estimates of the scattering corrected absorption coefficients and of the relative refractive index of the aggregates. The results obtained indicate that subsequent application of spectral deconvolution techniques can yield quantitative information on the spectral changes undergone by the intrinsic protein chromophores upon denaturation or renaturation     

An Interpretation Model For The UV-VIS Spectra Of Microorganisms

Multiwavelength UV-vis spectra of microorganisms and cell suspensions contain quantitative information on their properties such as number, size, shape, chemical composition, and internal structure. These properties are essential for the identification and classification of cells. The complexity of microorganisms in terms of their chemical composition and internal structure makes the interpretation of their spectral signature a difficult task. In this article a model is proposed for the interpretation of the multiwavelength spectra of microorganisms. The proposed interpretation model is based on light scattering theory, spectral deconvolution techniques, and the approximation of the frequency-dependent optical properties of the basic constituents of living organisms. The optical properties as functions of wavelength and available literature data on the size and chemical composition of E. coli cells and Bacillus globigii spores were used to explore the sensitivity of the calculated spectra to the model parameters. It is shown that the proposed model can reproduce the features of experimentally measured spectra. The sensitivity of the spectra to the model parameters suggests that the proposed model can be used for the quantitative deconvolution of the UV-vis spectra in terms of critical information necessary for the detection and identification of microorganisms.     

APPLICATION OF LASER DIAGNOSTIC TECHNIQUES FOR THE EXAMINATION OF LIQUID FUEL SPRAY STRUCTURE

Results are presented on the spatial distribution of droplet mean size and number density obtained from a hollow-cone kerosene spray, introduced into nonswirling and swirling flow fields. An ensemble light scattering technique, based on measurement of the polarization ratio, has been employed to determine local droplet characteristics in both dense and dilute regions of the spray. The measurements are complemented with Lorenz-Mie calculations of the scattering characteristics for a polydispersion of droplets; the calculations were carried out for different mean sizes and refractive indices. The results reveal that the degree of swirl imparted to the surrounding air flow has a strong influence on spray structure. For all conditions examined the droplet mean size is found lo be larger on the spray boundary than towards the centerline. Droplet mean size is also found to increase with axial distance at all radial positions of the spray; this trend is attributed to the vaporization of smaller droplets and/or possible coalescence between the droplets.

In addition to the ensemble technique, measurements have also been obtained with the phase/Doppler interferometry and light intensity deconvolution techniques under identical experimental conditions. The droplet mean sizes obtained with the ensemble approach are in general smaller than those measured with the phase/Doppler technique; however, general features of the radial profiles obtained with both techniques are similar. The deconvolution technique also indicates the presence of smaller size droplets and supports the results obtained with the ensemble technique. The selective sensitivity of these sizing techniques to different ranges of droplet size and number density is discussed.     

Laser Diagnostics of Combustion Processes: From Chemical Dynamics to Technical Devices

Laser-based in situ diagnostic techniques with high temporal, spectral, and spatial resolution have become valuable tools to study the molecular dynamics of gas-phase and heterogeneous reactions as well as complex technical combustion processes. Results of recent experiments will be presented in which laser-induced fluorescence, sum-frequency generation surface vibrational spectroscopy, Rayleigh scattering, excimer laser-induced fragmentation fluorescence, and high-resolution tunable diode laser absorption spectroscopy were applied to investigate elementary chemical gas-phase and catalytic combustion reactions, internal combustion engine processes, as well as coal combustion and waste incineration.     

Das verhalten von polyvinylalkohol in wäßriger lösung

Polyvinylalcohol in water was investigated by inelastic and elastic light scattering techniques and by viscosity measurements. In order to measure this system at high temperatures and at pressures up to 4000 bars we designed a light scattering photometer for use at elevated temperatures and pressures on the basis of the Malvern Photon Correlation Spectrometer. Our data suggest, that the diffusion coefficient increases slightly at low temperatures with increasing pressure and decreases at high temperatures. Both inelastic and elastic light scattering indicate a very high molar mass of the system. Viscosity measurements and light scattering on reacetylated Polyvinylalcohol indicate a much smaller molar mass. This is explained with association of the polymer molecules by hydrogen bonds with water molecules.     

