Department of Biochemistry University of Toronto Toronto, Ontario CANADA
Received
October
21,1975
SUMMARY Electrophoretic and sedimentation velocity studies on the histone H3-H4 complex show that provided the 113 cysteine residues remain reduced the complex reforms quantitatively when removed from a variety of denaturing conditions. If histone 113 is allowed to become intramolecularly oxidized while denatured only monomer and large aggregates are formed on return to native conditions. At pH 7 ionic strength 0.1 the complex remains with reduced sulfhydryl groups indefinitely suggesting a vital role for the sequence 96-110 in histone H3 in the tertiary structure of the complex. Histones solution
and indirect
these this
H3 and H4 have been shown
two histones complex
literature has a tertiary
extracted depends
2, 6-8)
to report complexes
exclusively
an irreversibly changes
occur
MATERIALS
which
or not that
or the presence
suggest
ULL thecysteine
these
there
structure
the results
altered in
in chromatin.
as to whether
or quanternary
to form
structure
a tetrameric
has been presented
to be elucidated
of pH, temperature
I wish
(4-6)
are associated
have yet (1,
extremes
evidence
(l-3)
which
Although appears
indicates
structural
the complex
as extracted
in
that details
to be a controversy
of
in from
can be irreversihly,altered
the
chromatic by
of denaturants.
of some experiments
carried
that
of the complex
the "nativity"
residues
complex
being
has been found
reduced. when only
out with
chromatin in solution
No evidence noncovalent
for structural
two histones.
AND METHODS
Whole histone was extracted from calf thymus chromatin (9) by protamine extraction (10) and was further fractionated (10) to yield a pure histone H3-H4 The urea-acetic acid gels of I'anyim and Chalkley (11) were used for complex. identifying the respective histones and for determining (12) the extent of oxidation of the histone H3 cysteine residues. The double gel was run according to Lewis et al. (13).
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
329
BIOCHEMICAL
Vol. 68, No. 2, 1976
a
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
b
+ 0
Figure
1.
(a)
Electrophoresis
complex
and (b)
extracted
was carried for
6 hrs.
gels
were
out
cnl E thynius
on 0.05M
phosphate
stained
Theschlieren reached
from
at 100 volts,
was sedimented
sedimentation
with
chromatin.
amido
H3-H4
Electrophoresis
pH 7 polyacrylamide
30 ug of protein
in a synthetic pattern
of the histone
black.
were
gels
loaded.
The same protein
boundary
shown was recorded
cell
The sample
at 20,000
rpm.
as soon as the rotor
speed.
The existence of a complex was monitored where indicated by sedimentation velocity measurements run at 20°C (5mgs/ml protein ) in a Beckman Node1 E analytical ultracentrifuge equipped with schlieren ortics and by electrophoresis on nondenaturing lo%, 0.05 M phosphate pH7 polyacrylamide gels (14) with cooling and buffer recirculation facilities. The various conditions to which the complex has been exposed are indicated in the next section. RESULTS AND DISCUSSION The histone as a single also