Protein ultraviolet absorption

My interest at that time revolved around evaluating optical rotary dispersion data [12]. The paired values of optical rotation vs. wavelength were used to fit a function called the Drude equation (later modified to the Moffitt equation for William Moffitt [Harvard University] who developed the theory) [13]. The coefficients of the evaluated equation were shown to be related to a significant ultraviolet absorption band of a protein and to the amount of alpha-helix conformation existing in the solution of it. 

Protein calculator for calculation of, for example, charge, molecular weight, ultraviolet absorption, and some other parameters. 

Smirnova et al. [5] have described a simple non-enzymatic method of quantitative determination of adenosine triphosphate in activated sludge from aeration tanks. Extraction of the nucleotides in boiling distilled water was followed by removal of the protein impurities by acidification. Barium salts of di- and triphosphates of the nucleotides were precipitated and the precipitate was washed and dissolved in acid to convert the barium salts to sodium salts. The quantity of adenosine triphosphate was determined quantitatively by inorganic phosphorus in the liquid over the precipitate before and after acid hydrolysis, and by ultraviolet absorption spectra. The method was tested in activated sludge from operational sewage works. There was good agreement between the adenosine triphosphate content determined spectrophotometrically and by phosphorus, despite the presence of small quantities of secondary impurities. 

In any chromatographic analysis the method of detection is determined by the nature of the analyte and the mobile phase used must not interfere with this system. The use of ultraviolet absorption detection systems is very common but the solvents used must not absorb significantly at the wavelength used. For instance, absorption at 280 nm is frequently used to detect protein but some solvents, e.g. acetone, absorb at this wavelength. Similarly the use of concentration gradients in the mobile phase may present problems with refractive index and electrochemical detection systems. 

G. H. Beaven and E. R. Holiday, Ultraviolet absorption spectra of proteins and amino acids, Adv. Protein Chem. 7, 319-386 (1952). 

A number of investigators have studied the effect of ozone on the ultraviolet absorption spectra of proteins and amino acids. A decrease in the absorption of 280-nm light in a number of proteins was originally reported ly Giese et aV to be a consequence of ozone exposure they suggested that this was due to an interaction of ozone with the ring structures of tyrosine and tryptophan. Exposure of a solution of tryptophan to ozone resulted in a decrease in 280-nm absorption, whereas the extinction coefficient of tyrosine increased. Similar results with tyrosine were reported by Scheel et who also noted alterations in the ultraviolet spectra of egg albumen, perhaps representing denaturation by ozone. 

The densities of the solvent and of / -lactoglobulin A ( -Lg) in 40% (v/v) 2-chloroethanol, in the presence of 0.01 M HC1 and 0.02M NaCl, were determined, with and without prior dialysis, in a 10-ml pycnometer at 20°C. Solutions were prepared as described previously (8, 24). The solutions were filtered through millipore filters in syringe adapters just before the density measurements. Protein concentrations were determined after filtration by ultraviolet absorption at 278 nm. The apparent partial specific volume, oapp, was calculated from the densities using the standard equation (21, 25) ... 

The procedure outlined here will provide a final product that has an ultraviolet absorption at 280 nm, indicating that protein material is likely present in solution. However several questions remain unanswered ... 

Amino acids do not give any very useful ultraviolet absorption spectra unless they possess aromatic groups as in phenylalanine, tryptophan, and tyrosine. The absorption characteristics of these groups are more useful in monitoring chemical and conformational changes in proteins than they are in the simple amino acids. 

Fig. 17. Comparison of the transition temperatures for RNase-A (circles), RNase-S (triangles), and S-protein (squares) as determined by optical rotation (open symbols) and ultraviolet absorption difference spectroscopy (filled symbols). Reproduced from Sherwood and Potts (387).

Ultraviolet absorption spectra of tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) at pH 6. The molar absorptivity is reflected in the extinction coefficient, with the concentration of the absorbing species expressed in moles per liter. (Source From D. B. Wetlaufer, Adv. Protein Chem. 17 303-390, 1962.)... 

Concentration changes are observed optically from time to time either by light absorption (e.g. ultraviolet absorption for protein solutions) or, more usually, by schlieren or interference methods. These optical methods for examining concentration changes in liquid columns (especially the schlieren technique in its various forms) are also employed in the ultracentrifuge and for studying movingboundary electrophoresis. The schlieren method is based on the fact that, at a boundary between two transparent liquids of different... 

It was pointed out previously that both bacterial and plant fer-redoxins are colored proteins in the oxidized state. Fig. 3 shows the visible and ultraviolet absorption spectra of a bacterial (C. pasteurianum) and plant (spinach) ferredoxin. Bacterial ferredoxin shows a single peak in the visible region at 390 m(r and a peak in the ultraviolet region at about 280 mp. with a shoulder at 300 mp. The relative height of the peak at 280 mp to the shoulder at 300 mp varies among preparations from different bacteria generally the peak at 280 mp predominates (Loven-berg, Buchanan, and Rabinowitz (65) Bachofen and Arnon (12)). Plant... 

Figure 14. Changes in ultraviolet absorption spectra of turkey ovomucoid after reduction and after various periods of reoxidation. Protein (0.7 mg/mL, containing 8-10% water) teas dissolved in 0.006M Tris buffer adjusted to pH 8.3. Incubation was at room temperature for the following times (hours) A, zero (starting) ...

As already described the enzyme-modified 7S protein retains a high molecular weight like the native protein it is excluded by Bio-Gel P-150 which has an exclusion limit of 150,000 daltons. The specificity of rennin is such that it is easy to control and limit the extent of digestion. When the enzyme action is monitored by ultraviolet absorption it is apparent that the rennin action is quite different from that obtained with an enzyme such as trypsin (Figure 1). Thus the UV difference spectrum for the rennin-modified protein shows an initial unfolding of the 7S protein chains as indicated by a negative peak at 236 nm. As rennin action continued this negative peak was replaced by a positive peak at about 237 nm characteristic of an ordered secondary structure. 

A solvent which has been foimd to be of great interest in connection with protein conformation studies is ethylene glycol. Sage and Singer (1958, 1962) have investigated in some detail the properties of RNase in pure ethylene glycol, containing added neutral electrolyte. They examined the ultraviolet absorption spectrum, the ionization behavior of the tyrosine residues by spectrophotometric titration experiments, and the optical rotatory dispersion of the system. 

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