Interfacial Aggregation of Bovine Serum Albumin Related to Crystallization Conditions Studied by Total Internal Reflection Fluorescence

Total internal reflection fluorescence (TIRF) was used to examine adsorption and interfacial aggregation of bovine serum albumin (BSA) from solvent conditions that are known to promote BSA crystallization. In the absence of a crystallizing agent (thermodynamically good solvent conditions), adsorption from a 0.1 mg/mL BSA solution in 50 mM phosphate buffer, pH 6.2, 25°C, results in formation of not more than monolayer protein films on both hydrophilic and hydrophobic surfaces. However, in the presence of ammonium sulfate, which is used as an agent to crystallize BSA, adsorption yields a relatively thick multilayer protein film. The interfacial aggregation is sensitive to hydrophobicity of the substrate as well as to the prehistory of adsorption. A hydrophilic substrate promotes formation of thicker protein films than a hydrophobic one. For example, in the presence of 2.13 M ammonium sulfate, BSA adsorption during approximately 10 h yields approximately a 20-monolayer film on the quartz surface, in contrast to a 6-7 monolayer film on the hydrophobic surface. Rates of multilayer adsorption and desorption and the ultimate thickness of the multilayer protein film critically depend on the concentration of crystallizing agent. The rates correlate with the osmotic second virial coefficient, B22, which has been shown to be a predictor for protein crystallization. In the range of B22 values known as a "crystallization slot," the multilayer adsorption demonstrates a uniform rate of film growth. Noncrystallizing solvents do not exhibit this feature. The TIRF technique can be used to investigate molecular mechanisms of crystal growth and growth cessation as well as epitaxial and pseudoepitaxial growth using protein crystals grown at or attached to the surface. TIRF provides a sensitive and powerful tool to probe in situ and in real time the interactions of dissolved protein with a growing protein solid phase. Copyright 1997 Academic Press. Copyright 1997Academic Press