X-Message-Number: 30125
Date: Wed, 12 Dec 2007 00:07:47 -0800 (PST)
Subject: no denaturation damage from glycerol

[Not much progress has been made in elucidating the nature of cryoprotectant
toxicity since Dr Fahy produced the following paper in 1986. It has been
proposed that the intrinsic toxicity of cryoprotectants is due largely to
their hydrophobicity, and consequence ability to denature DNA and proteins.
However the rank order of hydrophobicity is a poor predictor of overall
cryoprotectant toxicity. In particular, it does not explain glycerol
toxicity, since this solvent does not denature DNA or proteins.]

Cryobiology. 1986 Feb;23(1):1-13.
The relevance of cryoprotectant "toxicity" to cryobiology.
  Fahy GM.
Cryoprotective agents are essential for the cryopreservation of almost all
biological systems. These additives, however, do not usually permit 100%
survival after freezing and thawing, though from a theoretical point of view
they should be able to fully suppress all known types of freezing injury. In
view of the known biological and physicochemical effects of cryoprotectants,
it is suggested that the toxicity of these agents is a key limiting factor
in cryobiology. Not only does this toxicity prevent the use of fully
protective levels of additive, but it may also be manifested in the form of
cryoinjury over and beyond the cryoinjury due to classical causes. Evidence
for this extra injury ("cryoprotectant-associated freezing injury") is
reviewed. It is suggested that better suppression of toxicity is possible
and will lead to advances in cryopreservation.
PMID: 3956226

[Membrane proteins are not destabilized by glycerol. Hydrophobicity of the
solvents predicted destabilization tendancies. Rank order was glycerol,
ethylene glycol, methanol, ethanol, acetone, pyridine, ethyl acetate, with
diethyl maleate as the most damaging.]

Pharmazie. 2001 Oct;56(10):808-9.
Rapid method for comparing the cytotoxicity of organic solvents and their
ability to destabilize proteins of the erythrocyte membrane.
  Ivanov IT. Department of Physics & Biophysics, Medical Institute of
Thracian University, Stara Zagora, Bulgaria.
Cytotoxicities of a group of frequently used organic solvents were assessed
by their effect on thermal stability of erythrocyte membrane proteins. The
denaturation temperatures Tm of membrane proteins, peripheral and intrinsic,
were detected by the increase in the derivative of suspension impedance
during heating. These Tm linearly changed by delta Tm in the presence of
organic solvents indicating labilization (negative delta Tm) or
stabilization (positive delta Tm) of the structure of respective membrane
protein. The potency P of the solvent with molar concentration Cex to affect
the conformation stability of membrane protein was defined as delta Tm/Cex.
This potency decreased as both polarity of solvent and its capability to
form hydrogen bonds increased. In some solvents (dimethyl sulfoxide and
dimethyl formamide) the potencies to destabilized peripheric and intrinsic
proteins were equal. Formamide destabilized selectively peripheral proteins.
Some solvents (glycerol, especially erythritol) stabilised thermally
proteins. As the hydrophobicity of the solvents increased (ethylene glycol,
methanol, ethanol, acetone, pyridine, ethyl acetate, diethyl maleate) the
potency for destabilization of intrinsic proteins strongly increased. Thus,
the use of more polar solvents capable of forming more hydrogen bonds
appears preferable when low cytotoxicity should be attained.
PMID: 11683129

[DNA is denatured in many commonly used cryoprotectants, with the exceptions
of glycerol, and ethylene glycol.]

Biotechnol Bioeng. 2000 May 5;68(3):339-44.
Structural stability of DNA in nonaqueous solvents.
  Bonner G, Klibanov AM. Department of Chemistry, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139, USA.
One of the defining physicochemical features of DNA in aqueous solution is
its ability to maintain a double-helical structure and for this structure to
undergo a cooperative, heat-induced denaturation (melting). Herein we show
that a 21-mer synthetic DNA can form and maintain such a duplex structure
not only in water but even in 99% glycerol; moreover, this double-helical
structure reversibly and cooperatively melts in that solvent, with a T(m)
value of some 30 degrees lower than in water. Two much larger, natural DNAs,
from calf thymus and salmon testes, exhibit similar behavior in glycerol.
All three DNAs can also sustain a double-helical structure in 99% ethylene
glycol, although its thermostability (as reflected by the melting
temperature) is some 20 degrees lower than in glycerol. In contrast, no
duplex structure of any of the DNAs was detected in 99% formamide, methanol,
or DMSO. This solvent trend resembles that previously observed in studies of
protein structure and folding and underscores the importance of hydrophobic
interactions in both protein and DNA structure and stability. Our findings
suggest that water may not be unique as a suitable medium not only for
protein structure but also for that of nucleic acids. Copyright 2000 John
Wiley & Sons, Inc.
PMID: 10745202

[Lysozyme is denatured in many commonly used cryoprotectants, with the
single exception of glycerol. Ethylene glycol and methanol are less damaging
than DMSO, formamide, and dimethylformamide.]

