Eur. J. Biochem. 100, 285-293 (1979)
Reaction of Formaldehyde with Calf-Thymus Nucleohistone Yoshiki OHBA, Yoshimi MORIMITSU, and Atsuko WATARAI Ilepartment of Biology, Faculty of Pharmaceutical Sciences, Kandzawa University (Received January 4, 1979)
The reactions of formaldehyde with calf thymus nucleohistone were analyzed in the following ways : measurement with fluorescamine of the decrease in primary amino groups resulting from hydroxymethylation and crosslinking reactions, measurement with dodecylsulphate-gel electrophoresis of formation of histone oligomers, measurement of fixation of histones to the DNA in nucleohistone, and measurement of changes in the circular dichroism spectrum in the region of 250 - 300 nm. In the presence of formaldehyde, the primary amino groups of histones decreased very rapidly, attaining an equilibrium within 60 min, and successively intermolecular crosslinks were also formed between histone molecules, the resulting dimers and oligomers being separable by dodecylsulfate-gel electrophoresis. Whereas the fixation reaction proceeded much more slowly. The extent of fixation could be measured more accurately by dodecylsulfate/sucrose centrifugation analysis than by sulfuric acid extraction. After removal of f-ormaldehyde from the reaction mixture, the fraction of masked amino groups decreased, perhaps due to the reverse reaction, but the extent of fixation of histones continued to increase with time. No specificity was observed among five molecular species of histones in the fixation reaction. With increase in formaldehyde concentration, the ellipticity of nucleohistone decreased to a minimum with about 0.4 % formaldehyde, and then increased.
Formaldehyde is known to bind to various groups of proteins, including primary amino and imidazole groups [l -31. There formaldehyde has both monofunctional and bifunctional actions. Many studies on the interactions of formaldehyde with nucleic acids have also shown that formaldehyde can react with both the exocyclic amino groups and endocyclic imino groups of DNA bases [4 - 61. Formaldehyde has recently been used in physical characterization of chromatin [7 - lo]. The mechanism of the reaction between formaldehyde and nucleohistone has been reported by several workers [ l l - 161. In this process formaldehyde reacts with both histones and DNA, acting as a fixative. The treatment of nucleohistone with formaldehyde results in complex in which protein-DNA interaction is no longer ionic as it is in native nucleohistone. The treated complexes are not dissociated either by 4 M CsCl or 0.1 M HzS04. In this work, we investigated the interactions of formaldehyde with calf thymus nucleohistone in following ways : namely by measuring decreases in primary amino groups of histone, fixation of histones to DNA, and changes of the circular dichroism spectrum in the Abbreviation. CD, circular dichroism
region of 250 - 300 nm, due to conformational changes of the DNA in chromatin.
MATERIALS AND METHODS Preparation of Nucleohistone Nucleohistone was prepared from 20 - 40 g of frozen calf thymus glands by a modification of the method of Zubay and Doty [17]. Thus 20-40g of frozen calf thymus glands were minced with scissors and then homogenized in 300 ml of standard solvent (0.14 M NaC1, 0.01 M EDTA pH 7.0) with a homogenizer (Nihonseiki) for 5 min. The mixture was filtered through double-layered gauze, and the filtrate was centrifuged at 3000 rev./min for 10min. The precipitate was again homogenized with the standard solvent and centrifuged as before. This cycle was repeated more than ten times. The final precipitate was then homogenized in 50-100 ml of doubledistilled water and the mixture was dialyzed against 0.7 mM phosphate buffer (pH 7.2) for 3 days. All procedures were carried out at 4 "C. The final preparation contained no detectable non-histone proteins
286
when examined by sodium dodecylsulfate electrophoresis [18]. An aliquot of the concentrated nucleohistone gel was weighed and diluted with water or phosphate buffer (0.7 mM, pH 7.2) by homogenization, until pipetting became feasible (generally diluted to 0.05% DNA). DNA was estimated from the absorbance at 260nm, being taken as 200, or by Burton's method [19]. Histones were estimated by the method of Lowry et al. [20], with isolated histones as a standard. The ratio of protein to DNA was 1.20-1.25.
