Amino acid sequences absorption

Since aromatic amino acids and cysteine are absent, there is no protein absorption above 270 nm. Metallothioneins exhibit a broad absorption peak, with the maximum at 190 mn. Absorptions due to the metal-thiolate complexes show as shoulders at 250 nm (Cd), 220 nm (Zn) and 270 nm (Cu).1458,1459 Theoretical predictions based on the amino acid sequence of the peptide chain indicate that the or-helical conformation is forbidden, and /3-structure is almost impossible to attain. CD and NMR studies on both the metal-containing and metal-free protein confirmed the predictions.1459 1460 However, metallothioneins are stable to tryptic digestion and the slow exchange of many peptide hydrogens of metallothionein with those of the solvent suggest that the protein has a compact and well-defined tertiary structure. 

IR spectroscopy is not a very sensitive analytical tool and is, therefore, not well suited to the detection of small amounts of material. If, however, intermediates have intense and well-resolved IR absorptions, the progress of their chemical transformation can be followed by IR spectroscopy [83,88,91-93], Near-infrared spectroscopy, in combination with an acousto-optic tunable filter, can be sufficiently sensitive to enable the on-bead identification of polystyrene-bound di- and tripeptides, even if the peptides have very similar structures (e.g., Leu-Ala-Gly-PS and Val-Ala-Gly-PS) or differ only in their amino acid sequence (e.g., Leu-Val-Gly-PS and Val-Leu-Gly-PS) [94]. Special resins displaying an IR and Raman barcode have been developed, which may facilitate the deconvolution of combinatorial compound libraries prepared by the mix-and-split method [48]. 

Direct labeling of a biomolecule involves the introduction of a covalently linked fluorophore in the nucleic acid sequence or in the amino acid sequence of a protein or antibody. Fluorescein, rhodamine derivatives, the Alexa, and BODIPY dyes (Molecular Probes [92]) as well as the cyanine dyes (Amersham Biosciences [134]) are widely used labels. These probe families show different absorption and emission wavelengths and span the whole visible spectrum (e.g., Alexa Fluor dyes show UV excitation at 350 nm to far red excitation at 633 nm). Furthermore, for differential expression analysis, probe families with similar chemical structures but different spectroscopic properties are desirable, for example the cyanine dyes Cy3 and Cy5 (excitation at 548 and 646 nm, respectively). The design of fluorescent labels is still an active area of research, and various new dyes have been reported that differ in terms of decay times, wavelength, conjugatibility, and quantum yields before and after conjugation [135]. New ruthenium markers have been reported as well [136]. 

A ferric NHase from Rhodococcus sp. N-771 [54], which is probably the same as that from Rhodococcus sp. N-774 because of the coincidence in amino acid sequences, shows a photosensitive phenomenon both in vivo and in vitro [55,56], Its NHase activity is increased by exposure to near ultraviolet light. Absorption and fluorescence spectra indicated that the chromophore involved in the photoactivation is the iron complex. Crystallographic parameters of the Rhodococcus sp. N-771 NHase have been determined by precision X-ray diffraction studies [57]. 

Hemoglobin, absorption spectra, 317-319 Hemoprotein(s), amino acid sequences, 371... 

Figure 32.27. Comparison of the Amino Acid Sequences of the Green and Red Photoreceptors. Open circles correspond to identical residues, whereas colored circles mark residues that are different. The differences in the three black positions are responsible for most of the difference in their absorption spectra.

The amino-acid sequence deduced for the M. thermoautotrophicum SOD most closely resembles bacterial, archaeal and eucaryal Mn-SODs, however atomic absorption spectroscopy has established that this is a Fe-SOD [305]. In contrast to other Fe-SODs, including the Fe-SOD from Methanobrevibacter smithii [302], this enzyme is resistant to azide and hydrogen peroxide, features it has in common with Mn-SODs [305]. Amino-acid residues identified as metal ligands or active sites are, however, conserved in both Mn-SODs and Fe-SODs, implying that all these enzymes have a common ancestry and similar enzyme mechanisms [63]. 

For many applications it is important to know what the loading actually is (e.g. to compare activity of different enzymes immobilized to the same loading). Loading determination of an immobilized enzyme is often possible by methods such as quantitative amino acid analysis or active site titration. The latter has the advantage that only active enzyme is quantified. Typically, however, loading is determined indirectly during the immobilization process by measuring enzyme concentration in the aqueous solution before and after incubation with the carrier. The concentration of a pure enzyme in aqueous buffer can be determined from the absorption at 280 nm. The specific absorption can be calculated from the amino acid sequence of the enzyme. For less pure enzyme solutions, the total protein content can be determined with Bradford, BCA or other assays. 

Ultraviolet Absorption The ultraviolet absorption characteristics of the aromatic side chains of the primary amino acid sequence are often used to estimate an extinction coefficient for a protein. The extinction coefficient is calculated based upon the number of tyrosine, tryptophan, and phenylalanine (aromatic) residues in the protein. Phenylalanine has a relatively weaker absorptivity and may not be included in the calculations. One advantage of this approach is the fact that it is relatively nondisruptive, leaving protein conformation undisturbed. In addition, the same solution used for the concentration assessment can be removed from the cuvette and used for additional experiments. The measurement is based upon Beer s law ... 

The crystalline preparation of E. coli cytochrome 6-562 172,173) shows absorption peaks at 418 nm in the oxidized form and peaks at 427, 531.5, and 562 nm in the reduced form. The cytochrome does not combine with CO in the pH range of 3.0-10.5 but at a higher pH CO causes a small shift of the a maximum. It does not react with cyanide or azide. Its molecular weight is calculated to be 12,000, 11,900-12,700, and 11,954, based on the heme content, ultracentrifugal data, and amino acid analysis, respectively. The amino acid sequence of cytochrome 6-562 has been determined Wl). As shown in Table VII this protein is composed of 110 amino acid residues, and lacks CySH and tryptophan. The cytochrome molecule possesses only two histidines, both of which probable coordinate to heme. Therefore, the cytochrome may be said to contain minimal numbers of histidines which are necessary to keep protoheme in the cytochrome molecule. However, it is also possible that one of the two histidines, especially near the carboxyl terminus, does not par-... 

N-Terminal amino acid sequence of human serum transferrin M.-H. Metz-Boutigue el al. Biochrm. Biophys. Acta 670, 243 (I9S1). Absorption max of human serum Fe + -transferrin about 465 nm (E 0,57) uv max 280 nm (E1S, 14.3). 

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