*2.5. Surface Hydrophobicity (H0)*

The protein surface hydrophobicity was measured using the ANS fluorescence probe as described by Haskard and Li-Chan [23]. A 50 mL aliquot of 0.5% protein solution was added to 10 mmol/L phosphate buffer with pH values of 3.0, 5.0, 7.0, 9.0, and 11.0. Then, the mixtures were stirred for 1 h at room temperature, followed by centrifugation for 30 min at the rate 7500 rpm. The protein concentrations of the supernatant were measured using the Lowry method. After dilution with the same phosphate buffer solution (PBS) (concentration ranging from 0.005 to 0.5 mol/mL), different concentrations of sample solutions were collected (4 mL), to which 40 μL of 8 mmol/L ANS solution (10 mmol/L, prepared with pH 7.0 PBS) was added. After oscillation, the mixtures were held static for 3 min, and the fluorescence intensity of the samples was tested using a G9800A fluorescence spectrophotometer (Cary Eclipse, Agilent, Santa Clara, CA, USA). In this test, the excitation and emission wavelengths were λex = 370 nm and λem = 490 nm, respectively, and the crack was 5 nm. Graphs of fluorescence intensity relative to the protein concentration were plotted. The slope of the initial section is equal to the surface hydrophobicity (*H*0) of the protein molecules.
