X-Message-Number: 9755
Date: Fri, 22 May 1998 02:44:19 -0700 (PDT)
From: Doug Skrecky <>
Subject: freezing and drying denaturation of proteins Part II

Authors
  Prestrelski SJ.  Arakawa T.  Carpenter JF.
Institution
  Amgen Inc., Amgen Center, Thousand Oaks, California 91320.
Title
  Separation of freezing- and drying-induced denaturation of
  lyophilized proteins using stress-specific stabilization. II. Structural
  studies using infrared spectroscopy.
Source
  Archives of Biochemistry & Biophysics.  303(2):465-73, 1993 Jun.
Abstract
  The conformation of two labile enzymes, lactate dehydrogenase and
  phosphofructokinase, has been examined in the aqueous and lyophilized states,
  using infrared spectroscopy. In the preceding paper it was demonstrated that
  a stress-specific stabilization scheme, which employs a combination of a
  cryoprotectant (polyethylene glycol) and a compound which protects the dried
  protein (sugars or mannitol), can be used to optimize
  recovery of activity of these enzymes upon
  freeze-drying and rehydration. The purpose
  of the present study is to determine the effects of these additives on the
  conformation of these enzymes during lyophilization. Lyophilization in the
  absence of stabilizers was observed to induce significant conformational
  changes in both enzymes. Addition of 10 mM mannitol,
  lactose, or trehalose or 1% polyethylene glycol to the enzyme solutions
  attenuated the unfolding, but significant spectral differences for the
  enzymes in the dried state are still observed when compared to the aqueous
  conformation. Addition of any one of these stabilizers does not improve
  recovery of activity. However, when a combination of 1% PEG and either 10 mM
  mannitol, lactose, or trehalose is added, the native
  structure is preserved during lyophilization and essentially full enzymatic
  activity is recovered upon reconstitution. The ability of the stabilizers to
  preserve the native structure during lyophilization correlates directly with
  the recovery of enzymatic activity upon reconstitution. It appears that for
  labile proteins, preservation of the native structure during lyophilization
  is requisite for recovery of activity following rehydration. This study
  demonstrates that the infrared spectroscopic technique is a rapid and useful
  method for studying protein conformation in the dried state and can aid in
  determining the optimal conditions for stabilization of proteins during
  lyophilization.

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