Reaction with Formaldehyde Formaldehyde reagent grade (approximately 37 "/,) was obtained from Wako Chemicals. Contaminating formic acid present was removed by passing the cold solution through a small column of Dowex I in hydroxide form. The total formaldehyde content w7as calculated from the specific gravity measured with a hydrometer. For each experiment a sample of the stock solution was heated to 65 "C for 15 min and then diluted. The reaction was carried out at various concentrations of formaldehyde in 6 mM triethanolamine (pH 8.7), at a nucleohistone concentration of A260 1.0, unless otherwise mentioned. The reaction was allowed to proceed for various times at 4 " C ,or 20 "C, as indicated, and was usually stopped by adding either 0.1 vol. 10 % sodium dodecyl sulfate to remove the unfixed histones, or 0.5 vol. fluorescamine reagent, to estimate the amount of remaining primary amines of histones. Sometimes the reaction was stopped by rapid removal of excess formaldehyde. For this, 5 ml of the reaction mixture (A260 = 4.0) was applied to a Sephadex G-25 column (coarse, 2 x 30cm), and the column was eluted with 0.7 mM phosphate buffer (pH 6.7) at a flow rate of 5 ml/min at room temperature. Fractions of 5 ml were collected, and assayed first spectrophotometrically at 260 nm, and then by the Schiff test for formaldehyde. With the latter method 10 min was sufficient for complete separation of nucleohistone from formaldehyde.
Reaction with Fluorescarnine Fluorescamine (Fluram) was obtained from JapanRoche Diagonostics, and dissolved in anhydrous dioxane at a concentration of 0.1 mg/ml just before use. A sample of 1.0-20 pg protein in 2 ml of 0.2 M borate buffer (pH 9.5) was rapidly mixed with 1 ml of fluorescamine reagent, while holding the test tube on a Vortex mixer. The resulting fluorescence was measured at least 30 min later in a Hitachi spectrofluorometer, using an excitation wavelength of 340 nm and emission at a wavelength of 475 nm [21]. The extent of fluorescence remained unchanged for several hours at least. Under these conditions the amino
Reaction of Formaldehyde with Nucleohistone
groups of histones reacted quantitatively with fluoresamine, and the reaction v7as not influenced by the presence of DNA (Y. Ohba & K. Yoshii, unpublished results).
Separation oj Unfixed Histones After fixation of nucleohistone with formaldehyde, the unfixed histone fraction was separated by extraction with H 2 S 0 4 or centrifugation in dodecylsulfate/ sucrose. For extraction with HzS04, 2/3 vol. of 0.5 M HzS04 was added to the reaction mixture, containing partially fixed nucleohistone, and the mixture was stirred vigorously overnight at 4 "C, and then centrifuged at 10000 x g for 30 min and the supernatant was dialyzed against 0.7 mM phosphate. The volume of the dialyzed solution was calculated from the total weight, and then the total amount of dissociated protein was estimated. In the dodecylsulfate/sucrose method, 4-ml samples of the mixture of nucleohistone and formaldehyde were removed at intervals, mixed with 2 ml of 2 % sodium dodecylsulfate, and incubated at 37 "C for 30 min. Then the solution was layered on a discontinuous sucrose gradient consisting of layers of 2 ml of 60 % sucrose and 5 ml of 5 % sucrose, 0.1 % sodium dodecylsulfate. The gradient was centrifuged for 18 h at 35000 rev./min in the RPS 40 T rotor of a Hitachi P3 ultracentrifuge at 18 "C, and then 2-ml fractions were collected from the top of the tube with a microinfusion pump (LKB, Perpex). Their A260 was measured and the fractions in the peak position were combined and dialyzed against distilled water to estimate the ratio of protein to DNA. The DNA fraction obtained in this way from untreated nucleohistone contained less than 5 % of the total protein [23]. Sometimes the fractions of dissociated histones were lyophilized to analyze their amino acid composition or their dodecylsulfate-electrophoretic pattern.
