Cysteine absorption spectrum

The blue color of these "type 1" copper proteins is much more intense than are the well known colors of the hydrated ion Cu(H20)42+ or of the more strongly absorbing Cu(NH3)42+. The blue color of these simple complexes arises from a transition of an electron from one d orbital to another within the copper atom. The absorption is somewhat more intense in copper peptide chelates of the type shown in Eq. 6-85. However, the -600 nm absorption bands of the blue proteins are an order of magnitude more intense, as is illustrated by the absorption spectrum of azurin (Fig. 23-8). The intense blue is thought to arise as a result of transfer of electronic charge from the cysteine thiolate to the Cu2+ ion.520 521... 

The molecular weight of 320,000 obtained for the muscle enzyme from sedimentation-diffusion data at 2-6 mg/ml and v = 0.75 (132) is to be compared with 270,000 obtained by Wolfenden et al. from s20,w = 11.1 S and D2 ,w = 3.75 X 10 7 cm2 sec1, and v = 0.731 calculated from the amino acid content (92). The rabbit muscle enzyme has a normal amino acid content, that is, no unusually low or large amount of a particular amino acid was found. Of the 32 cysteine/half-cystine residues per mole based on a molecular weight of 270,000, 6.2 were rapidly titrated with p-mercuribenzoate (92). Typical protein absorption spectra were reported for elasmobranch fish (126), carp (125), rat (127), and rabbit muscle enzyme (68). An E m at 280 nm = 9.13 has been reported for the rabbit muscle enzyme (133). The atypical absorption spectrum with a maximum at 275-276 nm observed by Lee (132) is indicative of contaminating bound nucleotides. 

Bovine ROS membranes show a CD band at ca. 280 nm attributed to tt-tt transitions of aromatic residues and n-ir transitions of cysteine, as well as two maxima in the visible region at ca. 340 nm and 490 nm (the / and a bands) corresponding to the cis and main peaks of the absorption spectrum. The intensity of the a band in the CD is species-dependent, but is always somewhat blue-shifted relative to the Xmax in all species, and this shift is promoted by detergent solubilization. Strong micellar effects have been observed in the intensity of both a and / CD bands [44] (Fig. 4). 

Figure 19.31. Structure of a Phycobilisome Subuuit. This protein, a phycoerythrin, contains a phycoerythrobilin linked to a cysteine residue. The inset shows the absorption spectrum of a phycoerythrin. 

The visible absorption spectrum of a solution containing a known concentration of nitrated protein is measured in a solution buffered at pH 9.0, and the absorbance at the maximum (near 428 nm) used to calculate the nitrotyrosine content ( 428nm for the nitrophenoxide ion is 4200). The tyrosine and nitrotyrosine content of the modified protein should also be determined by amino acid analysis. If the sum of these values does not add up to the tyrosine content of the unmodified protein, intra- or intermolecular cross-linking may have occurred. The amino acid analysis may also reveal whether other side-reactions have taken place. Particular attention should be paid to the half-cystine, cysteine, methionine, histidine and tryptophan contents of the modified proteins. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate offers a rapid and highly sensitive way of detecting products of intermolecular cross-linking. Such products are readily removed by gel filtration. 

Lead bound to cysteine residues in proteins results in the appearance of several intense absorption bands in the ultraviolet (UV) region of the absorption spectrum (37, 50-53, 89, 90). These absorption bands have been used successfully in our laboratory and elsewhere to monitor the stability of lead-protein interactions (37, 53) (discussed in detail in Sections IV and VI) but there has not yet been a detailed study of the electronic origin of these bands. Here, we review previous work on the theory of CT transitions in related systems and then apply those ideas to the spectra observed for lead in proteins. 

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