Protein ultraviolet spectroscop

Demchenko AP (1986) Ultraviolet spectroscope of proteins. Springer-Verlag, Heidelberg, New York... 

Probing Metalloproteins Electronic absorption spectroscopy of copper proteins, 226, 1 electronic absorption spectroscopy of nonheme iron proteins, 226, 33 cobalt as probe and label of proteins, 226, 52 biochemical and spectroscopic probes of mercury(ii) coordination environments in proteins, 226, 71 low-temperature optical spectroscopy metalloprotein structure and dynamics, 226, 97 nanosecond transient absorption spectroscopy, 226, 119 nanosecond time-resolved absorption and polarization dichroism spectroscopies, 226, 147 real-time spectroscopic techniques for probing conformational dynamics of heme proteins, 226, 177 variable-temperature magnetic circular dichroism, 226, 199 linear dichroism, 226, 232 infrared spectroscopy, 226, 259 Fourier transform infrared spectroscopy, 226, 289 infrared circular dichroism, 226, 306 Raman and resonance Raman spectroscopy, 226, 319 protein structure from ultraviolet resonance Raman spectroscopy, 226, 374 single-crystal micro-Raman spectroscopy, 226, 397 nanosecond time-resolved resonance Raman spectroscopy, 226, 409 techniques for obtaining resonance Raman spectra of metalloproteins, 226, 431 Raman optical activity, 226, 470 surface-enhanced resonance Raman scattering, 226, 482 luminescence... 

The second recent spectroscopic improvement is second and fourth derivative spectroscopies in the ultraviolet region of proteins. Derivative spectroscopy is a new tool for analyzing the effects of pressure on proteins. It permits one to enhance selectively spectral changes due to the UV absorbance of phenylalanine, tyrosine and tryptophan. The solvent polarity affects the amplitude, the position and the shape of the second and fourth derivative spectral bands. 

RetinalS. The structure and photophysics of rhodopsins are intimately related to the spectroscopic properties of their retiny1-polyene chromophore in its protein-free forms, such as the aldehyde (retinal), the alcohol (retinol or vitamin A), and the corresponding Schiff bases. Since most of the available spectroscopic information refers to retinal isomers (48-55), we shall first center the discussion on the aldehyde derivatives. Three bands, a main one (I) around 380 nm and two weaker transitions at 280 nm and 250 nm (II and III), dominate the spectrum of retinals in the visible and near ultraviolet (Fig. 2). Assignments of these transitions are commonly made in terms of the lowest tt, tt excited states of linear polyenes, the spectroscopic theories of which have been extensively discussed in the past decade (56-60). In terms of the idealized C2h point group of, for example, all-trans butadiene, transitions are expected from the Ta ground state to B , A, and A" excited states... 

Spectroscopic methods for following the titration of other common titratable groups of protein molecules do not exist. The reason is that the peptide group and the aromatic rings of phenylalanine, tryptophan, and tyrosine side chains absorb strongly in the ultraviolet below 250 m/i, making it essentially impossible to observe the relatively small changes in absorb-... 

Spectrophotometric analyses are the most common method to characterize proteins. TTie use of ultraviolet-visible (UV-VIS) spectroscopy is t rpically used for the determination of protein concentration by using either a dye-binding assay (e.g., the Bradford or Lowry method) or by determining the absorption of a solution of protein at one or more wavelengths in the near UVregion (260-280 nm). Another spectroscopic method used in the early-phase characterization of biopharmaceuticals is CD. 

Over the range of serum protein levels usually encountered, the absorption of a 1 1000 dilution obeys Beer s law both at 225 nm (Wl), and at 210 nm (T14). Neither NaCl nor ammonium sulfate interferes. Direct measurement at a single low wavelength (T14) seems to be a satisfactory procedure for determining either serum protein or separated albumin (T14, W8). The method is applicable to electrophoretically separated albumin and globulins (M31), and to albumin in acid-alcohol mixtures, when suitable blanks are included (W8). With appropriate blanks, concentrations of acetate, citrate, succinate, phthalate, and barbiturate up to 0.005 M can be tolerated. Absorption in the far-ultraviolet is unaffected by pH in the range pH 4-8. Outside this range, an altered state of ionization may result in a new molecular form of protein with different spectroscopic characteristics (see Rosenheck and Doty, R24). 

