Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life
Authorized Users Only
2016
Authors
Rašković, BrankicaVatić, Saša
Anđelković, Boban D.
Blagojević, Vladimir A.
Polović, Natalija
Article (Published version)
Metadata
Show full item recordAbstract
Trypsin is a serine protease with widespread applications, including protein sequencing and typsin mass fingerprinting. In the present study, the storage of trypsin in acidic conditions significantly affected the recovery of activity (40%) after 7 freeze-thaw cycles. Further, trypsin lost parts of its native secondary structure elements, which resulted in a 10% increase in beta-sheet content (band maximum detected at a frequency of 1634 cm in the Fourier transform infrared (FT-IR) spectrum) indicative of freezing-induced denaturation of the protein. The cold storage of trypsin in ammonium bicarbonate (pH 8.2) with the addition of ayoprotectants, such as glycerol or lysine, led to protein stabilization (complete secondary structure content preservation was detected by FT-IR), higher activity recovery ( gt 90%) and modest autolysis ( lt 10%). High activity recovery ( gt 90%) was also detected with the addition of propylene glycol and polyethylene glycol, saccharides and arginine. Neverth...eless, trypsin stored at pH 8.2 with the addition of glycerol or lysine was as efficient as untreated trypsin in the trypsin mass fingerprinting analysis of BSA, suggesting that the cold storage of trypsin in slightly alkaline conditions with the addition of cryoprotectants could prolong its shelf life. (C) 2015 Elsevier B.V. All rights reserved.
Keywords:
Enzyme activity / Cold stability / Protein recovery / Protein denaturation / Proteolysis / ProteomicsSource:
Biochemical Engineering Journal, 2016, 105, 168-176Publisher:
- Elsevier Science Bv, Amsterdam
Funding / projects:
- Allergens, antibodies, enzymes and small physiologically important molecules: design, structure, function and relevance (RS-MESTD-Basic Research (BR or ON)-172049)
- Directed synthesis, structure and properties of multifunctional materials (RS-MESTD-Basic Research (BR or ON)-172057)
Note:
- Supplementary material: http://cherry.chem.bg.ac.rs/handle/123456789/3586
DOI: 10.1016/j.bej.2015.09.018
ISSN: 1369-703X
WoS: 000367776300019
Scopus: 2-s2.0-84942792521
Collections
Institution/Community
Hemijski fakultet / Faculty of ChemistryTY - JOUR AU - Rašković, Brankica AU - Vatić, Saša AU - Anđelković, Boban D. AU - Blagojević, Vladimir A. AU - Polović, Natalija PY - 2016 UR - https://cherry.chem.bg.ac.rs/handle/123456789/2024 AB - Trypsin is a serine protease with widespread applications, including protein sequencing and typsin mass fingerprinting. In the present study, the storage of trypsin in acidic conditions significantly affected the recovery of activity (40%) after 7 freeze-thaw cycles. Further, trypsin lost parts of its native secondary structure elements, which resulted in a 10% increase in beta-sheet content (band maximum detected at a frequency of 1634 cm in the Fourier transform infrared (FT-IR) spectrum) indicative of freezing-induced denaturation of the protein. The cold storage of trypsin in ammonium bicarbonate (pH 8.2) with the addition of ayoprotectants, such as glycerol or lysine, led to protein stabilization (complete secondary structure content preservation was detected by FT-IR), higher activity recovery ( gt 90%) and modest autolysis ( lt 10%). High activity recovery ( gt 90%) was also detected with the addition of propylene glycol and polyethylene glycol, saccharides and arginine. Nevertheless, trypsin stored at pH 8.2 with the addition of glycerol or lysine was as efficient as untreated trypsin in the trypsin mass fingerprinting analysis of BSA, suggesting that the cold storage of trypsin in slightly alkaline conditions with the addition of cryoprotectants could prolong its shelf life. (C) 2015 Elsevier B.V. All rights reserved. PB - Elsevier Science Bv, Amsterdam T2 - Biochemical Engineering Journal T1 - Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life VL - 105 SP - 168 EP - 176 DO - 10.1016/j.bej.2015.09.018 ER -
@article{ author = "Rašković, Brankica and Vatić, Saša and Anđelković, Boban D. and Blagojević, Vladimir A. and Polović, Natalija", year = "2016", abstract = "Trypsin is a serine protease with widespread applications, including protein sequencing and typsin mass fingerprinting. In the present study, the storage of trypsin in acidic conditions significantly affected the recovery of activity (40%) after 7 freeze-thaw cycles. Further, trypsin lost parts of its native secondary structure elements, which resulted in a 10% increase in beta-sheet content (band maximum detected at a frequency of 1634 cm in the Fourier transform infrared (FT-IR) spectrum) indicative of freezing-induced denaturation of the protein. The cold storage of trypsin in ammonium bicarbonate (pH 8.2) with the addition of ayoprotectants, such as glycerol or lysine, led to protein stabilization (complete secondary structure content preservation was detected by FT-IR), higher activity recovery ( gt 90%) and modest autolysis ( lt 10%). High activity recovery ( gt 90%) was also detected with the addition of propylene glycol and polyethylene glycol, saccharides and arginine. Nevertheless, trypsin stored at pH 8.2 with the addition of glycerol or lysine was as efficient as untreated trypsin in the trypsin mass fingerprinting analysis of BSA, suggesting that the cold storage of trypsin in slightly alkaline conditions with the addition of cryoprotectants could prolong its shelf life. (C) 2015 Elsevier B.V. All rights reserved.", publisher = "Elsevier Science Bv, Amsterdam", journal = "Biochemical Engineering Journal", title = "Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life", volume = "105", pages = "168-176", doi = "10.1016/j.bej.2015.09.018" }
Rašković, B., Vatić, S., Anđelković, B. D., Blagojević, V. A.,& Polović, N.. (2016). Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life. in Biochemical Engineering Journal Elsevier Science Bv, Amsterdam., 105, 168-176. https://doi.org/10.1016/j.bej.2015.09.018
Rašković B, Vatić S, Anđelković BD, Blagojević VA, Polović N. Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life. in Biochemical Engineering Journal. 2016;105:168-176. doi:10.1016/j.bej.2015.09.018 .
Rašković, Brankica, Vatić, Saša, Anđelković, Boban D., Blagojević, Vladimir A., Polović, Natalija, "Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life" in Biochemical Engineering Journal, 105 (2016):168-176, https://doi.org/10.1016/j.bej.2015.09.018 . .