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A new nmr technique to study disulfide reduction: Comparison of lysozyme and α-lactalbumin
Vol. 45, No. 6, 1971
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
A NEW NMR TECHNIQUE TO STUDY DISULFIDE REDUCTION: COMPARISON OF LYSOZYME AND a-LACTALBUMIN J. A. Magnuson
and N. S. Magnuson
Department of Chemistry Washington State University,
Received
September
and Biophysics Program Pullman, Washington 99163
30, 1971
SUMMARY 35 Cl resonances A nuclear magnetic resonance technique employing has been used to study the reduction of disulfides in lysozyme and a-lactalbumin The nmr technique allows rapid analysis of approximately 2 X 10L5 moles of disulfides. Reductions were carried out with dithiothreitol (DTT) in 4 M urea. From a time study of the amount of reduction for the two proteins, it appears that the disulfides of a-lactalbumin are more accessible for reduction than those of lysozyme. Because
of the close
and a-lactalbumin,
the
homology
in
two proteins
primary
structure
between
have been examined
lysozyme
for
other
for
a-lactalbumin
. similarities.
Browne
which
retain
Aune3
has shown
rotary
dispersion similarities
tinamide
chloride.
but
a large
that
the
for
that
has recently one-to-one
Given
the molecular
of the
two proteins
in disulffde
positions,
may reflect
differences
in tertiary
DTT, or Cleland's
a technique reagent,
for
1513
small
are quite
angle
different,
have predicted
and the apparent
structure. resonances
quantitating
from
regions.
of the rate
using
demon-
of N-methylnico-
and Sheraga'
in helical
8 a measure
with
conformations Lewis
optical
binding
have concluded
2
of lysozyme.
have recently
towards
and Kugler'
been disputed.6
the homology
have been developing
and Deranleau4
the two proteins
correspondence
features
have indistinguishable
Bradshaw
Krigbaum
models
and tertiary
two proteins
curves.
studies
this
have built
much of the secondary
strated
diffraction
and coworkers'
of reduction In our
similarity of these
laboratory
we
of 35C1 in conjunction disulfide
reduction.
BIOCHEMICAL
Vol. 45, No. 6, 1971
Because
of the
recent
a-lactalbumin
were
chosen
The technique
with
excess
4 M urea
near
all
pH 8.0 and is a 35Cl
unoxidized
under
resonance
carried
by lowering
the
and then
the 35 Cl resonance
from
free
line
width
conditions
and bound
studies.
and Cleland,' monothiols
We have used Hg*
is
signal,
and
reduction
Dl? and measured
Reduction
stopped
exchanging
initial of Zahler
DTT as a flocculent
rapidly
spectra
for
lysozyme
acid).
from HgC14= to the
contribution
of the
excess
DTT.
DTT has been scavenged,
which
systems
on the procedure
unoxidized
to provide
consume
as model
to scavenge
RESEARCH COMMUNICATIONS
the two proteins,
5,5'-dithiobis(Z-nitrobenzoic
scavenge
added
in
is based
who used arsenite formed
interest
AND BIOPHYSICAL
out with pH.
DTT in
NaCl is
Hg++ is
added
to
DTT-Hg++
adduct.
broadens
due to a large
When all
of the observed is
to
a weighted
spectra,
average
Cl-.lG
EXPERIMENTAL A typical
reduction
is
carried
in 5 ml of 4 M urea
containing
pH of approximately
3.0.
Reduction
0.75
which
is
ml of 4 M urea
brings time
the pH to 8.3. and the
NaCl is
reaction
added
supernatant based
is is
is
titrated
obtained. two acid were
checked
1% El cm 26.0
lysozyme,
by using for
is
I,
an El1%cm 20.5
adding
addition
for
the desired Then
up to 10 ml with and an aliquot
an aliquot
Amount
1.7 g water.
of the
of reduction of DTT-4
of this
is M urea
solution.
from Sigma was used as commercially
Nutritional
- (NH4)2S04
lyso~yme.~
This
The DTT concentration
Grade
from
base.