Amino Acid Analysis Amino acid analysis was carried out on fixed 10 7; histones or 20% histones without removal of DNA, and on 1 4 % histones remaining unfixed. When the fixation reaction was carried out in 0.2 %formaldehyde for 0.5 or 1 h, the partially fixed nucleohistones, including either 10% or 20% histones, were obtained by the dodecylsulfate/sucrose method described above. The 14 % histones remaining unfixed were obtained as the supernatant solution on dodecylsulfate centrifugation of reaction mixture treated with l % formaldehyde for 4 h at 4°C. When the reaction was carried out in 2 %, formaldehyde for 48 h, 100% fixation was achieved. These samples were hydrolysed in Nz-replaced sealed tubes with 6 M HC1 at 110 "C for 20 h, and analyzed in an amino acid analyzer (Joel 6SH). The
Y. Ohba, Y. Morimitsu, and A. Watarai
281
glycine content was not calculated, because glycine is produced during DNA degradation under these conditions of hydrolysis. Dodecylsuljate Electrophoresis
The unfixed histones in the fixation reaction were analyzed by dodecylsulfate electrophoresis. The samples were prepared by the same method as for dodecylsulfate/sucrose centrifugation method, and dissolved in the suitable amount of 2 sodium dodecylsulfate, 0.2 M phosphate buffer (pH 7.2), 50% glycerol to give a final concentration of 1 mg histone/ml. The samples were subjected to electrophoresis on 12.5 7; polyacrylamide gel in dodecylsulfate/phosphate buffer as previously described [24]. The gels were stained with Comassie brilliant blue, and scanned at 600 nm with a Gilford spectrophotometer model 2400s.
1 .o
0
"
f.
2.0
2 "D
610
do
1 o; Time (min)
I
18021h
Fig. 1 . Time course of formaldehyde reaction with primary amino groups of histones. Calf thymus nucleohistone ( A 2 6 0 = 1.0, final concentration) was treated with various concentrations of formaldehyde in 6 mM triethanolamine (pH 8.7) at 4°C as a function of time, and unchanged primary amino groups were measured by the fluorescamine method (see Materials and Methods). The relative fluorescence after normalization with native nucleohistone is plotted
Spectrophotometers
Ultraviolet-absorbance was measured with a Hitachi spectrometer, type 139, and spectra were obtained with a Hitachi recording spectrometer, type 323. Fluorescence was measured with a Hitachi spectrofluorometer, type MPF 4, and expressed in arbitrary units. Circular dichroism (CD) spectra were recorded in a Jeol spectropolarimeter model 5-20. Results were reported as molecular ellipticities. RESULTS Estimation of Free Amino groups of Histones after Reaction with Formaldehyde
Formaldehyde reacts in two ways with nucleoprotein : by masking primary amines with hydroxymethyl groups, and by crosslinking between two amino groups of histones, or between histones and DNA. The masking reaction is known to be reversible at equilibrium. In this work, the extent of the reaction was estimated by measuring the decrease in primary amino groups. A solution of calf thymus nucleohistone was mixed with various concentrations of formaldehyde at 4 ' C , and samples of the mixture were taken at intervals, and mixed with fluorescarnine solution. The amino groups free from hydroxymethylation with formaldehyde reacted immediately with fluorescamine to form a fluorophore, while unreacted fluorescamine was hydrolyzed to a non-fluorescent product. Fluorescence at 475 nm was measured after a 30-min incubation. As seen in Fig. 1, the amount of free amino groups decreased as a function of the formaldehyde concentration, attaining equilibrium within 30 min. On incubation for 21 h, however, the amount of free amino groups decreased further, possibly due to secondary crosslinking reactions. In Fig. 2 A, the
amount of free amino groups is plotted against the formaldehyde concentration. The extent of reaction decreased with increase in the nucleohistone concentration : on treatment with 1 % formaldehyde for 30 min, 15 % of the total free amino groups remained unmasked with nucleohistone at ,4260 = 1.0, while 35 % of the free amino grops remained unmasked with nucleohistone at A 2 6 0 = 4.0. The hydroxymethylation reaction of primary amines is known to be reversible. To confirm the reversibility of this reaction, reaction mixtures countaining various concentrations of formaldehyde were dialyzed to eliminate unreacted formaldehyde, and then the amount of free amino groups in nucleohistone was measured again. The results are also shown in Fig.2A. On treatment with 1 % formaldehyde most of the free amino groups of histones became masked, but on dialysis of the solution for 48 h, nearly 50% of