Protein optical rotation

My interest at that time revolved around evaluating optical rotary dispersion data [12]. The paired values of optical rotation vs. wavelength were used to fit a function called the Drude equation (later modified to the Moffitt equation for William Moffitt [Harvard University] who developed the theory) [13]. The coefficients of the evaluated equation were shown to be related to a significant ultraviolet absorption band of a protein and to the amount of alpha-helix conformation existing in the solution of it. 

The racemization of the phosphine (118) has been followed by optical rotation. The lack of a solvent effect indicates that there is little change in dipole moment in the formation of the planar transition state. Circular dichroism has been used to study the interactions of nucleotides with proteins and DNA with a histone. Faraday effects have been reviewed. Refraction studies on chloro-amino-phosphines, fluoro-amino-phosphines, and some chalcogenides are reported. 

For instance, one would like to know the types of structures actually present in the native and denatured proteins.. .. The denatured protein in a good solvent such as urea is probably somewhat like a randomly coiled polymer, though the large optical rotation of denatured proteins in urea indicates that much local rigidity must be present in the chain (pg. 4). 

Tanford (1968) reviewed early studies of protein denaturation and concluded that high concentrations of Gdm-HCl and, in some cases, urea are capable of unfolding proteins that lack disulfide cross-links to random coils. This conclusion was largely based on intrinsic viscosity data, but optical rotation and optical rotatory dispersion (ORD) [reviewed by Urnes and Doty (1961) ] were also cited as providing supporting evidence. By these same lines of evidence, heat- and acid-unfolded proteins were held to be less completely unfolded, with some residual secondary and tertiary structure. As noted in Section II, a polypeptide chain can behave hydrodynamically as random coil and yet possess local order. Similarly, the optical rotation and ORD criteria used for a random coil by Tanford and others are not capable of excluding local order in largely unfolded polypeptides and proteins. The ability to measure the ORD, and especially the CD spectra, of unfolded polypeptides and proteins in the far UV provides much more incisive information about the conformation of proteins, folded and unfolded. The CD spectra of many unfolded proteins have been reported, but there have been few systematic studies. 

See Section IV.1 for alternative methods of chiral resolution. Partial chemical hydrolysis of proteins and peptides with hot 6 M HC1, followed by enzymatic hydrolysis with pronase, leucine aminopeptidase and peptidyl D-amino acid hydrolase, avoids racemiza-tion of the amino acids281. The problems arising from optical rotation measurements of chiral purity were reviewed. Important considerations are the nonideal dependence of optical rotation on concentration and the effect of chiral impurities282. 

Kauzmann, W. and R.B. Simpson. 1953. Kinetics of protein denaturation. in. Optical rotations of serum albumin, fS-lactoglobulin, and pepsin in urea solutions. J Am Chem 75 5154-5157. 

Optical Rotation and the Conformation of Polypeptides and Proteins Peter Urnes and Paul Doty... 

Fia. 12. (a) The thermal transition of RNase-A (0.3-0.4 mg/ml) at various pH values. The solvent was either dilute HC1 or 0.04 M glycine buffer for the two most alkaline curves. The change in molar absorbancy at 287 nm is shown as a function of temperature. From Brandts and Hunt (336). (b) The thermal transition as monitored by the change in optical rotation at 436 nm. The protein concentration was 1.9 mg/ml in 0.16 M KC1. From Hermans and Scheraga (337). (c) Comparison of the data from (b) and from a set comparable to (a) where both the absorbance and rotation values have been normalized to fractional conversion to the denatured form. From Hermans and Scheraga (337). 

Fig. 17. Comparison of the transition temperatures for RNase-A (circles), RNase-S (triangles), and S-protein (squares) as determined by optical rotation (open symbols) and ultraviolet absorption difference spectroscopy (filled symbols). Reproduced from Sherwood and Potts (387).

Studies on irradiated solutions of phenolase showed that the enzyme was inactivated. The radiation-induced changes were found to be different from those reported to occur upon denaturation of proteins. Infrared absorption spectra revealed that deamination had occurred. Acid- and basebinding groups were reduced in number rather than increased, and the optical rotation became more dextrorotatory than levorotatory. It was fur-... 

The last subject in the discussion of inherently chiral compounds deals with the analysis of the aminoacids, peptides, and proteins. Most all of the remarks that were made about the steroids and carbohydrates regarding CD detection apply equally well to these. The enantiomeric purity of aminoacids is usually determined by their optical rotations at the sodium-D line. Rotations are normally so small that concentrated solutions and long pathlengths are needed. The detection is enhanced a little if laser illumination is used [66] or if ORD detection is done around 230 nm [71]. Without derivatization, only aminoacids with aromatic substituents are CD active in the near UV. Signals are generally weak and enantiomeric purity measurements are not quantitative. 

Among the properties of amino adds that are most pertinent to the biomedical scientist are their optical rotations, already discussed, which are listed for each amino acid in Table 4.1. Note the dramatic differences between optical rotations in the zwitterionic (water) and fully protonated (HC1) forms. Further, all amino acids absorb ultraviolet light in the range 190-220 nm. The C=0 bond in carboxyl residues is largely responsible. Moreover, aromatic amino acids, especially tryptophan, absorb in the 260-285 nm range. Protein concentrations in solutions are often determined via absorption at 210 or 280 nm. 

Table I shows the optical rotations of five proteins in trifluoroacetic acid solution compared to the optical rotations in aqueous solution. It is evident that most or all of the secondary and tertiary structure has been lost. In Fig. 6, the NMR spectra of 20% trifluoroacetic acid solutions of these proteins are shown. /5-Lactoglobulin, which shows the greatest levorotation, appears also to have the best resolved spectrum, although the others are nearly equivalent. From a knowledge of the...

Table 1. Optical Rotation of Five Globular Proteins in Water and in Trifluoroacetic acid...

Optical rotatory dispersion and circular dichroism (83, 84) can often be of great value, and the spectra are particularly sensitive to the conformation of the protein. Much work remains to be done in this field before the results can be definitely interpreted in terms of the electronic structure of the metal. Magneto-optical rotation and magnetic circular dichroism (MOR and MCD), which are beginning to be applied to porphyrins and haemoproteins, offer much greater promise (30, 85). 

Heat and alkaline treatments have been known since the early part of the century to raoemize amino acid residues in proteins (1,2,). Dakin and Dudley (3) also studied digestibility of casein in vitro and in vivo after hydroxide treatment. Heating casein with 0.5 N NaOH at 37° for about 30 days completely prevented enzymatic hydrolysis and intestinal absorption when the treated casein was fed to a dog. The kinetics of base-catalyzed racemization of proteins was investigated by Levene and Bass (4-6). In these early studies, the extent of racemization was measured by changes in optical rotation. 

From the unique amino acid composition of the gliadin proteins, one would expect a structure that is quite different from globular proteins. However, optical rotation studies have shown that the gliadin proteins possess compact tertiary structures similar to those of globular proteins (4,5). 

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