Analytical Sciences

Adsorption Behavior of Cytochrome c, Myoglobin and Hemoglobin in a Quartz Surface Probed Using Slab Optical Waveguide (SOWG) Spectroscopy

Jose H. SANTOS,* Naoki MATSUDA,*  Zhi-mei QI,* Takamitsu YOSHIDA,*^,** Akiko TAKATSU,*** and Kenji KATO***

*Nanoarchitectonics Research Center, AIST, 1-1-1 Higashi, Tsukuba 305-8565, Japan
**Institute of Materials Science, Tsukuba University, 1-1-1 Tennoudai, Tsukuba 305-8577, Japan
***National Metrology Institute of Japan, 1-1-1 Umezono, Tsukuba 305-8563, Japan

Slab optical waveguide (SOWG) spectroscopy was used to observe the adsorption behavior of three important heme proteins, namely cytochrome c, myoglobin and hemoglobin, in a quartz surface. Using prism-coupled polychromatic visible light propagated into a quartz waveguide by internal total reflection, the real-time monitoring of evanescent wave absorption revealed a strong dependence of the protein-surface interaction on the protein concentration, the solution pH and the ionic strength. For the three proteins studied, the absorbance-bulk concentration ratio was higher at low bulk concentrations, and decreased at higher concentrations. For cytochrome c and myoglobin, the absorbance approached a limiting value, but buffered hemoglobin surprisingly did not show any indication of forming a signal plateau. Moreover, the slow introduction of protein into the solution lessened the total adsorbed amount per unit area. These observations suggested a possible conformational transition of the protein molecules at the quartz surface after adsorption. For a bulkier protein, hemoglobin, adsorption onto the quartz surface was enhanced in the presence of a phosphate buffer, while the opposite effect was observed for the smaller cytochrome c and myoglobin molecules. The results of pH studies concurred with the electrostatic interactions predicted from the isoelectric data of proteins and the quartz surface.

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