Biotechnol Bioeng. 1999 Apr 20;63(2):242-8.
Structure of lysozyme dissolved in neat organic solvents as assessed by NMR
and CD spectroscopies.
  Knubovets T, Osterhout JJ, Klibanov AM. Department of Chemistry,
Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
The structure of the model protein hen egg-white lysozyme dissolved in water
and in five neat organic solvents (ethylene glycol, methanol,
dimethylsulfoxide (DMSO), formamide, and dimethylformamide (DMF)) has been
examined by means of 1H NMR and circular dichroism (CD) spectroscopies. The
NMR spectra of lysozyme reveal the lack of a defined tertiary structure in
all five organic solvents, although the examination of line widths suggests
the possibility of some ordered structure in ethylene glycol and in
methanol. The near-UV CD spectra of the protein suggest no tertiary
structure in lysozyme dissolved in DMSO, formamide, and DMF, while a
distinctive (albeit less pronounced than in water) tertiary structure is
seen in ethylene glycol and a drastically changed one in methanol. A highly
developed secondary structure was observed by far-UV CD in ethylene glycol
and methanol; interestingly, the alpha-helix content of the protein in both
was greater than in water, while the beta-structure content was lower.
(Solvent absorbance in the far-UV region prevents conclusions about the
secondary structure in DMSO, formamide and DMF.) Copyright 1999 John Wiley &
Sons, Inc.
PMID: 10099601

Proc Natl Acad Sci U S A. 1999 Feb 16;96(4):1262-7.
Structure, thermostability, and conformational flexibility of hen egg-white
lysozyme dissolved in glycerol.
  Knubovets T, Osterhout JJ, Connolly PJ, Klibanov AM. Department of
Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Hen egg-white lysozyme dissolved in glycerol containing 1% water was studied
by using CD and amide proton exchange monitored by two-dimensional 1H NMR.
The far- and near-UV CD spectra of the protein showed that the secondary and
tertiary structures of lysozyme in glycerol were similar to those in water.
Thermal melting of lysozyme in glycerol followed by CD spectral changes
indicated unfolding of the tertiary structure with a Tm of 76.0 +/- 0.2
degreesC and no appreciable loss of the secondary structure up to 85
degreesC. This is in contrast to the coincident denaturation of both
tertiary and secondary structures with Tm values of 74.8 +/- 0.4 degreesC
and 74.3 +/- 0.7 degreesC, respectively, under analogous conditions in
water. Quenched amide proton exchange experiments revealed a greater
structural protection of amide protons in glycerol than in water for a
majority of the slowly exchanging protons. The results point to a highly
ordered, native-like structure of lysozyme in glycerol, with the stability
exceeding that in water.
PMID: 9990012

[Glycerol even inhibits denaturation damage from hydrophobic solvents.]

J Biotechnol. 2006 Dec 15;127(1):45-53. Epub 2006 Jun 7.
Effects of water-miscible solvents and polyhydroxy compounds on the
structure and enzymatic activity of thermolysin.
  Pazhang M, Khajeh K, Ranjbar B, Hosseinkhani S. Department of Biochemistry
and Biophysics, Faculty of Sciences, Tarbiat Modares University, P.O. Box
14115-175, Tehran, Iran.
The effect of organic solvents (n-propanol, isopropanol, dimethylformamide
and dimethylsulfoxide) on the structure, activity and stability of
thermolysin was the focus of this investigation. Results show the ability of
the solvents to cause mixed inhibition of thermolysin, which was indicated
by kinetic and structural studies (near-UV CD spectra and intrinsic
fluorescence). Inhibitory effect of the solvents increased with increments
in solvents logP. Thermoinactivation of thermolysin was studied at 80
degrees C in 50% of solvents and showed that with the increase in solvent
hydrophobicity, thermal stability of the enzyme decreased. For the
stabilization of thermolysin at high temperature, additives such as
glycerol, sorbitol and trehalose were employed. In the presence of DMF with
a relatively low logP, trehalose was shown to be a good stabilizer, whereas
glycerol had a marked stabilization effect in the presence of n-propanol and
isopropanol with a relatively high logP. Consequently, it was concluded that
the stabilizing effect of additives can be correlated with the logP of
PMID: 16860424

[So why is glycerol toxic?]

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