0.5 ml CHCl2COOH. brought
to a
by immediately
to proceed
DTT.
by titrating
precipitation
free
allowed
unoxidized
values.
a-La&albumin
initiated
with
60 mg of protein
of DTT and adjusted
by centrifugation,
for
calibrated
Hen egg white
is
volume
removed
on 100% reduction
solution
is
1 M in Tris
Reduction
total
by dissolving
8 umoles/ml
is quenched
and the
The precipitate
out
Biochemicals
precipitation at 280 nm for
DTT, Cleland's
Calbiochem. 1514
reagent,
was purified
cycles."
by
Concentrations
a-lactalbumin was obtained
and an from
Vol.45,
No.6,
1971
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
I
68-
62-
rmoles
Hg’+
A typical titration showing 35C1 line width versus umoles Figur$+l. added for a 30 second a-lactalbumin reduction. Theoretical of Hg endpoint for no reduction and 100% reduction are 35.2 and 20.3 umoles, respectively.
35
Cl resonances
spectrometer Fabri-Tek
were
at 5.8762 1070 Signal
observed
MHz.
with
Signal
Averaging
a modified
enhancement
Varian
DP-60 nmr
was achieved
with
a
Computer.
RESULTS Figure remaining
1 presents after
width
occurs
width
increases
the
results
a disulfide
until
all markedly
reduction.
DTT has been with
of the DTT reducing
solution,
From the difference
of the
predicted reduction,
endpoint the
Complete within
several
The comparison
for
percentage reduction minutes.
of a typical
each
No change consumed. addition
predicted
reduction
of reduced of both
of the a-lactalbumin
for
the
and lysozyme
1515
the
line
From a titration of DTT present.
from
and the protein
can be calculated.
was achieved reduction
point
line
no reduction
in an aliquot
disulfides
proteins
In 4 M urea
At this
the amount
endpoint
of DTT
in chlorine
of HgC12.
one determines
complete
titration
in 8 M urea rate
is
reductions
reduced. is
given
Vol. 45, No. 6, 1971
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Minutes
Figure 2. lysozyme,O
Comparison of reduction of a-lactalbumin,a , in 4 M urea; % reduction versus reduction
in Figure
2.
Lysozyme
a-lactalbumin
is
is
reduced
while
the
several
minutes.
measurements
of similar
samples
give
values
the same within
*5X of total
reduction.
quite
stable
in
the solution
does decompose
4 M urea
Fresh
reagent
occurs the
but is
at higher
usually
DTT is
daily.
reduction
by addition
Removal
titration
prepared
pH so that
pH and stopped
the protein.
reduced
rate,
within
Repetitive
greatly
at a slower
, and time in minutes.
over
Reduction is easily
sulfhydryls
is necessary
compete
several
initiated
with
days. only
by raising
also
precipitates
before
mercury
DTT for
are
at pH 3.0
of protein
of CHC12COOH, which
of the protein
as protein
slowly
which
HgCC.
DISCUSSION Obviously DTT than
those
25% of the obtained reduction
the disulfides of lysozym.
lysozyme
by Atassi, in
of a-lactalbumin
various
is
Half
reduced.
Habeeb,
of the a-lactalbumin The overall
and Rydstedt"
concentrations
are more accessible
result
reduced
agrees
with
before that
who used HSCH2CH20H to effect
of guanidfne 1516
is
to
hydrochloride.
Vol. 45, No. 6, 1971
Presumably,
if
resistant
technique.
like
this
the experiments
of the disulfides.
are allowable.
is
easily
one for
reported,
the same,
analysis.
quantitating
data for
the
reduction
lysozyme
is more highly
accomplished
by using
this
is
conducted
of both
X low6
is
reduction.
proteins,
and should
moles
the
proteins
excess
in all
the
in conditions
of disulfides
then
a rapid
are
and sensitive
As can be seen from
technique
be applicable
45 minutes.
in twofold
many variations
The nmr method disulfide
two model
rates
lo-25
35C1 nmr
in approximately
DTT was initially
although
In general,
for
similar,
The concentrations
were
necessary
are
RESEARCH COMMUNICATIONS
by urea.