Absorption and scattering of light in nanodiamond hydrosols

Scattering and absorption of optical radiation in hydrosols of detonation nanodiamonds (DND) have been studied. Experimental data are presented on the spectral response of the optical density of DND hydrosols prepared by different techniques and in different concentrations. The size distribution of DND particles in these hydrosols was investigated by dynamic light scattering (DLS). The experimental data are compared with calculations. The calculations were performed on models including both the structure of a single DND particle made up of a diamond core and a thin graphite-like shell and the size distribution of the DND particles. A comparison of experiments with the calculation provided a possibility of refining the model of the DND particle and gaining insight into the nature of particle aggregation. It is demonstrated that the combined use of two methods of investigation, which deal with the spectral response of optical density and dynamic light scattering, offers valuable information on the nature of DND hydrosol coloring and the results of particle size determination.     

Lipid--protein interactions revealed by two-photon microscopy and fluorescence correlation spectroscopy

Cellular processes involve a multitude of chemical reactions that must be kept in delicate equilibrium to maintain cellular homeostasis. Powerful biophysical techniques are needed to measure the localization and concentration of target molecules as well as to quantify complex molecular processes in model and in vivo systems. Two-photon microscopy and fluorescence correlation spectroscopy (FCS) can measure association and dynamics of appropriate molecules under equilibrium conditions. FCS provides information on motility (diffusion coefficients), concentration (number of particles), association (molecular brightness), and localization (image) of the target molecules. All of this information, in conjunction with computational modeling techniques, can help us to better understand the network of complex molecular interactions, which are at the basis of cellular processes. Fluorescence imaging techniques add the beauty of visualization to the scientific information. Photons emitted by a fluorescent dye are digitized, and the associated spatial information and intensity can be translated into different colors and shades providing the researcher not only with quantitative intensity information but also with spatial resolution and visual comprehension of two- or three-dimensional images. In this Account, we review the use of two-photon excitation microscopy and FCS in the study of lipid-protein interactions. We discuss these new methodologies and techniques, and we present examples of different complexity from qualitative to quantitative, from simple model systems to studies in living cells.     

Analysis of polymer blend morphology by transmission and reflection light scattering techniques

Optical inspection is a potentially powerful and practical tool to collect information on polymer blend morphology for on-line quality and process control. This paper describes two approaches for the characterization of polymer blend materials by multiple light scattering. A quantitative evaluation of spectral turbidimetry (scattering of transmitted light) is presented and an adaptation of this technique for monitoring particle size, independent of the volume fraction is demonstrated. Such a procedure is applicable to materials in film form. A different approach is presented for the analysis of nearly spherical particles in optically thick samples: the diffuse reflection pattern surrounding the position of incidence of a focused light beam on the surface of the turbid medium is scanned by a single side imaging system. A spectral pseudo-transmittance signal processing technique provides particle size information again independent of the volume fraction: Both techniques have been tested using well characterized polystyrene microspheres suspended in water as well as poly(methyl) (methacrylate)/polystyrene polymer blends.     

Possible Impact of Heterogeneous Photocatalysis on the Global Chemistry of the Earth's Atmosphere

Photochemistry is recognized to be important for various physicochemical processes in the atmosphere, such as formation of the ozone layer and smogs, degradation of waste substances, etc. [1]. However, up to the present the emphasis in atmospheric photochemistry has been mainly on the study of photochemical reactions that occur with molecules directly excited by absorption of light quanta. However, the major components and impurities of the earth's atmosphere (such as nitrogen, oxygen, water, carbon dioxide, methane, methane halides, etc.) are totally transparent to most solar radiation. Electronically excited states of these molecules are formed only upon absorption of vacuum ultraviolet light quanta with energy hv ≥ 5 eV (i.e., with wavelength λ ≤ 200 nm). Only a small portion of the energy of solar light is found in this spectral region. In other words, most of the energy of the solar flux cannot participate in such direct photochemical reactions.     

