Multiple isoelectric forms of detergent-solubilized bovine rhodopsin. II. Spectral analysis.

Academic Article

Abstract

  • The absorption and circular dichroic spectra of three stable isoelectric forms of purified rhodopsin (in Emulphogene BC 720) with isoelectric points of 5.19, 5.58 and 6.14 were analysed over the accessible wavelength region of 190-800 nm. It was found from the spectral analysis of the 5.58 and 6.14 forms that the tertiary structure of these isoelectric forms was different, without any change being observed in the secondary structure of these proteins. The difference in structure was removed by denaturation with SDS. Specifically, the aromatic amino acid residues of the 6.14 isoelectric form were suggested to be in a more polar environment as compared to those for the 5.58 isoelectric form. The changes of structure in the microenvironment of retinal in these proteins were minor in comparison to the tertiary changes observed in the proteins. This indicated that the site of charge perturbation is not localized in the retinylidene microenvironment. Furthermore, the spectral features of these isoelectric forms were features common to the spectra of both purified unfocused rhodopsin and crude rhodopsin, suggesting that the isoelectric forms are not a consequence of purification. Spectral analysis of the 5.19 isoelectric form indicated that this form of rhodopsin had an increased tendency to aggregate after focusing. Based on the denaturation properties of these isoelectric forms, it was shown that the 5.19 and 5.58 isoelectric forms had different conformations. It was concluded from this study that preparations of 'purified' rhodopsin are a heterogeneous mixture of stable proteins with different tertiary structures and different isoelectric points.
  • Published In

    Keywords

  • Animals, Cattle, Circular Dichroism, Isoelectric Focusing, Retinal Pigments, Rhodopsin, Solubility, Spectrophotometry
  • Author List

  • Muccio DD; Plantner JJ; Kean EL
  • Start Page

  • 320
  • End Page

  • 327
  • Volume

  • 744
  • Issue

  • 3