these groups became unmasked again. Thus the reverse reaction must be slower than the forward reaction. The fact that the reverse reaction was not complete was partly due to the formation of crosslinking methylene bridges, which is known not to be reversible. In most of these experiments free amino groups were estimated with fluorescarnine in the presence of formaldehyde. The observed reaction might be due to the reaction of fluorescamine with formaldehyde. Therefore, to test whether formaldehyde affects the reaction of fluorescamine, we measured the amount of free amino groups in the presence of formaldehyde using two different concentrations of fluorescamine (0.1 mg/ml and 0.2 mgiml). As shown in Fig. 2 B, there was only a slight difference between the value with these two concentrations of fluorescamine. This slight difference is attributable to the competition between fluorescamine and formaldehyde
Reaction of Formaldehyde with Nucleohistone
288
1
.Ot
A
7-
loo
Ii C H O j
80 -
-
- - _ _- - 0 c '
"
0
1
2
3
4
Time (h) Formaldehyde (%)
Fig. 3 . Fixation reaction qfformaldehyde between proteins and DNA. The fixation reaction was measured with the indicated formaldehyde concentrations in nucleohistone solution of A260 = 10.0 in 6 mM triethanolamine (pH 8.7). The reaction was stopped by adding either 2/3 vol. 0.5 M HzSO4 or 1 vol. 2 % sodium dodecyl sulfate. Unfixed histone was separated by the dodecylsulfate/sucrose method (0 -0) or the sulfuric acid method (0-0) (see Materials and Methods)
3
1 .o
2.0
Formaldehyde
3 .O
(%)
Fig. 2. Reaction of primary nniino groups qf nucleohistone with increasing fijr?nddehj,de concentrations. (A) Effects of nucleohistone concentration, and reaction time. (a) ,4260 = 4.0. reaction time = 30 min; (b) i l ~=~1.0, ~ !reaction time = 30 min; (c) A260 = 2.0, reaction time = 66 h: (c') 3 ml of reaction mixture of (c) were dialyzed against I 1 of 6 mM triethanolamine buffer (pH 8.5) for 48 h at 4'C:. (d) A260 = 1.0, reaction time = 22 h. (B) Effect of fluorescamine concentration on the formaldehyde reaction. Nucleohistone final concentration of A260 = 2.0; reaction time, 60 min. Two con0.1 mg/ml centrations of fluorescamine were used: (00) dioxane; (O----o) 0.2 mg!ml dioxane. Other conditions were as described in the legend to Fig. 1
for the amino groups of histones, and thus the results indicate that fluorescamine does not seem to react directly with formaldehyde. Fimtion of Histonrs to D N A In the presence of formaldehyde the ionic bonds between histones and DNA are converted so that the treated complexes are not dissociable either by salt or acid. The extent of this fixation was estimated by separating unfixed histones from DNA by two methods. Dodecylsulfate treatment results in complete dis-
sociation of unfixed histones from DNA, and the D NA and fixed histone fraction can then be separated by centrifugation. Alternatively, after the fixation reaction unfixed histones can be extracted with sulfuric acid. The results in Fig. 3 show that in 1.OX formaldehyde more than SO:/;; of the histones were fixed to DNA within 4 h, whereas after treatment with 0.2% formaldehyde for 4 h, 60 % of the histones could still be dissociated by dodecylsulfate treatment. Using 0.2 M HzS04 instead of dodecylsulfate, a great difference was observed in values for the unfixed fraction of histones. From these results, it is concluded that histones became less soluble in sulfuric acid on reaction with formaldehyde not only by fixation, but also by hydroxymethylation of the amino groups, and the dodecylsulfate method is preferable to the sulfuric acid method for estimating the extent of fixation. Besides the fixation reaction of histone to DNA, intermolecular crosslinking reactions must occur between histone molecules in nucleohistone, which should be detectable as formation of dimers or oligomers of unfixed histones by dodecylsulfate electrophoresis. As shown in Fig.4, after treatment of nucleohistone with 2 % formaldehyde for 30 min, which resulted in 40% fixation, five peaks were observed in the region of higher molecular weight on dodecylsulfate electrophoresis. The molecular weight of each fraction was calculated from its mobility, although it is uncertain whether the molecular weights of formaldehyde-treated proteins are correlated with their mobilities on dodecylsulfate/gel electrophoresis. The fraction of M , = 29000, which is presumably
Y. Ohba. Y. Morimitsu, and A. Watarai 2% HCHO
289 0.2 n/o
HCHO
Table 1. Amino acidanalysis ojfised OP unfixed histones after fi)rmaIdehyde treatment No corrections were made for hydrolytic loss. Values for glycine are omitted Amino acid