Each nmr titration
experiments
AND BIOPHYXAL
the conformations
to denaturing
A study
In all
BIOCHEMICAL
the
can he used to study
to many systems.
ACKNOWLEDGMENTS This and medical
research research
was supported
in part
by State
of Washington
by funds Initiative
provided
for
Measure
biological No.
171.
REFERENCES 1. 2. 3. 4. 5. 6.
7.
J. W. Browne, and C. D. K. R. W. E. P.
A. C. T. North, D. C. Phillips, K. Brew, T. C. Vanaman, R. L. Hill, J. Mol. Biol., 42, 65 (1969). C. F. Blake, D. F. Koenig, G. A. Mair, A. C. T. North, C. Phillips, and V. R. Sarma, Nature, 206, 757 (1965). C. Aune, Ph.D. Thesis, Duke University, Durham, N. C., 1968. A. Bradshaw and D. A. Deranleau, Biochemistry, 9, 3310 (1970). R. Krigbaum and F. R. Kugler, Biochemistry, 9, i-216 (1970). K. Achter and I. D. A. Swan, Biochemistry, E, 2976 (1971). N. Lewis and H. A. Sheraga, Arch. Biochem. Btophys., 144, 584,
8
R. L. Hill, (1g71)’ K. Brew, T. C. Vanaman, I. P. Trayer, and P. Mattock, ' Brookhaven Symp. Biol., 21, 139 (1968). 9. W. L. Zahler and W. W. Cleland, J. Biol. Chem., 243, 716 (1968). 10. T. R. Stengle and J. D. Baldeschwieler, J. Amer.xem. Sot., z, 3045 (1967). 11. W. G. Gordon and J. Ziegler, Biochem. Prep., Q, 16 (1955). 12. M. Z. Atassi, A. F. S. A. Habeeb, and L. Rydstedt, Biochim. Biophys. Acta, 200, 184 (1970).
1517
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
A NEW NMR TECHNIQUE TO STUDY DISULFIDE REDUCTION: COMPARISON OF LYSOZYME AND a-LACTALBUMIN J. A. Magnuson
and N. S. Magnuson
Department of Chemistry Washington State University,
Received
September
and Biophysics Program Pullman, Washington 99163
30, 1971
SUMMARY 35 Cl resonances A nuclear magnetic resonance technique employing has been used to study the reduction of disulfides in lysozyme and a-lactalbumin The nmr technique allows rapid analysis of approximately 2 X 10L5 moles of disulfides. Reductions were carried out with dithiothreitol (DTT) in 4 M urea. From a time study of the amount of reduction for the two proteins, it appears that the disulfides of a-lactalbumin are more accessible for reduction than those of lysozyme. Because
of the close
and a-lactalbumin,
the
homology
in
two proteins
primary
structure
between
have been examined
lysozyme
for
other
for
a-lactalbumin
. similarities.
Browne
which
retain
Aune3
has shown
rotary
dispersion similarities
tinamide
chloride.
but
a large
that
the
for
that
has recently one-to-one
Given
the molecular
of the
two proteins
in disulffde
positions,
may reflect
differences
in tertiary
DTT, or Cleland's
a technique reagent,
for
1513
small
are quite
angle
different,
have predicted
and the apparent
structure. resonances
quantitating
from
regions.
of the rate
using
demon-
of N-methylnico-
and Sheraga'
in helical
8 a measure
with
conformations Lewis
optical
binding
have concluded
2
of lysozyme.
have recently
towards
and Kugler'
been disputed.6
the homology
have been developing
and Deranleau4
the two proteins
correspondence
features
have indistinguishable
Bradshaw
Krigbaum
models
and tertiary
two proteins
curves.
studies
this
have built
much of the secondary
strated
diffraction
and coworkers'
of reduction In our
similarity of these
laboratory
we
of 35C1 in conjunction disulfide
reduction.
