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Two efficient enzyme conjugation techniques have been explored by exploiting the reactions of bromomaleimides. The conjugations utilize monobromo-and dibromomaleimides, which have been reacted with reduced disulfide bonds or terminal amines in -chymotrypsin and human lysozyme. These reactions allow the formation of dithio-, monoamino-and aminobromomaleimides, which are solvent-dependent fluorophores and have a handle for further functionalization, which allowed fluorogenic PEGylation via this technique. In this work, the efficiency of these maleimide conjugations was monitored and the fluorescence of the resulting conjugates was examined. The quantum yields of the small-molecule maleimide conjugates were calculated, but are low due to solvent quenching effects. Catalytic activities of the conjugate enzymes were compared to those of their respective native enzymes, which show no discernible effect of the modifications on enzymatic activity or stability at room temperature.

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