4.6. Insulin Aggregation Assay

The insulin aggregation assay was performed according to the method described previously with a slight modification [49,50]. Two milligrams of human insulin (FUJIFILM Wako Chemicals, Osaka, Japan) was dissolved in 1 mL of 10 mM HCl and kept at −20 °C. The assay mixture was prepared by mixing 150 µL of acid-denatured insulin (2 mg/mL) and 750 µL of 100 mM sodium phosphate buffer (pH 6.8) containing 400 mM NaCl and 30 µL of 20 mM DTT, rPfPV1-Strep, and H₂O up to 1.5 mL in a quartz cell. The absorbance was measured by observing absorbance at 360 nm at 25 °C using a UV-VIS spectrophotometer (V-650, JASCO, Tokyo, Japan).