title: Fourier transform infrared spectroscopy provides an evidence of papain denaturation and aggregation during cold storage.
abstract: 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±0.3 kJ/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 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. 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.
authors.affiliation: Department of Biochemistry, University of Belgrade - Faculty of Chemistry, Studentski trg 12 - 16, 11000 Belgrade, Serbia.
authors.forename: Brankica
authors.initials: B
authors.lastname: Rašković
authors.affiliation: Department of Biochemistry, University of Belgrade - Faculty of Chemistry, Studentski trg 12 - 16, 11000 Belgrade, Serbia.
authors.forename: Milica
authors.initials: M
authors.lastname: Popović
authors.affiliation: Institute of General and Physical Chemistry, Studentski trg 12, 11000 Belgrade, Serbia.
authors.forename: Sanja
authors.initials: S
authors.lastname: Ostojić
authors.affiliation: Department of Organic Chemistry, University of Belgrade - Faculty of Chemistry, Studentski trg 12 - 16, 11000 Belgrade, Serbia.
authors.forename: Boban
authors.initials: B
authors.lastname: Anđelković
authors.affiliation: Department of Organic Chemistry, University of Belgrade - Faculty of Chemistry, Studentski trg 12 - 16, 11000 Belgrade, Serbia.
authors.forename: Vele
authors.initials: V
authors.lastname: Tešević
authors.affiliation: Department of Biochemistry, University of Belgrade - Faculty of Chemistry, Studentski trg 12 - 16, 11000 Belgrade, Serbia. Electronic address: polovicn@chem.bg.ac.rs.
authors.forename: Natalija
authors.initials: N
authors.lastname: Polović
mesh_terms: D000595:Amino Acid Sequence; D003080:Cold Temperature; D004355:Drug Stability; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D010206:Papain / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011309:Preservation, Biological / Q000379:methods; D066329:Protein Aggregates; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D066329:Protein Aggregates; D010206:Papain
keywords: Aggregation; Cold denaturation; Cold inactivation; Cold stability; FT-IR; Papain