Electronic spectra proteins

Figure 97 Blue copper protein (a) electronic spectrum (b) ESR spectrum (-------------- blue copper ------- normal copper) ...

We explored the detection and quantification of specific functional groups in proteins using quantitative specific chemical modifications that contain elements with high detection sensitivity. Fluorine was introduced specifically into Bovine Serum Albumin (BSA) by N-trifluoracetylation of the e-amino group of lysine using ethyl thiotrifluoracetate (47). The fluorine electron spectrum from N-trifluoracetylated BSA is shown in Figure 18. 

The physical characteristics reported for 5 are striking, especially in their seemingly close relationship to those observed for the protein oxy-Hc. The Cu...Cu distance in 5, its diamagnetism (normal H NMR Evans susceptibility), and the electronic spectrum with 349 ran (8 = 21,000) and 551 ran (8 = 800) bands closely resemble the properties of oxy-Hc. The particularly low value (even for peroxide) of the O—O stretch of 741 cm1 (resonance Raman) in 5 also matches corresponding values seen in oxy-Hcs (—750 cm-1) this latter property has been accounted for in theoretical studies [110], which show that the unoccu-... 

Figure 1 Spectral characteristics of oxidized Cu, Zn SOD (a) X-band EPR spectrum, 77 K (Used with permission from Ref 1. 1985 Division of Chemical Education, Inc) (b) room-temperature electronic absorption spectrum [protein] = 35mgmL ...

The electronic spectrum of the dimanganese(III) form of catalase from Thermus thermophilus shows an intense absorption at 450 nm with a shoulder at 500 nm. EPR studies show that Mn Mn , Mn Mn , and Mn Mn forms are also accessible and the Mn Mn protein exhibits a 16-line spectrum, characteristic of a p-oxo bridged system 202). Comparison with the model complexes leads to prediction of p-oxo-bis(/[i-carboxylato)dimanganese(III) structure for the catalase. X-ray crystal data (203) have indicated an Mn—Mn distance of 3.6 3 A for the enzyme this appears to be inconsistent with the above proposal but, as the X-ray data are at rather low (3 A) resolution, this may be the least reliable piece of data. 

Type 1 600 nm 1000-4000 Responsible for the blue color of blue oxidases and electron-transfer proteins, L > M charge transfer spectrum of Cu—S bond... 

For spectroscopic determinations of the amounts of protein material it should be noted that SOD absorbs, in the UV region, with a maximum at 265 nm. Different extinction coefficients were found for various isoenzymes, as a consequence of the different primary sequence. For example, Emax is 15,900 M cm, for the human enzyme, and 10,300 Af cm for the bovine enzyme (2, 103). In the visible region of the electronic spectrum the holoenzyme has an absorption maximum at 680 nm [e = 300 M cm (2)]. The CU2E2 enz5nme has a typical absorption with A ,ax at 700 nm, at pH 6 (183). The two metal binding sites in SOD are largely determined by the tertiary structure of the protein, so that a number of M2N2SOD derivatives can be prepared (Fig. 25) with coordination properties similar to those seen for Cul Zn SOD and Cu Zn SOD. 

The electronic spectrum of the B2 subunit ( ax = 455, 485, and 615 nm) closely resembles those of Mn-catalase and synthetic tribridged Mn 0 complexes (8). The metal site was thus proposed (229) to be analogous to the diiron center of the enzyme from E. coli. This analogy may be reasonable as iron restores 50-70% of the activity in protein derived from Mn-deprived cells (230). Similar to the enzyme from E. coli, the Mn-containing ribonucleotide reductase is inhibited by hydroxyurea and au-... 

This method was also used to study the electronic spectrum of imidazole. Section III.A.7 is devoted to the excited states of chromo-phores in proteins, and the imidazole results are presented there. 

The high sensitivity of the ER method benefits bioelectrochemists in the detection of the redox reaction of the electron transfer proteins. Even for an adsorption monolayer of proteins, the superficial density of the electroactive center is much smaller than that of small molecules, especially when the molecular weight of the protein is several kilo-Daltons. The redox reaction of adsorbed protein buried in the double-layer charging current in the voltammogram can be detected by the ER method. Ikeda and coworkers succeeded in the clear observation of the ER spectrum and ER voltammogram of a heme c in an adsorbed protein (alcohol dehydrogenase) of ca. 140 kD containing hemes and PQQ, while direct redox reaction could not be detected by cyclic voltammetry [90]. 

Fig. 6. Cyclic voltammograms (fourth scan, 20 mVs" ) of horse cytochrome c at various types of pyrolytic graphite electrode. Protein is 0.15 mM in 5 mM Tricine and 0.10 M NaCI at pH 8. Temperature 20 °C. In each case the corresponding X-ray photo-electron spectrum of the graphite surface is shown. The scale enlargement for the O, peak is x 3...

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