Protein ultraviolet-visible

Optical Spectroscopy General principles and overview, 246, 13 absorption and circular dichroism spectroscopy of nucleic acid duplexes and triplexes, 246, 19 circular dichroism, 246, 34 bioinorganic spectroscopy, 246, 71 magnetic circular dichroism, 246, 110 low-temperature spectroscopy, 246, 131 rapid-scanning ultraviolet/visible spectroscopy applied in stopped-flow studies, 246, 168 transient absorption spectroscopy in the study of processes and dynamics in biology, 246, 201 hole burning spectroscopy and physics of proteins, 246, 226 ultraviolet/visible spectroelectrochemistry of redox proteins, 246, 701 diode array detection in liquid chromatography, 246, 749. 

Using ultraviolet/visible (UV/Vis) absorption spectroscopy, it is possible to measure the protein concentration using Beer s Law A = e c, where A is the measured absorbance of a solution, e is the absorptivity of the protein, is the pathlength of the cell used to determine the absorbance, and c is the protein concentration. Proteins typically exhibit two strong, broad absorption bands in the UV/Vis part of the spectrum. The first and most intense band is centered at 214 nm and arises from absorption of light by the peptide backbone. The second absorption band is typically found at 280nm. This band arises from absorbance from the aromatic side chains of Trp, Tyr, and Phe. Disulfide bonds may exhibit weak absorption in this range as well. 

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. 

Another approach developed to increase the solubility of proteins in a bulk aqueous phase is the use of reverse microemulsions. Zhang et al. (84) reported the GAS-based precipitation of lysozyme solubilized in AOT reverse micelles in iso-octane using pressurized CO2. Comparing the on-line UV-vis (ultraviolet-visible) spectra of processed and unprocessed lysozyme, the authors concluded that the lysozyme was not denatured. The use of reverse micelles to dissolve proteins in a bulk organic phase is a promising variation of the GAS technique. The use of reverse micelles could potentially increase the stability of proteins because they would be in a primarily aqueous local environment until precipitation. 

Many other interaction energies come from the electromagnetic frequency spectrum. They can come from outside of the ultraviolet, visible, and infrared frequency ranges. All that is required is an element of the model protein in water that is able to take up the energy. The dipole moment of the peptide group with its positive end at the NH and its negative end at the oxygen of the CO provides one site of inter-... 

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. 

The absorption characteristics have been used to estimate the number of nitroazidophenyl groups that are attached to protein derivatized with 4-azido-2-nitrofluorobenzene. The ultraviolet-visible absorption or the characteristic infrared band at approximately 2100 cm-, often broad or a doublet, can be used to follow photolyses. 

HPLC has been used for measuring various compounds, for example, carbohydrates, vitamins, additives, mycotoxins, amino acids, proteins, triglycerides in fats and oils, lipids, chiral compounds, and pigments. Several sensitive and selective detectors such as ultraviolet-visible, FL, electrochemical, and diffractometric are available to utilize with HPLC depending on the compound to be analyzed. Various HPLC methods based on these detectors have been published for the measurement of vitamin E in biological and pharmaceutical samples and food products. Excellent literature reviews of HPLC based on various detectors in the analysis of vitamin E content in various matrices have been reported (Abidi, 2000 Aust et al., 2001 Ruperez et al., 2001 Lai and Franke, 2013). Table 19.5 reports several recent HPLC methods for the analysis of vitamin E and similar compounds in various matrices. 

HPLC is used for nonvolatile compounds and is well suited for the analysis of low- and high-molecular weight compounds such as peptides and proteins. HPLC is mostly coupled with ultraviolet visible (UV-VIS) wavelength spectroscopy or mass spectrometric detection. 

The literature on the effect of photooxidation of proteins in somewhat confusing, since it is apparent that insufficient attention has been paid to the factors that control photooxidation of amino acid side chains. A wide variety of light sources has been used by different authors. Clearly the pathways of photoreactions will be related to the wavelength of light used and it is particularly difficult to interpret results of irradiation of proteins when mixed ultraviolet/visible light sources such as sunlight have been used. 

Luminescence measurements on proteins occupy a large part of the biochemical literature. In what surely was one of the earliest scientific reports of protein photoluminescence uncomplicated by concurrent insect or microorganism luminescence, Beccari (64), in 1746, detected a visible blue phosphorescence from chilled hands when they were brought into a dark room after exposure to sunlight. Stokes (10) remarked that the dark (ultraviolet) portion of the solar spectrum was most efficient in generating fluorescent emission and identified fluorescence from animal matter in 1852. In general, intrinsic protein fluorescence predominantly occurs between 300 nm and 400 nm and is very difficult to detect visually. The first... 

Amino acids do not absorb visible hght and thus are colorless. However, tyrosine, phenylalanine, and especially tryptophan absorb high-wavelength (250—290 nm) ultraviolet light. Tryptophan therefore makes the major contribution to the abihty of most proteins to absorb hght in the region of 280 nm. 

Purines absorb only ultraviolet light and they contribute to structural colors (white and silver) in animals. Pterines are generally yellow, orange, or red pigments. Because they are amphoteric molecules, the absorption spectra depend on the pH and present three or two absorption maxima, usually one in the visible region. Sepiapterin has an absorption maximum at 340 nm in O.IM NaOH and at 410 nm in O.IM HCl." Leucopterin has three maxima 240, 285, and 340 nm. Xanthopterin has two 255 and 391 nm. Because they are conjugated with proteins, pterins show bathochromic shifts in vivo. They also present fluorescence when excited with UV light. 

Hapten density is important for both immunization and assay performance, and hence the extent of conjugation or hapten density should be confirmed by established methods. A characteristic ultraviolet (UV) or visible absorbance spectrum that distinguishes the hapten from the carrier protein or use of a radiolabeled hapten can be used to determine the degree of conjugation. If the hapten has a similar A. iax to the protein, the extent of incorporation can still be estimated when the concentration of the protein and the spectral characteristics of the hapten and protein are known. The difference in absorbance between the conjugate and the starting protein is proportional to... 

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