Amount in histones 10%
fixed
l h
A
JV
I\
I
8888 8'L P%Xd R
100"" fixed
isolated H1
6.1 6.0 3.9 9.7 5.2 15.9 7.9
2.7 6.0 6.0 4.0 9.9 26.2 5.8
mol/lO0 mol Aspartic acid Threonine Serine Glutamic acid Proline Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Arginine Lysine
0 0 00
Migration
16% unfixed ~~
n 0.5 h
20"/, fixed
-
Fig. 4. Dodecylsulfate electrophoresis of foemaldehyde-treated histones. Nucleohistone solution (AZ6"= 7.0, final concentration) was treated with either 2 % o r 0.2% formaldehyde for 0.5, 1, 2 and 4 h at 4" C. After the reaction the unfixed histone was extracted by the dodecylsulfate/sucrose method. The released histone was subjected to electrophoresis, and the gels were stained and scanned at 650 nm. Molecular weights were calculated from the mobilities with reference to marker proteins
histone dimer, has a slightly higher mobility than H I histones. The mobility of this supposed dimer in 2 % formaldehyde was higher than that of the reaction product in 0.2 "/, formaldehyde, which had a mobility corresponding to Mr = 32000. This finding suggests that increase in intramolecular crosslinks may affect the interaction of the proteins with dodecylsulfate. After treatment with 2 % formaldehyde for 4 h, more than 90 % the histones had become fixed to DNA, and the remaining unfixed fraction gave nearly the same electrophoretic pattern as that observed after 30-min treatment, but with more of the dimer fraction. Even after treatment with 0.2 % formaldehyde for 30 min, when only 5 % of the histones were fixed to DNA, these oligomer peaks were detectable in the unfixed histone fraction, and after treatment for 4 h, which resulted in 45% fixation, the amount of the dimer fraction was slightly h o r e than after 30-min treatment. Thus some, but not all, crosslinking reactions between histones themselves seem to proceed much faster than the fixation reactions. Therefore, the decrease of primary amino groups shown in Fig. 1 must be due to intra-crosslinking and inter-crosslinking of
6.1 5.1 3.9 9.5 6.2 16.7 8.0 0.7 4.1 8.2 1.5 2.1 2.0 7.5 16.2
1.9 5.6 3.1 9.4 6.3 18.3 7.3
7.1 5.6 3.0 10 7 4.1 15.2 8.2
-
-
0.1
4.5 8.5 0.2 2.1 3.4 7.3 16.3
5.5 9.8 0.4 2.1 2.5 9.9 14.8
5.3 9.6 -
2.1 1.8 10.1 16.3
-
1.0 4.8 1.0 1 .0 -
1.9 28.9
histone molecules themselves, as well as to hydroxymethylation. The electrophoretic pattern of these oligomer fractions remains unchanged during the fixation reaction, and the dimer is not converted to higher oligomer, but the amount of dimer fraction gradually increases with time. Therefore, the rates of fixation of each of these oligomers to DNA must be nearly the same as the rate of fixation of histone monomer. Is there any difference in the rates of fixation of the five histone components to DNA? To answer this question, we prepared three samples of histones : the 10 % and 20 % histone fractions fixed first, and the final 14 % histone fraction remaining unfixed. These fractions were subjected to amino acid analysis. As shown in Table 1, no difference was found in the amino acid compositions of these three fractions, which are identical to that of the hydrolyzate of completely fixed nucleohistone. Thus we tentatively conclude that there is no specificity in the fixation of different molecular species of histone to DNA. In the fixation reaction, which is much slower than the hydroxymethylation of primary amino groups, the hydroxymethyl group bound to amino groups of histone seems further to react with DNA. The following experiment was carried out to confirm that hydroxymethylation and fixation were successive reactions. A solution of nucleohistone (A260 = 4.0) was treated with 5 % formaldehyde for 15 min. During this treatment free amino groups decreased to 5 % of the initial value. Then unchanged formaldehyde was removed by passing the mixture through a column of
Reaction of Formaldehyde with Nucleohistone
290 Table 2. Relation hetueen hydroxymethylution and the fixation reaction
-~
-
Sample
Primary ammo group
Fixation
[8]275x
-
% Control nucleohistone
100
deg.cm2dmol
0
3.9
after elimination of formaldehyde: immediately 4 h later 35 h later
011
0.5
' 2' 1
I
1
5
10
100
Triethanolamine (mM)
7.5 20 40
46 60 63
0 2.6 2.2
Fig. 5. Influence of ionic strength on the ,formaldehyde reaction. Nucleohistone (A*,," = 10.0, final concentration) was allowed to react with 1 7; formaldehyde at various triethanolamine concentrations (pH 8.7). After 30 min, changes primary amino groups were and after 1 h the extent of estimated with fluorescamine (-0) fixation was measured by the dodecylsulfate/sucrose method (O-.)
