Multilayer adsorption of lysozyme on a hydrophobic substrate

Abstract

Macromolecular adsorption is known to occur as a complex process, often in a series of steps. Several models are discussed in the literature which describe the microscopic structure of the adsorbate. In the present study we investigated the adsorption of hen egg white lysozyme on alkylated silicon oxide surfaces. A combination of fluorescence excitation in the evanescent field and fluorescence recovery after photobleaching allowed us to measure the amount of adsorbed fluorescent lysozyme and the equilibrium exchange kinetics with molecules in solution. We found that a model with at least three classes of adsorbed molecules is necessary to describe the experimental results. A first layer is formed by the molecules which adsorb within a short time after the beginning of the incubation. These molecules make up approximately 65% of the final coverage. They are quasi-irreversibly adsorbed and do not measurably exchange with bulk molecules within one day even at temperatures up to 55 degrees C. A second layer, which reaches equilibrium only after several hours of incubation, shows a pronounced exchange with bulk molecules. The on-off kinetics show a distinct temperature dependence from which an activation barrier of delta E approximately 22 kcal/mol is derived. A third layer of molecules that exchange rapidly with the bulk can be seen to comprise approximately 10% of the total coverage. The exchange rate is on the order of fractions of a second. The binding of the latter two classes of adsorbed molecules is exothermic. From the temperature dependence of the coverage, the binding enthalpy of the slowly exchanging layer was estimated to be delta Hads approximately 3.8 kcal/mol. The second and third class of molecules remain enzymatically active as a muramidase, which was tested by the lysis of the cell walls of Micrococcus lysodeiktikus. The molecules in the first layer, on the other hand, showed no enzymatic activity.

Reduced protein adsorption at solid interfaces by sugar excipients

“…The HEWL adsorbed amount continued to increase even after 24 h where the adsorbed amount was greater than that predicted for monolayer coverage. This is in agreement with the data of (Schmidt et al, 1990), who found that HEWL formed multilayers on alkylated silicon oxide surfaces. RNase A clearly reached a plateau in less than 30 min, and no further increase in adsorbed protein was detected.…”

Quantification of the surface density of a fluorescent label with the optical microscope

“…However, as we previously mentioned, calibration of TIRFM can be achieved by other methods. [14][15][16] Our data on protein adsorption to glass illustrate the utility of FM in surface analysis. In addition to measuring the average amount of bound protein, we observed protein clustering.…”

“…Evaporation of a fluorescent solution on the surface 13 does not guarantee preservation of the quantum yield. In TIRFM 5 calibration methods were developed, [14][15][16] but these methods cannot be extended to conventional microscopy. These and some other methods were previously reviewed.…”

Formation of Adsorbed Protein Layers

“…Note, however, that in this seemingly simple system there are numerous complications, since this protein has been reported to display a side-on to end-on transition (65), conformational changes on adsorption (68), pH-dependent interfacial aggregation (67), and concentration-dependent multilayer adsorption (64 -66). In relation to the latter it is interesting to note that there seem to be large differences in the state of adsorption for early and late adsorbing proteins, since the former are poorly exchangable, whereas the latter are more extensively exchangable (69,70). Interestingly, this is coupled to a large loss in enzymatic activity for the initially adsorbing proteins, whereas the enzymatic activity of the proteins adsorbing later in the adsorption process display an effectively higher activity (69).…”

“…In relation to the latter it is interesting to note that there seem to be large differences in the state of adsorption for early and late adsorbing proteins, since the former are poorly exchangable, whereas the latter are more extensively exchangable (69,70). Interestingly, this is coupled to a large loss in enzymatic activity for the initially adsorbing proteins, whereas the enzymatic activity of the proteins adsorbing later in the adsorption process display an effectively higher activity (69). It is interesting to note that the readily exchangable fraction increases and the irreversibly adsorbed fraction decreases with an increasing bulk protein concentration (70).…”