Absorption of human lysozyme and adsorbate enzyme activity as quantified by means of total internal reflection fluorescence, 125I labelling and ESCA

• The adsorption of human lyozyme to bare mica and mica hydrophobised with a C20NH2/C20OH Langmuir-Blodgett film was quantified by means of ESCA, radiotracer technique and total internal retlection 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 rougly corresponded to a side-up monolayer, 2.2-2.5 mg m-2. In contrast, on hydrophobised mica, a gradual increase in adsorption was obseved over the whole concentration range studied, 10-3-10 mg ml-1. The adsorbed amount in equilibrium with a 1 mg ml -1 slution was 1.4 mg m -2. At concentrations above phobised mica surface. These results are consistent with a strong electrostatic force dominationg the adsorption on bare mica and a weak hydrophobic driving force for adsorption on hydrophoised 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, can be rationalized by a change in 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 125 I labelling measurements can be explained by an apparent sample area which is lager 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 hydrophilised 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-B-chitotrioside, on the lysozyme adsorbate layers.

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