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Polymers protect lactate dehydrogenase during freeze-drying by inhibiting dissociation in the frozen state.

Abstract：

:Enzymes subjected to freeze-thawing are known to be protected by polymers that are preferentially excluded from the hydrated surface of proteins [reviewed in Carpenter et al. (1994) ACS Symp. Ser. 567, 134-147]. Preferentially excluded solutes are also known to stabilize quaternary structure, which enhances the thermostability of multimeric proteins in aqueous systems. Also, it has been suggested that retention of quaternary structure may play a role in the protection of multimeric proteins by polymers during freeze-drying (lyophilization). Although preferential solute exclusion cannot occur in the absence of water, we reasoned that polymers could protect multimeric proteins during freeze-drying by stabilizing quaternary structure in the frozen state. Our results are consistent with this hypothesis and demonstrate that bovine serum albumin and polyvinylpyrrolidone stabilize lactate dehydrogenase by inhibiting dissociation in the frozen solution, during the initial phase of the sublimation step of lyophilization. Dissociation at this critical step correlated directly with decreased recovery of enzyme activity after rehydration. The damage to the protein, under conditions where dissociation was studied, was due to a large decrease in pH in the frozen state (e.g., from pH 7.5 to 4.5), which was attenuated by protective levels of polymers. Thus, inhibition of freezing-induced pH shifts, in addition to stabilization by the preferential exclusion mechanism, plays an important role in the protection conferred by polymers. Furthermore, high concentrations of these polymers were capable of maintaining quaternary structure during subsequent drying and rehydration. We suggest that the proximate cause for increased recovery of active, native protein after lyophilization is that the holoenzyme is more resistant to the stresses of drying/rehydration than unassociated monomers.

journal_name

Arch Biochem Biophys

journal_title

Archives of biochemistry and biophysics

authors

Anchordoquy TJ,Carpenter JF

doi

10.1006/abbi.1996.0337

subject

Has Abstract

pub_date

1996-08-15 00:00:00

pages

231-8

issue

eissn

0003-9861

issn

1096-0384

pii

S0003-9861(96)90337-9

journal_volume

332

pub_type

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Polymers protect lactate dehydrogenase during freeze-drying by inhibiting dissociation in the frozen state.