Cryoprotection mechanisms of polyethylene glycols on lactate dehydrogenase during freeze-thawing

Yanli Mi, George Wood, Laura Thoma

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

The purpose of this study was to explore the cryoprotection mechanisms of high molecular weight polyethylene glycols (PEGs) (eg, PEG 4000 and PEG 8000) on lactate dehydrogenase (LDH.). Ultraviolet activity assays, circular dichroism (CD) spectroscopy, gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 14C-PEG 4000 labeling and binding, and cryostage microscopic study were conducted. Different molecular weights and concentrations of PEGs in LDH formulations were treated by freeze-thawing. Higher molecular weights and concentrations of PEGs in LDH-PEG formulations obtained better activity and secondary structure recoveries of LDH after freeze-thawing. Insoluble aggregation of LDH was not observed in gel filtration studies. SDS-PAGE results suggested surface characteristic modifications of LDH by the larger molecular weight PEGs. The 14C-PEG 4000 labeling and binding study showed extensive nonspecific interactions between the PEG 4000 and LDH molecules in a concentration-dependent manner. The bound LDH-PEG 4000/free PEG 4000 ratio increased when LDH or PEG 4000 concentrations increased. Cryostage microscopic study showed that PEG 8000 delayed the ice crystallization and eutectic transition of LDH formulation. It appeared that multiple mechanisms were at work during PEGs' cryoprotection of LDH. It was unclear whether the delayed eutectic characteristics of PEGs contributed to LDH cryoprotection. The favorable interaction, rather than preferential exclusion, between LDH and PEGs (eg, 4000) cryoprotected LDH.

Original languageEnglish (US)
Article number22
JournalAAPS Journal
Volume6
Issue number3
DOIs
StatePublished - Sep 7 2004

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L-Lactate Dehydrogenase
Molecular Weight
Sodium Dodecyl Sulfate
Gel Chromatography
Polyacrylamide Gel Electrophoresis
Ice
Circular Dichroism
Crystallization
polyethylene glycol 4000
Spectrum Analysis

All Science Journal Classification (ASJC) codes

  • Pharmaceutical Science

Cite this

Cryoprotection mechanisms of polyethylene glycols on lactate dehydrogenase during freeze-thawing. / Mi, Yanli; Wood, George; Thoma, Laura.

In: AAPS Journal, Vol. 6, No. 3, 22, 07.09.2004.

Research output: Contribution to journalArticle

Mi, Yanli ; Wood, George ; Thoma, Laura. / Cryoprotection mechanisms of polyethylene glycols on lactate dehydrogenase during freeze-thawing. In: AAPS Journal. 2004 ; Vol. 6, No. 3.
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