0-Structural Study of κ-casein During Amyloid Formation at Different Temperature

K-Casein is a milk protein with a unique pattern of disulfide binding. The protein exhibits varying molecular sizes, ranging from monomer to octamer and above in the absence of a reducing agent. This study investigates the fibril-forming propensities and polymerization of reduced K-casein at temperatures between 25 and 48°C, by Thioflavin T binding assay, Trp fluorescence intensity, Transmission electron microscopy (TEM) CD spectroscopy and HPLC. ThT and TEM data demonstrate that reduced K-casein readily forms amyloid fibrils following reduction of its disulfide bonds which increase with increasing temperature. Our findings suggest that reducing agents and temperature induce fibril formation of K-casein possibly by raising the monomeric state of K-casein and accelerating its interaction with other K-casein molecules and eventually forming fibrils. This is also shown by intrinsic fluorescence experiments on reduced K-casein which reveal that temperature causes an increase in the polarity of the environment of the tryptophans residue. CD spectroscopy also revealed that temperature caused change in the tertiary structure of the protein while the secondary structure showed no considerable change consistent with the hypothesis that the core structure of K-casein is stable. Consistent with this HPLC data, it can be seen that increasing temperature increases aggregation of reduced K-casein