Sephadex G-25 (see Materials and Methods). In this way nucleohistone was completely separated from formaldehyde in 10 min. The excluded fraction was collected to measure the changes in the amount of free amino groups and in the extent of fixation with time (Table 2). Just after passing through the column, the nucleohistone preparation contained 7.5 "/, free amino groups, presumably formed by the reverse reaction after removal of formaldehyde. The unchanged amount of free amino group after removal of excess formaldehyde also supports the conclusion that formaldehyde does not react directly with fluorescamine. After 4 h, the amount of free amino group increased to 20% and after 35 h to 40%, due to the slow reverse reaction. In spite of the decrease of hydroxymethyl groups, the extent of fixation increased from 46 (I/, to 60 % during the first 4 h after removal of formaldehyde. This increase was probably due to a further reaction of hydroxymethyl groups bound to amino groups of histone to make crosslinks with DNA. The above experiments were carried out in 6 mM triethanolamine (pH 8.7). How is the formaldehyde reaction affected by ionic strength? To examine this question, we measured the extents of hydroxymethylation and fixation with 1.O % formaldehyde in various concentrations of triethanolamine, measuring the amount of free amino groups after 30min, and the extent of fixation after 60 min. As shown in Fig. 5 , the amount of free amino groups decreased with increase in ionic strength, showing a critical point with about 7 mM triethanolamine, while the fixation reaction was not influenced by ionic strength over the range tested. Clmizge of Circulur Dichroism Ellipticity The conformational characteristics of DNA can be estimated by measuring the positive ellipticity in the range of 250-300 nm in the C D spectrum, The positive ellipticity is known to decrease when DNA
0
% -10 E
D N
5 m
g
-20
b
30
-1 Wavelength (nrn)
Fig. 6. Circular dichroic. specrru of calf thymus nucleohistone, after 66 / I of reaction at 4 ' C with various concentrations of'formaldehj&. Formaldehyde concentrations: (......) 0 % ; (- - ) O.lYo; (.-.-.) 1 (----) 5 "4. CD measurements were made at 20 'C on the reaction mixture (AX,,) = 1.0) in a cuvette of 1.0-cm light path
x;
interacts with histones [25 - 281. On reaction of nucleohistone with formaldehyde the C D ellipticity decreased further. The CD spectra of nucleohistone with various concentrations of formaldehyde are shown in Fig. 6. The ellipticity values at 275 nm, [ H I 2 7 5 were 7.5 x lo3 deg . cm2 . (dmol nuc1eotide)-' for calf thymus DNA and 3.5 x lo3 deg . cm2 . dmol-I for the nucleohistone.
Y. Ohba, Y. Morimitsu, and A. Watarai
I
I
I
,
0.01
0.1
1
10
Formaldehyde 10
('lo)
I
I
0
9
P
29 1
'r
100
-
X'
60 .x LL
O
0
0.01
0.1
1
10
Formaldehyde (%)
Fig. 7. Variation of' ellepticity with formaldehyde concentration. (A) [HI275 as a function of formaldehyde concentration in a solution of nucleohistone ( A 2 6 0 = 1.0) after 2 h ( x), 8 h (0)and 20 h (0)at 4 "C. Nucleohistone concentration in the reaction mixture; (-) ,4260 = 1.0; (----) A260 = 5.0. (B) Comparison of [0]275 (-) with the fixation reaction (----), as a function of the formaldehyde concentration for nuclehistone (0) and for DNA (O), treated for 48 h at 4°C. The concentrations of nucleohistone or DNA were ,4260 = 1.0 for ellipticity measurements, and A260 = 10.0 for the fixation reaction. Conditions for the fixation reaction were as described in the legend to Fig. 3
In a solution of 0.1 % formaldehyde, the positive ellipticity at 275 nm decreases to 1.5x l o 3 deg.cm2 .dmol-l, the maximum shifts slightly to 280 nm, and the crossover point shifts from 260 nm to 263 nm. Furthermore a negative ellipticity appears in the region of 287300 nm. Surprisingly the positive ellipticity increases with increases in the concentration of formaldehyde. In Fig.7, is plotted as a function of formaldehyde concentration. The ellipticity begins to decrease with above 0.01 % formaldehyde, reaches a minimum with about 0.4% formaldehyde, and then increases again to a constant value above 2 formaldehyde. This change of ellipticity can be explained as follows. As seen in Fig. 2A, more than 80 % of the primary amino groups are masked with formaldehyde at concentrations above 0.4%, and -NH2 groups are converted to -NH2CH20H.The extent of this reaction depended on the nucleohistone concentration, but the decrease of with increase in formaldehyde concentration up to 0.5 % did not depend on the nucleo-
