SURFACE-LINKED MOLECULAR MONOLAYERS OF AN ENGINEERED MYOGLOBIN - STRUCTURE, STABILITY, AND FUNCTION

The maintenance of active conformation and biological function is impo rtant for the development of solid substrate immobilized biomacromolec ules for material applications. A protein monolayer was obtained on Si O2 substrates by chemically linking a site-directed mutant of sperm wh ale myoglobin, A126C, which has a unique and reactive cysteine residue on its surface, to a thiol specific functional group on the silane-de rivatized substrates. Fourier transform infrared (FTIR) spectroscopy o f this protein monolayer suggests that a native-like secondary structu re is retained for the immobilized myoglobin. In addition, the immobol ized myoglobin retains its ability to bind carbon monoxide. Structural changes in the surface-bound protein were examined under variation of temperature, pH, urea concentration, and ethanol content by UV-vis ab sorption spectroscopy. The densely packed protein, roughly 60% surface coverage of the substrate, is as resilient to high temperature, low-p H environment, and high concentration of urea as myoglobin solution is at low concentration. Surprisingly, we found that after immobilizatio n, the protein resists ethanol denaturation more efficiently than a di luted solution of 1 mu M myoglobin with ethanol as the cosolvent. Intr iguingly, the secondary structure of the immobilized myoglobin was pre served after 30 min incubation at 150 degrees C, as determined by FTIR at room temperature.