BIOCHEMICAL
Vol. 45, No. 6, 1971
Because
of the
recent
a-lactalbumin
were
chosen
The technique
with
excess
4 M urea
near
all
pH 8.0 and is a 35Cl
unoxidized
under
resonance
carried
by lowering
the
and then
the 35 Cl resonance
from
free
line
width
conditions
and bound
studies.
and Cleland,' monothiols
We have used Hg*
is
signal,
and
reduction
Dl? and measured
Reduction
stopped
exchanging
initial of Zahler
DTT as a flocculent
rapidly
spectra
for
lysozyme
acid).
from HgC14= to the
contribution
of the
excess
DTT.
DTT has been scavenged,
which
systems
on the procedure
unoxidized
to provide
consume
as model
to scavenge
RESEARCH COMMUNICATIONS
the two proteins,
5,5'-dithiobis(Z-nitrobenzoic
scavenge
added
in
is based
who used arsenite formed
interest
AND BIOPHYSICAL
out with pH.
DTT in
NaCl is
Hg++ is
added
to
DTT-Hg++
adduct.
broadens
due to a large
When all
of the observed is
to
a weighted
spectra,
average
Cl-.lG
EXPERIMENTAL A typical
reduction
is
carried
in 5 ml of 4 M urea
containing
pH of approximately
3.0.
Reduction
0.75
which
is
ml of 4 M urea
brings time
the pH to 8.3. and the
NaCl is
reaction
added
supernatant based
is is
is
titrated
obtained. two acid were
checked
1% El cm 26.0
lysozyme,
by using for
is
I,
an El1%cm 20.5
adding
addition
for
the desired Then
up to 10 ml with and an aliquot
an aliquot
Amount
1.7 g water.
of the
of reduction of DTT-4
of this
is M urea
solution.
from Sigma was used as commercially
Nutritional
- (NH4)2S04
lyso~yme.~
This
The DTT concentration
Grade
from
base.
0.5 ml CHCl2COOH. brought
to a
by immediately
to proceed
DTT.
by titrating
precipitation
free
allowed
unoxidized
values.
a-La&albumin
initiated
with
60 mg of protein
of DTT and adjusted
by centrifugation,
for
calibrated
Hen egg white
is
volume
removed
on 100% reduction
solution
is
1 M in Tris
Reduction
total
by dissolving
8 umoles/ml
is quenched
and the
The precipitate
out
Biochemicals
precipitation at 280 nm for
DTT, Cleland's
Calbiochem. 1514
reagent,
was purified
cycles."
by
Concentrations
a-lactalbumin was obtained
and an from
Vol.45,
No.6,
1971
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
I
68-
62-
rmoles
Hg’+
A typical titration showing 35C1 line width versus umoles Figur$+l. added for a 30 second a-lactalbumin reduction. Theoretical of Hg endpoint for no reduction and 100% reduction are 35.2 and 20.3 umoles, respectively.
35
Cl resonances
spectrometer Fabri-Tek
were
at 5.8762 1070 Signal
observed
MHz.
with
Signal
Averaging
a modified
enhancement
Varian
DP-60 nmr
was achieved
with
a
Computer.
RESULTS Figure remaining
1 presents after
width
occurs
width
increases
the
results
a disulfide
until
all markedly
reduction.
DTT has been with
of the DTT reducing
solution,
From the difference
of the
predicted reduction,
endpoint the
Complete within
several
The comparison
for
percentage reduction minutes.
of a typical
each
No change consumed. addition
predicted
reduction
of reduced of both
of the a-lactalbumin
for
the
and lysozyme
1515
the
line
From a titration of DTT present.
from
and the protein
can be calculated.
was achieved reduction
point
line
no reduction
in an aliquot
disulfides
proteins
In 4 M urea
At this
the amount
endpoint
of DTT
in chlorine
of HgC12.
one determines
complete
titration
in 8 M urea rate
is
reductions
reduced. is
given
Vol. 45, No. 6, 1971
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Minutes
Figure 2. lysozyme,O
Comparison of reduction of a-lactalbumin,a , in 4 M urea; % reduction versus reduction
in Figure
2.
