Fourier transform infrared spectroscopy provides an evidence of papain denaturation and aggregation during cold storage

• Published in: Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy Molecular and biomolecular spectroscopy, 2015-11, Vol.150, p.238-246
• Main Authors: Rašković, Brankica, Popović, Milica, Ostojić, Sanja, Anđelković, Boban, Tešević, Vele, Polović, Natalija
• Format: Article
• Language: English
• Subjects: Aggregation; Cold denaturation; Cold inactivation; Cold stability; FT-IR; Papain
• ISSN: 1386-1425
• DOI: 10.1016/j.saa.2015.05.061

[Display omitted] •Papain was the most stabile from 45 to 60°C with ΔG°321 13.9kJ/mol and Tm value 84°C.•Cold storage of papain gave an increase of 40% of intermolecular β-sheet content.•Six freeze–thaw cycles induced 75% activity loss due to denaturation and aggregation.•Autoproteolysis of papain did not cause significant loss of the protein activity. Papain is a cysteine protease with wide substrate specificity and many applications. Despite its widespread applications, cold stability of papain has never been studied. Here, we used differential spectroscopy to monitor thermal denaturation process. Papain was the most stabile from 45°C to 60°C with ΔG°321 of 13.9±0.3kJ/mol and Tm value of 84±1°C. After cold storage, papain lost parts of its native secondary structures elements which gave an increase of 40% of intermolecular β-sheet content (band maximum detected at frequency of 1621cm−1 in Fourier transform infrared (FT-IR) spectrum) indicating the presence of secondary structures necessary for aggregation. The presence of protein aggregates after cold storage was also proven by analytical size exclusion chromatography. After six freeze–thaw cycles around 75% of starting enzyme activity of papain was lost due to cold denaturation and aggregation of unfolded protein. Autoproteolysis of papain did not cause significant loss of the protein activity. Upon the cold storage, papain underwent structural rearrangements and aggregation that correspond to other cold denatured proteins, rather than autoproteolysis which could have the commercial importance for the growing polypeptide based industry.