Protein optical constants

To quantify the orientation of a pulmonary surfactant-specific protein SP-C incorporated into simple and mixed-lipid monolayers at the AW interface, Ger-icke et al. [848] applied the spectrum fitting procedure using the optical constants expressed by Eq. (3.56). This protein is a promoter of the spreading... 

At the time of writing this article, a fluorescence optical system is under development by Aviv Biomedical Inc., and will be available as an accessory to the XL-I and XL-A instruments. Fluorescence optics provide very high sensitivity for studies of labeled or naturally fluorescent compounds. Test systems have proved useful with concentrations as low as SOOpmol, allowing determination of protein equilibrium constants below 10 moll Studies with fluorescent tracers may also be used to characterize macromolecules at higher concentrations. 

In dye-binding tests, milk is mixed with excess acidic dye solution where the protein binds the dye in a constant ratio and forms a precipitate. After the dye—protein interaction takes place, the mixture is centrifuged and the optical density of the supernatant is determined. Utilization of the dye is thus measured and from it the protein content determined. Several methods for appHcation of dye-binding techniques to milk are given (24,25). 

In the M. trichosporium OB3b system, a third intermediate, T, with kmax at 325 nm (e = 6000 M-1cm 1) was observed in the presence of the substrate nitrobenzene (70). This species was assigned as the product, 4-nitrophenol, bound to the dinuclear iron site, and its absorption was attributed primarily to the 4-nitrophenol moiety. No analogous intermediate was found with the M. capsulatus (Bath) system in the presence of nitrobenzene. For both systems, addition of methane accelerated the rate of disappearance of the optical spectrum of Q (k > 0.065 s-1) without appreciatively affecting its formation rate constant (51, 70). In the absence of substrate, Q decayed slowly (k 0.065 s-1). This decay may be accompanied by oxidation of a protein side chain. 

Selected entries from Methods in Enzymology [vol, page(s)] Association constant determination, 259, 444-445 buoyant mass determination, 259, 432-433, 438, 441, 443, 444 cell handling, 259, 436-437 centerpiece selection, 259, 433-434, 436 centrifuge operation, 259, 437-438 concentration distribution, 259, 431 equilibration time, estimation, 259, 438-439 molecular weight calculation, 259, 431-432, 444 nonlinear least-squares analysis of primary data, 259, 449-451 oligomerization state of proteins [determination, 259, 439-441, 443 heterogeneous association, 259, 447-448 reversibility of association, 259, 445-447] optical systems, 259, 434-435 protein denaturants, 259, 439-440 retroviral protease, analysis, 241, 123-124 sample preparation, 259, 435-436 second virial coefficient [determination, 259, 443, 448-449 nonideality contribution, 259, 448-449] sensitivity, 259, 427 stoichiometry of reaction, determination, 259, 444-445 terms and symbols, 259, 429-431 thermodynamic parameter determination, 259, 427, 443-444, 449-451. 

Chance and co-workers have designed a flow system where the protein is continuously pumped optically using a tungsten or xenon flash lamp (764 nm). Using continuous illumination for various times and temperatures. Chance et al. have observed three intermediate states upon MbCO photolysis. At 40 K, a state with a recombination rate constant of 2 x 10 /s has been identified from two slower states with rate constants of 10 /s. 

Adsorption of putidaredoxin on gold electrodes has been studied using dynamic spectroscopic ellipsometry and differential capacitance measurements [307]. In Ref. 307, a method for the measurement of metal surface optical perturbation during protein adsorption at a constant potential has been described. The method is based on the concept that the charged transition layer develops between the electrode substrate and the adsorbate. 

Fig. 2c. It can be seen that at 530 nm, the fluorescence decays mono-exponentially with the fluorescence lifetime of 3.24 ns. The rise of the emission seen below 50 ps in the corresponding FlUp data is obviously not resolved here. In contrast, the TCSP data at 450 nm is described by a triple-exponential decay whose dominant component has a correlation time well below the time resolution. This component is obviously equivalent to the fluorescence decay observed in the FlUp experiment. A minor contribution has a correlation time of about 3.2 ns and reflects again the fluorescence lifetime that was also detected at 530 nm. The most characteristic component at 450 nm however has a time constant of about 300 ps. It is important to emphasize that this 300 ps decay does not have a rising counterpart when emission near the maximum of the stationary fluorescence spectrum is recorded. In other words, the above mentioned mirror image correspondence of the fluorescence dynamics between 450 nm and 530 nm holds only on time scales shorter than 20 ps. Finally, in contrast to picosecond time scales, the anisotropy deduced from the TCSPC data displays a pronounced decay. This decay is reminiscent of the rotational diffusion of the entire protein indicating that the optical chromophore is rigidly embedded in the core of the 6-barrel protein.

Changes in biomass concentration throughout the fermentation process were followed by optical density (OD) measurement at 580 nm using an Ultrospec 2000 Spectrophotometer. Quantitative biomass concentration was assayed applying microbiuret cell protein determination (16). Biomass concentration was expressed in grams of dry matter per liter of fermentation broth by assuming a twofold multiplication constant for microbial protein to cell mass. For carbon balance calculations, the elemental composition of C. saccharolyticus was assumed to be CH1 8O0 5N0 2 (24.6 mg/mmol). 

Fig. 2.3 Solvent screening of electronic couplings between chromophores in the four photo-syntletic proteins PE545 (pink triangles), PC645 (blue inverted triangles), PSII/LHCII (green circles). The protein medium is modeled as a dielectric continuum medium with a relative static constant of estat = 15 and optical dielectric constant of n2 = 2. Calculated values for the solvent screening factor s for the various chromophores pairs. The Forster value I jnl and the Onsager value 3(2n1 + 1) are indicated by the horizontal line...

In Eq. (8), e is the electron charge, rD and rA are the van der Waals radii of the donor and acceptor, rDA is the center-to-center separation distance, and and es are, respectively, the optical and static dielectric constants of the medium (e.g., the solvent or protein matrix). 

While the redox titration method is potentiometric, the spectroelectrochemistry method is potentiostatic [99]. In this method, the protein solution is introduced into an optically transparent thin layer electrochemical cell. The potential of the transparent electrode is held constant until the ratio of the oxidized to reduced forms of the protein attains equilibrium, according to the Nemst equation. The oxidation-reduction state of the protein is determined by directly measuring the spectra through the tranparent electrode. In this method, as in the redox titration method, the spectral characterization of redox species is required. A series of potentials are sequentially potentiostated so that different oxidized/reduced ratios are obtained. The data is then adjusted to the Nemst equation in order to calculate the standard redox potential of the proteic species. Errors in redox potentials estimated with this method may be in the order of 3 mV. 

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