histone concentration. Thus the decrease of ellipticity is probably due to other reactions of formaldehyde than hydroxymethylation of histone moiety : that is presumably crosslinks or fixation reaction. It is not likely that the DNA double-helix structure in nucleohistone is unwound in the presence of formaldehyde, because there was no hyperchromism at 260 nm. As shown in Fig. 7B there is a good correspondence between the fixation reaction and decrease of [O]275. If the decrease of [fl]275 is caused by the fixation reaction, the change must occur slowly and gradually. As shown in Fig.7A, even 2 h after the reaction started, there was only a slight change in ellipticity. After 8 h the minimum point began to appear with about 0.4% formaldehyde, when more than 60% of the total histones were fixed to DNA. This decrease of ellipticity with formaldehyde is a unique character of nucleohistone: with isolated DNA, [6]2,5 did not change with increase in concentration of formaldehyde until 1 and then decreased by 10 % (Fig. 7 B). Thus we conclude tentatively that the decrease of C D ellipticity at 275 nm observed with 0.01 -0.4:< formaldehyde is due to the fixation reaction. It is uncertain why the ellipticity increased with formaldehyde concentrations of more than 0.4 %. With formaldehyde concentration of about 1 %, the changes in values of nucleohistone and isolated DNA both showed a critical point. This finding suggests that formaldehyde at concentrations of above 1 % may react directly with DNA bases of nucleohistone.
(x,
DISCUSSION Formaldehyde has been extensively used in studies on protein and protein/nucleic acid complex. It has long been known to react with the amino groups of amino acids and proteins. The reaction of formaldehyde with primary amines can be decribed by following two steps [4]. R-NH2
+ HCHO
R-NH-CH20H
+ R'NH2 -+ R-NH-CHz-NH-R' + H2O. R-NH-CH20H
The first step is rapid and reversible, and the second step is slow and irreversible. We estimated the extent of hydroxylation with fluorescarnine, which reacts very rapidly with primary amines only ( t l , z = 0.1 s). Fluorescamine can be used to estimate the amount of free amines in the presence of formaldehyde, because the rate of the reverse reaction to hydroxymethylation is slow and remaining fluorescamine decomposes rapidly ( f l p = 5 s) [29]. Moreover this fluorescent dye does not react with native DNA. Immediately after the nucleohistone is exposed to formaldehyde, a rapid reaction will occur between the
292
formaldehyde and the amino groups of histones. The hydroxymethyl group formed can react further with other binding sites, if present, to form methylene crosslinks between two neighbouring amino groups of the same or different molecules of histones. The crosslinked products are detectable as dimers or oligomers of histones on dedecylsulfate-gel electrophoresis. Histone oligomers formed by treatment of chromatin with formaldehyde was first reported by Van Lente et al. [30]. They observed five sorts of specific histone-crosslinked products, and determined two major fractions as a dimer of histone 2 A and 2B, and that of histone 2B and 3. In this report also, five sorts of histone oligomers have been detected (Fig. 4), and these crosslinks must be formed much faster than the fixation reaction. Regardless of the reaction time and concentration of formaldehyde, approximatively two-thirds of histones remained as monomers. Most of the amino groups of histones are hydroxymethylated immediately after the reaction starts, as shown in Fig. 1, and these masked forms of all reactive sites may interfere the formation of crosslinks. Chalkley and Hunter [15] reported that formaldehyde cannot induce intermolecular crosslinking of histones. Recently Jackson has succeeded in the selective reversal of formaldehyde crosslinkings in both histone-histone and histone-DNA [31]. He analyzed ten possible dimers of the four sorts of histones after formaldehyde treatment and HzS04 extraction, though he could not detect oligomer complex. These discrepancies are probably because we used the dodecylsulfate method but they used sulfuric acid for