Lysozyme
a-lactalbumin
is
is
reduced
while
the
several
minutes.
measurements
of similar
samples
give
values
the same within
*5X of total
reduction.
quite
stable
in
the solution
does decompose
4 M urea
Fresh
reagent
occurs the
but is
at higher
usually
DTT is
daily.
reduction
by addition
Removal
titration
prepared
pH so that
pH and stopped
the protein.
reduced
rate,
within
Repetitive
greatly
at a slower
, and time in minutes.
over
Reduction is easily
sulfhydryls
is necessary
compete
several
initiated
with
days. only
by raising
also
precipitates
before
mercury
DTT for
are
at pH 3.0
of protein
of CHC12COOH, which
of the protein
as protein
slowly
which
HgCC.
DISCUSSION Obviously DTT than
those
25% of the obtained reduction
the disulfides of lysozym.
lysozyme
by Atassi, in
of a-lactalbumin
various
is
Half
reduced.
Habeeb,
of the a-lactalbumin The overall
and Rydstedt"
concentrations
are more accessible
result
reduced
agrees
with
before that
who used HSCH2CH20H to effect
of guanidfne 1516
is
to
hydrochloride.
Vol. 45, No. 6, 1971
Presumably,
if
resistant
technique.
like
this
the experiments
of the disulfides.
are allowable.
is
easily
one for
reported,
the same,
analysis.
quantitating
data for
the
reduction
lysozyme
is more highly
accomplished
by using
this
is
conducted
of both
X low6
is
reduction.
proteins,
and should
moles
the
proteins
excess
in all
the
in conditions
of disulfides
then
a rapid
are
and sensitive
As can be seen from
technique
be applicable
45 minutes.
in twofold
many variations
The nmr method disulfide
two model
rates
lo-25
35C1 nmr
in approximately
DTT was initially
although
In general,
for
similar,
The concentrations
were
necessary
are
RESEARCH COMMUNICATIONS
by urea.
Each nmr titration
experiments
AND BIOPHYXAL
the conformations
to denaturing
A study
In all
BIOCHEMICAL
the
can he used to study
to many systems.
ACKNOWLEDGMENTS This and medical
research research
was supported
in part
by State
of Washington
by funds Initiative
provided
for
Measure
biological No.
171.
REFERENCES 1. 2. 3. 4. 5. 6.
7.
J. W. Browne, and C. D. K. R. W. E. P.
A. C. T. North, D. C. Phillips, K. Brew, T. C. Vanaman, R. L. Hill, J. Mol. Biol., 42, 65 (1969). C. F. Blake, D. F. Koenig, G. A. Mair, A. C. T. North, C. Phillips, and V. R. Sarma, Nature, 206, 757 (1965). C. Aune, Ph.D. Thesis, Duke University, Durham, N. C., 1968. A. Bradshaw and D. A. Deranleau, Biochemistry, 9, 3310 (1970). R. Krigbaum and F. R. Kugler, Biochemistry, 9, i-216 (1970). K. Achter and I. D. A. Swan, Biochemistry, E, 2976 (1971). N. Lewis and H. A. Sheraga, Arch. Biochem. Btophys., 144, 584,
8
R. L. Hill, (1g71)’ K. Brew, T. C. Vanaman, I. P. Trayer, and P. Mattock, ' Brookhaven Symp. Biol., 21, 139 (1968). 9. W. L. Zahler and W. W. Cleland, J. Biol. Chem., 243, 716 (1968). 10. T. R. Stengle and J. D. Baldeschwieler, J. Amer.xem. Sot., z, 3045 (1967). 11. W. G. Gordon and J. Ziegler, Biochem. Prep., Q, 16 (1955). 12. M. Z. Atassi, A. F. S. A. Habeeb, and L. Rydstedt, Biochim. Biophys. Acta, 200, 184 (1970).
1517