Extrinsic cotton effects in dye-bovine plasma albumin adducts.

With the growing realization that many of the unique properties of macromolecules can be attributed to their specific three-dimensional structure, various chemical and physical techniques have been utilized to study macromolecular conformation. Of these techniques, spectropolarimetry has proved itself to be particularly useful. Studies of the optical rotatory dispersion of macromolecular solutions, effects which arise from the intrinsic asymmetry of the macromolecules, have yielded a wealth of information.l3 Occasionally, a low-molecular-weight, optically symmetric chromophore may be rendered virtually asymmetric when it is bound by an optically active macromolecule. A number of planar dyes without optical activity in the free state bind to macromolecules and give rise to complexes which exhibit Cotton effects in the neighborhood of the absorption maximum of the dye.4-7 The dye-poly-a,L-glutamic acid and dye-poly-a,D-glutamic acid complexes studied by Stryer and Blout4' I showed an induced Cotton effect when the polypeptide was in the helical form, but not when it assumed a random-coil conformation. The induced Cotton effect was positive for a right-handed helix and negative for a left-handed helix. Similar extrinsic Cotton effects were observed in dyepolynucleotide complexes6 and in dye-mucopolysaccharide complexes.7 In the case of naturally occurring proteins, extrinsic Cotton effects have only been observed when the protein contained a chromophoric prosthetic group such as heme, or bound an absorbing coenzyme such as NADH2.8 Extrinsic Cotton effects induced in dyes covalently linked to bovine plasma albumin are reported in this communication. Studies of such dye-protein complexes should extend the usefulness of spectropolarimetry by providing a means for evaluating the conformation of a restricted region of naturally occurring proteins, and for studying the conformation of a given protein in a mixture of proteins. Materials and Methods.-Commercial preparations of acriflavine (National Aniline), rhodamine-B (National Aniline), fluorescein isothiocyanate (Nutritional Biochemicals), and lissamine rhodamine-B (Calbiochem) were used. Zinc-free acridine orange was a gift of General Aniline Corp. p-Dimethylaminobenzeneazophenylmercuriacetate and p-hydroxybenzeneazophenylmercuriacetate were synthesized as described previously.9 The mercurated derivatives of fluorescein were synthesized by reacting fluorescein with mercuric acetate in a molar ratio of 1: 1.6.10 The monomercurated and dimercurated derivatives of fluorescein were isolated by chromatography on a cellulose column using 0.5 N NH40H as developer. Chromatographically pure monomercurated dibromofluorescein was a gift of Dr. H. A. Brown Dunning, Jr. Bovine plasma albumin was purchased from Pentex, Inc. Poly-a,L-glutamic acid was purchased from Pilot Chemicals. Optical rotatory dispersion measurements were obtained using a Cary model 60 spectropolarimeter. Results and Discussion.-While complexes of acridine orange, acriflavine, and rhodamine-B with polyglutamic acid in the helical form showed the Cotton effects