Soret CD band

The CD spectra of peroxidases in the heme absorption region are highly sensitive to the conformation of the heme group inside the protein cavity and to the dipole-dipole coupling interactions between the porphyrin and the surrounding aromatic amino acid side chains [122, 123]. In this region, GS horseradish A2 peroxidase presents a band at 311 and the intense CD Soret band at 412 nm with a shoulder at 354 nm (Fig. 11.3). The main differences between the CD spectrum of CIII compared to that of GS are a reduction of the CD band at 311 nm and a red-shift of the Soret CD band to 430 nm with the disappearance of the 354 nm shoulder. Additionally a broad band, possibly composed by a series of shoulders, appears from 350 to 400 nm. During spontaneous decay, the CD spectrum of CIII slowly returns to that of GS, with the characteristic reduction of the Soret band due to porphyrin destruction (see above). 

CD can be used to monitor the phenomenon of heme isomerism in heme proteins. " The isomerism, discovered by LaMar and co-workers, results from the presence of two forms of myoglobin which differ by 180° in the heme orientation about the a, y-methine carbon axis. The two isomers are present in a 9 1 ratio at equilibrium in carbonmonoxymyo-globin (MbCO) but are in a 1 1 ratio in a sample freshly reconstituted from apomyoglobin and heme. Freshly reconstituted MbCO has a Soret CD band with only about half the amplitude of the native form, " suggesting that the minor isomer (at equilibrium) has only a weak Soret CD spectrum. The major form has a strong positive Soret CD band, whereas the minor form has a weakly negative Soret CD band. These results and the CD of the heme undecapeptide from cytochrome indicate that the origin of the Soret CD band must be more complex than proposed by Hsu and Woody, ... 

The intensities of CD bands in the near-UV and visible region of proteins are small compared to those in the far-UV region. They are often about 100 or less (the CD in the Soret band is a notable exception see Fig. 7.14 ). The use of a concentrated protein solution or a cell of long pathlength is limited by the absorbance of the solution (A is one or less). Furthermore, the bands usually overlap and are not easy to delineate. Therefore, Table 7.V lists only the positions and signs of some, but not all, bands of proteins. The near-UV CD spectra may be greatly affected by both the solvent and temperature. Only native proteins in aqueous solutions are listed in the table usually the solvent is phosphate or other buffers or water alone, the pH is neutral or at the isoelectric point of the protein, and the temperature is room temperature or 25°C. Accordingly, experimental conditions are not stated in the table they may be found in Table 7.IV if both far-UV and near-UV spectra of the proteins are available. 

Figure B3.5.5 Near-UV CD spectra. (A) Bovine a -casein peptide under a variety of conditions (data from Alaimo et al., 1999). Peptide concentration 0.631 mg/ml in 2 mM PIPES, 4 mM KCI, pH 6.75 scan rate 40 sec/nm path length 10 mm bandwidth 1.5 nm. The loss of aromatic dichroism with increasing temperature indicates denaturation, which is, however, not complete at 70°C or in 6 M guanidine hydrochloride. The shift in maximum wavelength indicates loss of tryptophan asymmetry, but less so of tyrosine. (B) Seed coat soybean peroxidase under native and denaturing conditions (data from Kamal and Behere, 2002). Protein concentration 15 pM and path length 10 mm. The negative aromatic band centered around 280 nm and the Soret band around 410 nm both disappear at 90°C, indicating the loss of net conformational asymmetry of the aromatic and heme chromophores.

Before discussing the remarkable kinetic inertia of these ternary species it is useful to present the main characteristic of the CuT4-poly-glutamate binary system. The binary complex is chiral and kinetically labile. This is shown by (1) the presence of an induced band in the Soret region of the circular dichroism (CD) spectra (inset of Fig. 3, curve a) and (2) the inversion (in about 10 min) of the same... 

However, these supramolecular ternary complexes behave quite differently compared to the parent binary species. In fact, the addition of a fourfold excess of poly-L-glutamate to ternary complexes built on the poly-D-glutamate does not lead to the inversion of the induced CD signal in the Soret region (even after 5 days following the addition). The only indication of the L-form excess is the inversion of the helix marker bands at 222 nm and 208 nm (Fig. 4). The same behaviour has been observed for the ternary L -supramolecular species upon addition of an excess of... 

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