Reappraisal of the binding processes of N-(3-pyrene)maleimide as a fluorescent probe of proteins.

N-(3-Pyrene)maleimide is known to form fluorescent adducts when reacted with sulfhydryl groups of proteins. Fluorescence properties of the probe, free or conjugated with N-acetylcysteine, were investigated. When conjugated with bovine serum albumin, pyrenemaleimide displayed several lifetime values and heterogeneous quenching effects. This appears to be due to the fact that it can be either covalently attached to -SH groups or adsorbed on hydrophobic areas of proteins. The product (bovine serum albumin labeled with N-(3-pyrene)maleimide) exhibited time-dependent spectral changes related to hydrolysis of the probe; this reaction occurred mainly with adsorbed probe. These results show that several spectroscopically different products, namely, covalently bound probe, adsorbed probe (2 to 8 mol/mol of protein), and hydrolyzed probe contribute collectively to the emission of the labeled protein. It is concluded that the lack of specificity limits the use of pyrenemaleimide as a fluorescent probe of proteins.