Fourier transform infrared spectroscopy provides an evidence of papain denaturation and aggregation during cold storage
Ostojić, Sanja B.
Anđelković, Boban D.
Article (Accepted Version)
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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 degrees C to 60 degrees C with Delta G degrees(321) of 13.9 +/- 0.3 kJ/mol and T-m value of 84 +/- 1 degrees C. After cold storage, papain lost parts of its native secondary structures elements which gave an increase of 40% of intermolecular beta-sheet content (band maximum detected at frequency of 1621 cm(-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. Autoproteolys...is 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.
Keywords:Papain / FT-IR / Cold stability / Cold denaturation / Cold inactivation / Aggregation
Source:Spectrochimica Acta. Part A: Molecular and Biomolecular Spectroscopy, 2015, 150, 238-246
- Pergamon-Elsevier Science Ltd, Oxford
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- This is the peer-reviewed version of the following article: Rašković, B.; Popović, M.; Ostojić, S.; Ancrossed D Signelković, B.; Tešević, V.; Polović, N. Fourier Transform Infrared Spectroscopy Provides an Evidence of Papain Denaturation and Aggregation during Cold Storage. Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy 2015, 150, 238–246. https://doi.org/10.1016/j.saa.2015.05.061