Protein conformation in cell membrane preparations as studied by optical rotatory dispersion and circular dichroism.

To elucidate the basic structure of biological membranes, experimental evidence concerning the detailed molecular organization of the proteins of membranes is required. This evidence has been almost totally lacking or ambiguous up to the present time. The Cotton effects due to the peptide bond ultraviolet absorption bands, as studied by ORD and CD, have recently proved extremely valuable in characterizing the molecular conformations of polypeptides and soluble proteins. In this paper, we report the results of ORD and CD studies in the ultraviolet on membrane preparations from human red blood cells and from B. subtilis in several solvent media. Materials and Methods.-Membrane preparations: Red blood cell membranes were prepared by the method of Dodge et al.1 from fresh human blood. Heme analyses' of several preparations showed uniformly that no more than 0.5% of the total protein present was hemoglobin. Membrane preparations from B. subtilis, strain 168, were prepared essentially by the procedure of Salton and Ehtisham-Ud-Din.' A low-speed centrifugation step was inserted to remove bacterial debris before the 39,000 X g centrifugation that brought down the membranes.3 Microscopic examination of both of these membrane preparations showed them to consist almost entirely of whole membranes. Solutions of each membrane preparation in 2-chloroethanol were prepared at 40 from lyophilized portions of the preparations. Lipids were removed from red cell membranes by an isopropanol-chloroform extraction procedure.4 The precipitate which remained (mainly the protein portion of the membrane) was about 50% soluble in 2-chloroethanol. After removal of the solvent from the extracted membrane lipids, these were dissolved in 2-chloroethanol for ORD study. Lipid concentrations were determined by the organic phosphorus assay of Bartlett.' Reagents: Reagent grade 2-chloroethanol from several commercial sources was purified by distillation under reduced pressure. Sufficient concentrated hydrochloric acid was added to the purified 2-chloroethanol to give a pH of 2.69-2.71 when 1 vol of 2-chloroethanol was diluted with 9 vol of water." ORD and CD measurements and calculations: ORD and CD measurements were carried out at room temperature with a Durrum-Jasco UV-5 instrument.7 To calculate [m'], the reduced mean residue rotation, and 0, the mean molar ellipticity, the protein concentration is required. This was determined from the OD280 of 2-chloroethanol solutions prepared from measured amounts of the original membrane suspension. The OD280 of the 2-chloroethanol solution was related to the protein content by total acid hydrolysis and amino acid analysis on a modified Beckman 120 analyzer.8 The protein concentration was then calculated by summing all the amino acid residues and assuming a mean residue weight of 114. The refractive indices of the aqueous media were assumed to be identical to pure water.9 For 2-chloroethanol, the refractive index measurements of Foss and Schellman'0 were extrapolated into the wavelength region of interest. Results: The ORD spectrum of intact red cell membranes in dilute phosphate buffer is shown in Figure 1. For comparison, the spectra are shown of poly-L-lysine in the a-helix, ,B, and random coil conformations." The membrane spectrum shows a peak and trough at wavelengths similar to the a-helix spectrum with the interesting exception that these wavelengths are shifted about 2 mg toward the red. Parameters of the ORD spectra are listed in Table 1. The CD spectrum of intact