ABSORPTION OF HUMAN LYSOZYME AND ADSORBATE ENZYME-ACTIVITY AS QUANTIFIED BY MEANS OF TOTAL INTERNAL-REFLECTION FLUORESCENCE, I-125 LABELING AND ESCA

The adsorption of human lysozyme to bare mica and mica hydrophobized with a C20NH2/ C20OH Langmuir-Blodgett film was quantified by means of ESCA, radiotracer technique and total internal reflection fluorescence (TIRF). The ESCA isotherm on bare mica showed a sigmoidal shape with a transition region in the concentration range 0.02-0.04 mg ml-1 and a semiplateau region above 0.05-0.2 mg ml-1. The adsorption values here roughly corresponded to a side-up monolayer, 2.2-2.5 mg m-2. In contrast, on hydrophobized mica, a gradual increase in adsorption was observed over the whole concentration range studied, 10-3-10 mg ml-1. The adsorbed amount in equilibrium with a 1 mg ml-1 solution was 1.4 mg m-2. At concentrations above 2 mg ml-1, where dimers appear in solution, multilayers of lysozyme were formed on the hydrophobized mica surface. These results are consistent with a strong electrostatic force dominating the adsorption on bare mica and a weak hydrophobic driving force for adsorption on hydrophobized mica. Although the isotherm shapes were similar, the absolute adsorption values obtained from TIRF and radioactive measurements were considerably different from those obtained from ESCA. The lower apparent adsorption values obtained from TIRF using fluorescein-labelled lysozyme, e.g., roughly half of the ESCA values, can be rationalized by a change in fluorescence quantum yield of the fluors on adsorption or possibly by preferential adsorption of unlabelled lysozyme. The apparent two to three times higher adsorption values obtained from 125I labelling measurements can be explained by an apparent sample area which is larger than the geometric area due to adsorption of labelled lysozyme in the interstices between mica layers. The normalized adsorption of fluorescein-labelled polyclonal anti-lysozyme antibodies was similar on bare and hydrophilized mica, around 0.9 mole/mole for a surface equilibrated in a lysozyme solution at 0.02 mg ml-1, and 0.3 mole/mole at 2.0 mg ml-1 (size exclusion). The steady-state binding and steady-state enzymatic cleavage of a fluorogenic lysozyme substrate, 4-methylumbelliferyl N,N′,N′' -triacetyl-β-chitotrioside, on the lysozyme adsorbate layers were studied in a qualitative manner. Relative binding and relative cleavage rate was larger on the hydrophobized mica than on bare mica, similar at low and high surface concentrations. © 1990.