FLUORESCENCE POLARIZATION OF HUMAN GAMMAG-IMMUNOGLOBULINS

The fluorescence polarization of 1-dimethylaminonaphthalene-5-sulfonyl conjugates of human [gamma]G-im-munoglobulins (DNS-IgG) has been measured under various conditions. Rotational relaxation times at 25 degrees were calculated from experiments in which the viscosity of the solvent was varied by altering the temperature. Different rotational relaxation times were obtained when the solvent viscosity was altered by addition of sucrose at fixed temperatures. The results are explained by assuming that DNS groups covalently bound to the immunoglobulin undergo thermally activated rotations independent of the rotation of the macromolecule. This hypothesis was tested by experiments which showed that addition of a DNS conjugate of a-aminocaproic acid to solutions of DNS conjugates of immunoglobulin mimics the effect of temperature on the immunoglobulin conjugates in the absence of any fluorescent small molecules. More-over, on the basis of this hypothesis, the polarizations in heated solutions were calculated from polarizations of DNS-IgG measured in sucrose solutions at fixed temperatures (sucrose isotherms). A rota-tional relaxation time of approximately 221 nsec was obtained both for human [gamma]G-immunoglobulin and human [gamma]G-myeloma protein. This value is in agreement with reported estimates obtained by other relaxation methods and is considerably higher than values previously obtained from fluorescence polarization measurements. An effect of the wavelength of excitation on the polarization of fluorescence was also noted. This effect appeared to be related to the presence of different environments of covalently bound DNS groups. The present findings are consistent with rodlike models of the IgG and incompatible with extreme molecular flexibility.