Salt effects on the aggregation of reduced-denatured lysozyme

doi:10.3969/j.issn.1000-1565.2018.06.006

Abstract

Abstract: Using reduced-denatured lysozyme as a model protein, the effects of sodium sulfate(Na₂SO₄), sodium perchlorate(NaClO₄), sodium nitrate(NaNO₃), and sodium chloride(NaCl)on protein aggregation were investigated. Turbidity assay were used to study the critical salt concentration on protein aggregation and the aggregation kinetics. It was found that the critical salt concentrations increased by following the order of SO₄^2- > ClO₄^- > NO₃^- > Cl^- and the aggregation rates decreased by following the order of SO₄^2- > ClO₄^- > NO₃^- > Cl^-.The fitted kinetic parameters showed a linear relationship with salt concentrations. All of the four salts induced the formation of amorphous protein aggregates as revealed by scanning electron microscopy. This work should be beneficial for a better understanding of protein aggregation.

Key words: lysozyme, protein aggregation, salt effect, turbidity assay

CLC Number:

O643

JIA Baohuan, YU Yang, WANG Yue, LIU Siyang, DU Baoan, MA Gang. Salt effects on the aggregation of reduced-denatured lysozyme[J]. Journal of Hebei University (Natural Science Edition), 2018, 38(6): 588-595.

References

[1] CHITI F, DOBSON C M. Protein misfolding, functional amyloid, and human disease[J]. Annu Rev Biochem,2006, 75: 333-366. DOI: 10.1146/annurev.biochem.75.101304.123901.
[2] 徐萍,刘超.胰淀素、胰岛淀粉样蛋白沉积与2型糖尿病[J]. 医学综述,2005, 11: 500-502.
[3] 金玲,杨慧.α-Synuclein聚集与帕金森病影响α-synuclein蛋白聚集的因素[J]. 生物化学与生物物理进展,2006, 33(4): 321-328. JIN L,YANG H.α-Synuclein aggregation and parkinson’s disease: factors affecting the aggregation of α-synuclein[J]. Prog Biochem Biophys, 2006, 33(4): 321-328.
[4] 张岚,杨延莲,王琛.调节分子对β淀粉样蛋白的聚集调控及细胞毒性抑制效应[J]. 生物物理学报, 2010, 26(8): 649-661. ZHANG L,YANG Y L,WANG C.Regulation of aggregation and cytotoxicity of β-Amyloid by molecular modulators[J]. ACTA Biophysica Sinica, 2010, 26(8): 649-661.
[5] LIU W R, LANGER R,KLIBANOV A M. Moisture-induced aggregation of lyophilized proteins in the solid state[J]. Biotechnol Bioeng, 1991, 37: 177-184. DOI:10.1002/bit.260370210.
[6] COSTANTINO H R, LANGER R, KLIBANOV A M. Moisture-induced aggregation of lyophilized insulin[J]. Pharm Res,1994, 11: 21-29. DOI: 10.1023/A:1018981208076.
[7] KATAKAM M, BELL L N, BANGA A K. Effect of surfactants on the physical stability of recombinant human growth hormone[J]. J Pharm Sci, 1995, 84: 713-716. DOI:10.1002/jps.2600840609.
[8] KREILGAARD L,FROKJAER S,FLINK J M, et al. Effects of additives on the stability of Humicola lanuginosa lipase during freeze-drying and storage in the dried solid[J]. J Pharm Sci, 1999, 88: 281-290. DOI:10.1021/js980399d
[9] GEORGIOU G, VALAX P. Isolating inclusion bodies from bacteria[J]. Methods Enzymol, 1999, 309: 48.
[10] ROBERTS C J.Kinetics of irreversible protein aggregation: analysis of extended Lumry-Eyring models and implications for predicting protein shelf life[J]. J Phys Chem B,2003, 107: 1194-1207. DOI: 10.1021/jp026827s.
[11] FROKJAER S, OTZEN D E.Protein drug stability: a formulation challenge[J]. Nat Rev Drug Discovery,2005, 4: 298-306. DOI: 10.1038/nrd1695.
[12] WANG W. Protein aggregation and its inhibition in biopharmaceutics[J]. Int J Pharm,2005, 289: 1-30. DOI: 10.1016/j.ijpharm.2004.11.014.
[13] ROSENBERG A S. Effects of protein aggregates: an immunologic perspective[J]. The AAPS journal,2006, 8: E501-E507. DOI:10.1208/aapsj080359.
[14] CROMWELL M E, HILARIO E, JACOBSON F. Protein aggregation and bioprocessing[J]. The AAPS Journal,2006, 8: E572-E579. DOI: 10.1208/aapsj080366.
[15] MAHLER H C, FRIESS W, GRAUSCHOPF U, et al. Protein aggregation: pathways, induction factors and analysis[J]. J Pharm Sci, 2009, 98: 2909-2934. DOI: 10.1002/jps.21566.
[16] WANG W, NEMA S, TEAGARDEN D. Protein aggregation-Pathways and influencing factors[J]. Int J Pharm,2010, 390: 89-99. DOI: 10.1016/j.ijpharm.2010.02.025.
[17] ENGELSMAN J, GARIDEL P, SMULDERS R,et al. Strategies for the assessment of protein aggregates in pharmaceutical biotech product development[J]. Pharm Res,2011, 28: 920-933. DOI: 10.1007/s11095-010-0297-1.
[18] CAO A, HU D, LAI L H. Formation of amyloid fibrils from fully reduced hen egg white lysozyme[J]. Protein Sci,2004, 13: 319-324. DOI: 10.1110/ps.03183404.
[19] 李昊一, 李冬敏, 马刚, 等. 牛血清白蛋白聚集体的分解动力学[J]. 河北大学学报(自然科学版), 2016, 36(1): 35-42. DOI: 10. 3969 /j. issn. 1000-1565. 2016. 01. 007. LI H Y, LI D M, MA G, et al. Structural insights into bovine serum albumin aggregates by a dissociation kinetic investigation[J]. Journal of Hebei University(Natural Science Edition), 2016, 36(1): 35-42. DOI: 10. 3969 /j. issn. 1000-1565. 2016. 01. 007.
[20] SUMIE YOSHIOKA, YUKIO ASO, KOJIMA S. Usefulness of the kohlrausch-williams-watts stretched exponential function to describe protein aggregation in lyophilized formulations and the temperature dependence near the glass transition temperature[J]. Pharm Res,2001, 18: 256-260. DOI:10.1023/A:1011082309058.
[21] JUÁREZ J, LÓPEZ S G, CAMBÓN A, et al. Influence of electrostatic interactions on the fibrillation process of human serum albumin[J]. J Chem Phys, 2009, 113: 10521-10529. DOI: 10.1021/jp902224d.