Possible Impact of Heterogeneous Photocatalysis on the Global Chemistry of the Earth's Atmosphere

Photochemistry is recognized to be important for various physicochemical processes in the atmosphere, such as formation of the ozone layer and smogs, degradation of waste substances, etc. [1]. However, up to the present the emphasis in atmospheric photochemistry has been mainly on the study of photochemical reactions that occur with molecules directly excited by absorption of light quanta. However, the major components and impurities of the earth's atmosphere (such as nitrogen, oxygen, water, carbon dioxide, methane, methane halides, etc.) are totally transparent to most solar radiation. Electronically excited states of these molecules are formed only upon absorption of vacuum ultraviolet light quanta with energy hv ≥ 5 eV (i.e., with wavelength λ ≤ 200 nm). Only a small portion of the energy of solar light is found in this spectral region. In other words, most of the energy of the solar flux cannot participate in such direct photochemical reactions.     

Spectrophotometric color matching based on two-constant kubelka-munk theory

An algorithm is proposed to minimize the spectral difference between the reflectance of a standard and that of a match formed from a mixture of scattering colorants. This method applies linear least-squares techniques and Kubelka-Munk two-constant theory to the calculation of colorant concentrations required to match the K/S curve of a standard. Several advantages may be gained over traditional tristimulus matching: the spectral reflectance of the standard and predicted formulation exhibit lower spectral difference, the use of spectrally similar colorants is enhanced, and formulations can be determined for standards measured over wavelength regions other than the visible spectrum.     

Advanced Oxidation Processes in Pharmaceutical Formulations: Photo-Fenton Degradation of Peptides and Proteins

Formulations of therapeutic proteins are sensitive to photo-degradation by near UV and visible light. Mechanistically, especially the processes leading to protein modification under visible light exposure are not understood. Potentially, these processes may be triggered by a ligand to metal charge transfer in excipient-metal complexes. This article summarizes recent analytical and mechanistic work on such reactions under experimental conditions relevant to pharmaceutical formulations.     

Drugs targeting protein-protein interactions

Most biological processes involve permanent and nonpermanent interactions between different proteins, and many protein complexes play a key role in various human diseases. Therefore, molecules that prevent the formation of these protein complexes could be valuable new therapeutic agents to treat these diseases. Protein interfaces have not evolved to bind low-molecular-weight molecules, as is the case with enzyme catalytic sites. It is therefore difficult to identify small compounds that inhibit protein-protein interactions. However, there is considerable diversity in the structure of protein interfaces, some of which may be more attractive than others for medicinal chemistry. One of the main challenges in drug discovery is to identify these interfaces and to exploit their properties to make marketable drugs. Herein, the properties of protein interfaces are discussed in light of their use as drug targets.     

Spectral‐reflectance‐factor deconvolution and colorimetric calculations by local‐power expansion

The experimental data of the spectral‐reflectance factor are considered as dependent on the instrument‐spectral‐bandwidth function in order to perform their deconvolution and to compute the tristimulus values. The deconvolution is performed by local‐power expansion. In the case that the spectral‐bandpass dependence regards only the spectral transmittance of the monochromator, the goodness of this technique is evaluated by simulation (1325 reflectance factors of the Munsell samples are considered as trial functions) and compared with other usual techniques: Stearns and Stearns method for bandpass error, ASTM‐weighting function interpolation, and Venable‐ASTM weighting function. The zero order of the deconvoluted spectral‐reflectance factor can be related to the Stearns and Stearns method for bandpass error. With respect to any other technique, the second‐order deconvolution, for the CIE standard illuminants, gives color differences lower by a factor 0.1 or more for a bandpass Δλ = 10 nm, color differences lower by a factor 0.3 or more for a bandpass Δλ = 20 nm and, for the CIE fluorescent illuminants, color differences generally lower. © 2000 John Wiley & Sons, Inc. Col Res Appl, 25, 176–185, 2000     

