Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life
Abstract
Trypsin is a serine protease with widespread applications, including protein sequencing and trypsin 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 β-sheet content (band maximum detected at a frequency of 1634 cm−1 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 cryoprotectants, such as glycerol or lysine, led to protein stabilization (complete secondary structure content preservation was detected by FT-IR), higher activity recovery (>90%) and modest autolysis (<10%). High activity recovery (>90%) was also detected with the addition of propylene glycol and polyethylene glycol, saccharides and arginine. Nevertheless, t...rypsin 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.
Keywords:
Enzyme activity / Cold stability / Protein recovery / Protein denaturation / ProteolysisSource:
Biochemical Engineering Journal, 2016, 105, A, 168-176Publisher:
- Elsevier
Funding / projects:
- Allergens, antibodies, enzymes and small physiologically important molecules: design, structure, function and relevance (RS-172049)
- Directed synthesis, structure and properties of multifunctional materials (RS-172057)
DOI: 10.1016/j.bej.2015.09.018
ISSN: 1369-703X
WoS: 000367776300019
Scopus: 2-s2.0-84942792521
Institution/Community
Институт техничких наука САНУ / Institute of Technical Sciences of SASATY - JOUR AU - Rašković, Brankica AU - Vatić, Saša AU - Anđelković, Boban AU - Blagojević, Vladimir A. AU - Polović, Natalija PY - 2016 UR - https://dais.sanu.ac.rs/123456789/16003 AB - Trypsin is a serine protease with widespread applications, including protein sequencing and trypsin 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 β-sheet content (band maximum detected at a frequency of 1634 cm−1 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 cryoprotectants, such as glycerol or lysine, led to protein stabilization (complete secondary structure content preservation was detected by FT-IR), higher activity recovery (>90%) and modest autolysis (<10%). High activity recovery (>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. PB - Elsevier T2 - Biochemical Engineering Journal T1 - Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life SP - 168 EP - 176 VL - 105 IS - A DO - 10.1016/j.bej.2015.09.018 UR - https://hdl.handle.net/21.15107/rcub_dais_16003 ER -
@article{ author = "Rašković, Brankica and Vatić, Saša and Anđelković, Boban and Blagojević, Vladimir A. and Polović, Natalija", year = "2016", abstract = "Trypsin is a serine protease with widespread applications, including protein sequencing and trypsin 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 β-sheet content (band maximum detected at a frequency of 1634 cm−1 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 cryoprotectants, such as glycerol or lysine, led to protein stabilization (complete secondary structure content preservation was detected by FT-IR), higher activity recovery (>90%) and modest autolysis (<10%). High activity recovery (>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.", publisher = "Elsevier", journal = "Biochemical Engineering Journal", title = "Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life", pages = "168-176", volume = "105", number = "A", doi = "10.1016/j.bej.2015.09.018", url = "https://hdl.handle.net/21.15107/rcub_dais_16003" }
Rašković, B., Vatić, S., Anđelković, B., 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., 105(A), 168-176. https://doi.org/10.1016/j.bej.2015.09.018 https://hdl.handle.net/21.15107/rcub_dais_16003
Rašković B, Vatić S, Anđelković B, 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(A):168-176. doi:10.1016/j.bej.2015.09.018 https://hdl.handle.net/21.15107/rcub_dais_16003 .
Rašković, Brankica, Vatić, Saša, Anđelković, Boban, 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, no. A (2016):168-176, https://doi.org/10.1016/j.bej.2015.09.018 ., https://hdl.handle.net/21.15107/rcub_dais_16003 .