Pseudoreference state

ORD data, however, can be obtained for a number of systems of interest where little or no data are possible by circular dichroism, or when the absorption bands cannot be reached due to the high absorbance of a solvent or because the absorption bands of interest are beyond the usual 180-190 nm limit of solution studies. Also, most of the optical rotation data in the literature prior to the mid-1960s are reported in terms of optical rotatory dispersion. Additionally, when correcting the distortions in the CD spectra of biomembranes (see below), it is convenient to have the ORD curve of what is referred to as the pseudoreference state. 

Because of the approximation that = Aobsc this approach will give rise to minimal correction values and an iterative procedure can be used to obtain the desired accuracy [112]. If the particulate system is best described as a vesicle, which is generally the case for membranes, an equivalent A for Qa (vesicles) [114] can also be obtained from Table 3. Of course, the problem at this stage is that in general an equivalent solution absorption spectrum will not be known for the suspension of interest. By also utilizing the circular dichroism data, however, an equivalent solution absorption spectrum can be determined as outlined below. This is called the pseudoreference state approach. 

Figure 45. Circular dichroism spectra of purple membrane. Curve a, suspension of previously frozen purple membrane. Curve b, suspension of curve a after sonication for 1 minute. Note the large increases in ellipticity that accompany decrease in particle size due to sonication. Curve c, purple membrane dissolved in 80% trifluoroethanol (TFE)/20% water. Characteristic a-helix CD spectrum is obtained. This is a molecularly dispersed state. Curve d, corrected ellipticities using the pseudoreference state of Figure 46B with differential scatter correction using proportionality to A[0]Js for PGA given as curve c of Figure 42. Adapted, with permission, from [125].

This value of [0]ci is an underestimate of about 10-30%. When a molecularly dispersed state is found which obeys both equations 84 and 86, then it is called the pseudoreference state (prs) and its absorption curve is to be used to calculate the values of Ga and Q for the corrected ellipticity curve. This is sufficient to provide reasonable estimates of the mean residue ellipticities at 224 and 190 nm. Since these are the important values for estimating the amount of a-helix, the result is sufficient. The differential light scattering term, however, can be estimated by using the ORD curve of the pseudoreference state as outlined in equations 81 and 82. This is more of a cosmetic effect to see that the entire curve generally conforms to the a-helix pattern implied by the mean residue ellipticities at 224 and 190nm using equation 44. 

Figure 46. A, Absorption curves for the purple membrane sonicated 1 minute (curve b) and the pseudoreference state as defined in equations 84 and 86, curve a. Reproduced, with permission, from [125], B, Circular dichroism spectra of purple membrane. Curve a, suspension of fresh purple membrane preparation sonicated for 1 minute. Curve b, pseudoreference state, 20% triiluoroethanol (TFE)/80% water with 0.4% SDS. This state was defined by equations 84 and 86. Curve c, pseudoreference state optical rotatory dispersion curve plotted on the right-hand ordinate. Dashed curve d, corrected ellipticities using 10" as the...

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