煤基粗油轻质组分定性定量分析现状与展望

煤基粗油是指原煤经各种热化学反应、化工过程加工处理后得到的初级液体产物,包括煤焦油、煤直接液化油和煤间接液化油等。以生产过程全流程物料衡算为约束条件,对煤基粗油全组分组成的准确定性定量分析,不仅可以全面掌握煤基粗油的组成组分性质,而且可实现对煤基粗油的进一步精准加工、提质生产。但由于煤基粗油组分组成的复杂性,不仅难以实现进样组分及分析结果之间的物料衡算,而且也不能最大程度地实现对所有组分的组成及含量的准确分析,至今尚未见到对此内容相关文献资料报道,也没有相应分析方法的国家或行业标准。因此,以物料衡算为约束,建立全面、系统的分析方法,对煤基粗油进行精准定性定量分析是目前亟需解决的问题。本文指出利用气相色谱与红外光谱联用、液相色谱分离后与紫外光谱、同步荧光光谱联用等光谱分析法可快速获得煤基粗油族组分含量的测定,或是对煤基粗油性质做定性的描述;核磁共振波谱法可实现酚类化合物准确的定性、定量;化学分析法仅适合于煤基粗油具体成分的定性定量分析;红外光谱、核磁共振波谱、凝胶渗透色谱和元素分析结合可系统分析与表征煤基粗油及其组分,得到一些重要的物质结构参数信息;色谱分析法可通过色谱的分离功能使复杂混合物分离,并利用各种检测器工作原理的不同来实现定性和定量。其中定性主要采用质谱仪、光谱仪、核磁共振波谱仪等;定量的方法有归一化法、内标法、外标法、响应因子预测法。针对煤基粗油轻质组分（沸点＜350℃）组成的定性定量,气相色谱-质谱联用（GC-MS）技术为此分析过程提供了可能,本文提出并推导了火焰离子化检测仪（FID）响应因子预测公式,将该公式与面积归一化法联用建立FID定量方法体系,加标回收实验（回收率为99.07%,质量分数）表明该方法体系准确、有效,可以被广泛应用于复杂有机混合物的定量分析中,并指出煤基粗油轻质组分定性定量分析过程中的关键在于对复杂混合物中各组分响应因子的准确获得。     

Electrochemical behavior of blood coagulation proteins—factors V and VIII

The electrochemical reactions of two blood coagulation proteins, factors V and VIII (AC globulin and antihemophila factor) at platinum -NaCl interface were investigated using cyclic voltammetric techniques. Voltammograms were recorded for three concentrations of each of the proteins (0·1, 1·0 and 10 times the physiologic concentration) at different scan rates. The results of this preliminary study suggest that these compounds take part in more than one electron transfer reaction at the interface. Some of these may be coupled with chemical reactions. With both the proteins, particularly at lower concentrations, their adsorption has a significant influence on the peak current functions. Prediction as to the detailed type or nature of these reactions is not feasible at the present time due to the complexity of the molecules and to the lack of structural information of the proteins.     

Application of stimuli responsive polymers for sustainable ion exchange chromatography

Ion exchange processes are widely used in the food, bioprocessing and related industries for the isolation of proteins and other ionic species. Traditional ion exchange resins require salts, acids or bases for releasing adsorbed molecules creating a strong saline waste stream with negative environmental and economic impact. Stimuli responsive polymers (SRPs) with ion exchange functional groups can be used to selectively capture and release charged molecules from a complex mixture using physical stimuli to trigger conformational transitions in the polymer. The structural change of the polymers in response to a stimulus may lead to reduced ligand–target molecule interaction resulting in the release of the captured molecule without the use of chemical reagents, thereby reducing the environmental burden associated with ion exchange processes. The use of temperature responsive polymers has already been demonstrated for such applications at analytical scale. However, little progress has been made to extend these discoveries to the development of materials and methods amenable to industrial scale processing. So far, other SRPs such as, electric, magnetic and light responsive polymers remain largely unexplored for such application. This article discusses the potential of temperature responsive and other SRPs for developing sustainable ion exchange processes. It also highlights the material science and engineering challenges that need to be overcome to bring such processes to industrial application.     

