Fluorescence Polarization of Pyrenebutyric-Bovine Serum Albumin and Pyrenebutyric-Human Macroglobulin Conjugates

Pyrenebutyric acid when conjugated with proteins has a long fluorescence lifetime in the range of 100 nsec as measured both directly and indirectly. The fluorescence life-times as measured indirectly by the fluorescence depolarization of pyrenebutyric-bovine serum albumin conjugates appears to be a linear function of quantum yield. This dependence was used to predict the lifetimes of the other protein conjugates. The rotational relaxation time of the conjugate of rabbit immunoglobulin G agrees with other determinations with a different chromophore. Conjugates were prepared with human immunoglobulin M, and the measured rotational relaxation times of the conjugate before and after reduction and alkylation correlated with the molecular weights of the 2 molecules. In addition, the reduction of the conjugate is followed, and evidence is presented supporting the concept that a greatly disordered conformation is an intermediate.