Studies of the Helix-Coil Transition and Aggregation in Polypeptides by Fluorescence Techniques
The technique of polarization of fluorescence using dye-macromolecule conjugates is a very sensitive hydrodynamic method for studying intramolecular transitions and intermolecular interactions. Several considerations influence the choice of the dye in such conjugates: extinction coefficient, lifetime, effect of pH, and interaction with the macromolecule to which it is coupled. Fluorescein has a higher molar extinction coefficient (3.4 × 104) than DNS (1-dimethylaminonaphthalene-5-sulfonyl chloride) (4.3 ×103); therefore, it is useful when working with small amounts of material. The lifetime of DNS is constant from approximately pH 2 to 14 (2), whereas that of fluorescein is strongly dependent upon pH. The latter varies in an approximately linear fashion from pH 2 (<1 ns) to pH 8 (6 ns); thereafter, it gradually increases (7 ns at pH 11.8). In addition, the lifetime of the dye can also be influenced by the molecule to which it is coupled. The degree of polarization of DNS and its conjugates is not significantly influenced by pH over the range 2.5 to 14; hence, DNS is useful for studying the effect of pH on intra-molecular transitions. On the other hand, fluorescein and its conjugates are quite sensitive to the effects of acid and base, so fluorescein is not useful for such studies (3). Finally, the interaction between the dye and the molecule to which it is attached is different for fluorescein and DNS conjugates. This factor influences measurement of the rotational relaxation time more than that of the transition temperature, i.e., the temperature at which internal rotation or intermolecular aggregation occurs. This effect may be due to the capabilities of the various dyes to interact with different portions of the macromolecule; thus, they would reflect the behavior of different rotational units (3).
KeywordsIntrinsic Viscosity Helical Conformation Glutamic Acid Residue Helix Formation Helical Segment
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