Since Hg(II) has a closed-shell electronic structure, it is often considered spectroscopically silent in optical and EPR spectroscopy. Recent advances in a variety of spectroscopic techniques have made this description of Hg(II) complexes obsolete. Relativistic effects tend to lower energy of the Hg 6s orbital (156), and consequently the LMCT spectra of Hg(II) tetrahalides and three- or four-coordinate alkyl thiolate complexes are distinctly different than those obtained for two-coordinate complexes. As described below, ultraviolet spectroscopy has provided details about the Hg(II) coordination in the MerR protein (202, 210). 

Several factors must be considered for a particular biomacromolecular structure application that will affect the choice of spectroscopic methods. These include structural resolution necessary, chemical nature of biomacromolecule (protein, nucleic add, or glycan), amount/concentration of biopolymer available, sample preparation (solid or solution), solvents of interest, and desired structure information (secondary or tertiary structure). Structural resolution varies considerably for the various spectroscopic methods, with X-ray diffraction and NMR providing atomic resolution (high resolution) and ultraviolet (UV) absorption revealing merely information about the polarity of the chromophore s environment (low resolution). X-ray studies require crystals while NMR experiments prefer solutions in deuterated solvent. Solvent preferences can affect the choice of spectroscopic method as, for example, infrared (IR) encoimters strong interference from water, while optical rotatory dispersion (ORD) and circniar dichroism (CD) do not. Some of the commonly used spectroscopic methods in structural analyses of biomacromolecules will be discussed. 

Spectroscopic analysis of proteins is nearly as old as spectrophotometers themselves. In the 1930s the first generation of laboratory-built ultraviolet-visible (UV-Vis) spectrophotometers were used to study proteins even before spectrophotometers were commercialized. In the earliest work, strong absorbance at 280 nm, due to the aromatic content of proteins, was the basis of detection in many protein-containing samples. The commercial availability of scanning spectrophotometers in the early 1950s provided an important tool for fundamental studies of protein structure and function. Quantitation of protein aromatic content via spectrophotometry was first proposed by Goodwin and Morton [1]). Beaven [2,3], Wetlaufer [4], and Donovan [5] have written comprehensive reviews of the early spectroscopic studies of peptides and proteins. 

Raman spectroscopy is a vibrational spectroscopic technique which can be a useful probe of protein structure, since both intensity and frequency of vibrational motions of the amino acid side chains or polypeptide backbone are sensitive to chemical changes and the microenvironment around the functional groups. Thus, it can monitor changes related to tertiary structure as well as secondary structure of proteins. An important advantage of this technique is its versatility in application to samples which may be in solution or solid, clear or turbid, in aqueous or organic solvent. Since the concentration of proteins typically found in food systems is high, the classical dispersive method based on visible laser Raman spectroscopy, as well as the newer technique known as Fourier-transform Raman spectroscopy which utilizes near-infrared excitation, are more suitable to study food proteins (Li-Chan et aL, 1994). In contrast the technique based on ultraviolet excitation, known as resonance Raman spectroscopy, is more commonly used to study dilute protein solutions. 

Amino acids are the structural units of protein, and some of them have been used as drugs or food additives, so the determination of amino acids is useful for biochemical research and for commercial product analysis. Among essential amino acids, there are three aromatic amino acids, phenylalanine, tyrosine and tryptophan, which exhibit fluorescence when they are excited by ultraviolet rays. So it is possible to determine them by the fluorescence spectroscopic method. The A ax of phenylalanine, tyrosine and tryptophan are 282 nm, 303 nm and 348 nm respectively, but their fluorescence spectra are partially overlapped. Since the separation operation of these three amino acids is tedious and... 

Thamann TJ (1996) Probing local protein structure with ultraviolet resonance Raman spectroscopy. In Havel HA (ed) Spectroscopic Methods for Determining Protein Structure in Solution, pp 96-134. New York VCH. 

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