Infrared spectroscopy, enzyme structure

Although the majority of the lipids in M. laidlawii membranes appear to be in a liquid-crystalline state, the system possesses the same physical properties that many other membranes possess. The ORD is that of a red-shifted a-helix high resolution NMR does not show obvious absorption by hydrocarbon protons, and infrared spectroscopy shows no ft structure. Like erythrocyte ghosts, treatment with pronase leaves an enzyme-resistant core containing about 20% of the protein of the intact membrane (56). This residual core retains the membrane lipid and appears membranous in the electron microscope (56). Like many others, M. laidlawii membranes are solubilized by detergents and can be reconstituted by removal of detergent. Apparently all of these properties can be consistent with a structure in which the lipids are predominantly in the bilayer conformation. The spectroscopic data are therefore insufficient to reject the concept of a phospholipid bilayer structure or to... 

Pharmaceutical companies are increasingly interested in developing products based on proteins, enzymes, and peptides. With the development of such products comes the need for methods to evaluate the purity and structural nature of these biopharmaceuticals. Proteins, unlike traditional pharmaceutical entities, rely on a specific secondary structure for efficacy. Methods to monitor the secondary structure of pharmaceutically active proteins, thus, is necessary. Infrared spectroscopy provides a way to study these compounds quickly and easily. Byler et al. (65) used second-derivative IR to assess the purity and structural integrity of porcine pancreatic elastase. Seven different lyophilized samples of porcine pancreatic elastase were dissolved in D20, placed in demountable cells with CaF2 windows, and IR spectra obtained. The second derivatives of the spectra were calculated and the spectral features due to residual water vapor and D20 removed. 

Information about the secondary structures (a-helices, /5-sheets, random coil) can be useful for understanding conformation changes of proteins upon the immobilization process. More specifically, circular dichroism (CD) [70] and FT-IR spectroscopy [56, 58, 61, 71-73] have been applied to study the structural characteristics of various proteins adsorbed on mineral surfaces. Kondo and coworkers [70] have studied the modification in a-helix content of proteins adsorbed on ultrafine silica particles with CD and found a decrease upon immobilization. Circular dichroism is not usually used because this technique is applicable only for the study of enzymes immobilized on nano-sized mineral particles due to problems arising from light scattering effects. On the other hand, infrared spectroscopy does not suffer from light scattering perturbations and has thus been used for the study of the conformation of proteins when they are immobilized on solid supports [57, 58]. 

As mentioned above, the catalytic activity of an enzyme is determined by its conformation in reverse micelles, so the characterization of the enzyme conformation is an important aspect of the somicellar enzymology. Infrared spectroscopy is a powerful technique for structural characterization [76-78]. For a protein hosted in reverse micelles, the spectral features of the protein are always interfered by the IR absorption bands of the medium in addition to the congestion in their IR spectra. Fortunately, there is a transparent window in the 2500-2200 cm region. Incorporation of a vibrational probe with IR absorption frequencies in this region into proteins represents a promising strategy for the study of the conformation of a protein in a reverse micelle. Huang et ul. [79] incorporated a 4-cyanobenzyl... 

The differently produced conductive polymer structures described above all have enhanced conductivity, which can be employed in microelectronics [44] and as sensors using immobilized enzymes [46, 47[. Martin and coworkers used polarized infrared absorption spectroscopy to access the alignment of the polymer fibers on the outer surface of the nanotubes [48[. The study showed that the enhancement of the conductivity is due to the alignment of the polymer fibers on the outer surface of the tubes. 

MCD spectroscopy in range 300 to 2000 nm at both ambient and liquid helium (4.2 K) temperatures can yield information about the spin, oxidation, and coordination states of each heme in a multiheme protein such as CCP (75). This technique, in combination with low-temperature X-band EPR spectroscopy, was used to great effect in characterizing the properties of the fully oxidized and MV forms of the P. aeruginosa CCP in solution. At 4.2 K, both hemes in the oxidized enzyme are low-spin ferric, with diagnostic features in the near infrared-MCD (NIR-MCD) spectrum consistent with one heme with His/Met axial coordination and the other with bis-histidine axial coordination this is entirely consistent with the crystal structure. In contrast, at room temperature only the low-potential (bis-histidine coordinated) heme in the C-terminal domain remains completely low-spin, whereas the high-potential (His/Met coordinated) heme exists as mixture of high- and low-spin forms 58). 

Adsorption of the enzymes subtilisin BPN and lysozyme onto model hydrophilic and hydrophobic surfaces was examined using adsorption isotherm experiments, infrared reflection-absorption spectroscopy (IRRAS), and attenuated total reflectance (ATR) infrared (IR) spectroscopy. For both lysozyme and BPN, most of the enzyme adsorbed onto the model surface within ten seconds. Nearly an order-of-magnitude more BPN adsorbed on the hydrophobic Ge surface than the hydrophilic one, while lysozyme adsorbed somewhat more strongly to the hydrophilic Ge surface. No changes in secondary structure were noted for either enzyme. The appearance of carboxylate bands in some of the adsorbed BPN spectra suggests hydrolysis of amide bonds has occurred. 

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