Vol. 68, No. 2, 1976

BIOCHEMICAL

O?I TIIE NATIVE

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SI'RL'CTURE OF TlIE HISTONE ii?-H4 Peter

COMPLEX

N. Lewis

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

band

agreement

no (

On the native structure of the histone H3-H4 complex.

Vol. 68, No. 2, 1976 BIOCHEMICAL O?I TIIE NATIVE AND BIOPHYSICAL RESEARCH COMMUNICATIONS SI'RL'CTURE OF TlIE HISTONE ii?-H4 Peter COMPLEX N. Lew...
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