The new chemical biology of nitrite reactions with hemoglobin: R-state catalysis, oxidative denitrosylation, and nitrite reductase/anhydrase

Because of their critical biological roles, hemoglobin and myoglobin are among the most extensively studied proteins in human history, while nitrite tops the list of most-studied small molecules. And although the reactions between them have been examined for more than 140 years, a series of unusual and critical allosterically modulated reactions have only recently been characterized. In this Account, we review three novel metal- and nitrite-catalyzed reaction pathways in the context of historical studies of nitrite and hemoglobin chemistry and attempt to place them in the biological framework of hypoxic signaling. Haldane first described the reaction between nitrite and deoxymyoglobin, forming iron-nitrosylated myoglobin, in his analysis of the meat-curing process more than a century ago. The reaction of nitrous acid with deoxyhemoglobin to form nitric oxide (NO) and methemoglobin was more fully characterized by Brooks in 1937, while the mechanism and unusual behavior of this reaction were further detailed by Doyle and colleagues in 1981. During the past decade, multiple physiological studies have surprisingly revealed that nitrite represents a biological reservoir of NO that can regulate hypoxic vasodilation, cellular respiration, and signaling. Importantly, chemical analysis of this new biology suggests a vital role for deoxyhemoglobin- and deoxymyoglobin-dependent nitrite reduction in these processes. The use of UV-vis deconvolution and electron paramagnetic resonance (EPR) spectroscopy, in addition to refined gas-phase chemiluminescent NO detection, has led to the discovery of three novel and unexpected chemistries between nitrite and deoxyhemoglobin that may contribute to and facilitate hypoxic NO generation and signaling. First, R-state, or allosteric, autocatalysis of nitrite reduction increases the rate of NO generation by deoxyhemoglobin and results in maximal NO production at approximately 50% hemoglobin oxygen saturation, which is physiologically associated with greatest NO-dependent vasodilation. Second, oxidative denitrosylation of the iron-nitrosyl product formed in the deoxyhemoglobin-nitrite reaction allows for NO formation and release in a partially oxygenated environment. Finally, the deoxyhemoglobin-nitrite reaction participates in a nitrite reductase/anhydrase redox cycle that catalyzes the anaerobic conversion of two molecules of nitrite into dinitrogen trioxide (N(2)O(3)). N(2)O(3) may then nitrosate proteins, diffuse across hydrophobic erythrocyte membrane channels such as aquaphorin or Rh, or reconstitute NO via homolysis to NO and NO(2)(*). Importantly, the nitrite reductase/anhydrase redox pathway also represents a novel mechanism of both anaerobic and metal-catalyzed N(2)O(3) formation and S-nitrosation and may thus play a vital role in NO-dependent signaling in a hypoxic and heme-rich environment. We consider how these reactions may contribute to physiological and pathological hypoxic signaling.     

Goniophotometric studies of film deterioration processes: I. Introduction and instrumentation

The use of reflectance techniques for the study of surface coatings is discussed and the applications of goniophotometry briefly reviewed. The necessary modifications to enable the Brice-Phoenix light scattering photometer to be used as a high sensitivity goniophotometer are described together with details of the application of the instrument to the study of point films. Results obtained with different types of coatings are presented in order to show typical effects of variation in, for example: pigmentation, application technique and environmental deterioration. The results are discussed in terms of the subjectively assessed “gloss” of the different coatings and the ways in which the features of the goniophotometric curves may be most effectively and compactly described. Goniophotometry is suggested as a potentially valuable and sensitive technique for the quantitative study of the early stages of deterioration of polymer films.