extraction of unfixed histones, and presumably intermolecular and intramolecular crosslinkings reduce the solubility of histones in sulfuric acid. The fixation reaction seems to occur much more slowly than the reaction of hydroxymethylation or crosslinks of histones themselves. Brutlag et al. [ l l ] first reported that formaldehyde-treated chromatin contained covalently linked histone-DNA complexes, which could not be dissociated by treatment with acid or a high concentration of cesium chloride. They represented protein-DNA crosslinks by formaldehyde which remains attached to the complex after pronase treatment. Pronase treatment of the fixed chromatin removes 96 of the protein. The formatin of crosslinks between histones and DNA results in a conformational change of DNA, which is detectable as a change of positive ellipticity in the region of 250-300 nm. Decrease of positive ellipticity of nucleohistone on reaction with formaldehyde has been reported by Senior and Olins [16], but their results do not agree with our results. They reported that the positive ellipticity above 240 nm progressively decreased with increase in the formaldehyde concentration, finally attaining a negative value with 3 % formaldehyde. They did not observe
Reaction of Formaldehyde with Nucleohistone
a minimum point on increasing the formaldehyde concentration. In an attempt to find out why they obtained different results, we exanlined calf thymus nucleohistone under their reaction conditions, but we found that in either water of 5 mM NaC1,0.035 mM phosphate buffer (pH 6.8) it gave the same pattern as that in Fig. 7. Thus this discrepancy may be attributed to the different chromatins used: that is, a difference between calf thymus and chicken erythrocyte chromatin. In fact, they reported that the [0]z75 value of chicken erythrocyte chromatin is more than 5000 deg . cm2 . dmol-' in water, while that of our calf thymus chromatin was 3500 deg . cm2 . dmol-', as indicated in this paper. The reason for the increase of ellipticity at 275 nm with concentrations of over 0.4 7; formaldehyde is unkown. Two possible explanations for this change may be considered. One is the direct reaction of formaldehyde with DNA bases in this concentration range, causing a conformational change in the secondary structure of DNA. The results in Fig.7 support this consideration. The other possibility is that the fraction of formaldehyde diadduct in histones may increase with over 0.4 % formaldehyde, and this increase in the amount of diadduct may reduce the interaction of histones with DNA. This work was supported in part by Scientific Research Grants from the Ministry of Education, Science and Culture of Japan.
REFERENCES 1. French, D. SC Edsall, J. T. (1945) Adv. Protein Chem. 2, 277. 2. Fraenkel-Conrat, H. & Olcott, H. (1948) J . Am. Chem. Soc. 70, 2673 - 2684. 3. Fraenkel-Conrat, H. SC Olcott, H. (1948) J . Bid. Chem. 174. 827 - 843. 4. Feldman, M. Y. (1973) Prog. Nucleic Acid Res. Mol. Biol. 13, 1-49. 5. McGhee, J. D. & von Hippel, P. H. (1975) Biochemistry, 14, 1281 - 1296. 6. McGhee, J. D. & von Hippel, P. H. (1975) Biochemistry, 14, 1297- 1303. 7. Ilyin, Y. V. & Georgiev, G . P. (1969) J . Mol. Biol. 41,299 - 303. 8. Hancock, R. (1970) J . Mo/. Biol. 48, 357-360. 9. Olins, A. L. & Olins, D. E. (1974) Science (Wash. D.C.) 183, 330 - 332. 10. Varshavsky, A. J., Ilyin, Y. V. & Georgiev, G. P. (1974) Nature iLond.) 250, 602- 606. 11. Brutlag, D., Schlehuber, C. Sr Bonner, J. (1969) Biochemistry, 8, 3214-3228. 12. Li, H. J. (1972) Biopolymers, 11, 835-847. 13. Siomin, Y. A., Siminov, V. V. & Poverenny, A. M. (1973) Biochim. Biophys. Acta, 331, 27-32. 14. Jackson, V. J. & Chalkley, R. (1974) Biochemistry, 13, 39523956. 15. Chalkley, R. & Hunter, C. (1975) Proc. Natl Acad. Sci. U.S.A. 72, 1304-1308. 16. Senior, M . B. & Olins, D. E. (1975) Biochemistry, 14, 33323337. 17. Zubay. G. & Doty, P. (1959) J . Mol. B i d . I , 1-20.
Y. Ohba, Y . Morimitsu, and A. Watarai
18. Ohba, Y. Sr Hayashi. M. (1972) Eur. J . Biochem. 29. 461 -468. 19. Burton, K. (1956) Biochem. J . 62, 315-323. 20. Lowry, 0. H., Rosebrough, N. J.. Farr, A. L. Sr Randall, R. J. (1951) J . B i d . Chem. 139, 265-275. 21. Bohlen, P., Stein, S., Dairman, W. Sr Udenfriend, S. (1973) Arch. Biochem. Biophys. 155, 213. 22. Reference deleted. 23. Hayashi, K . Sr Ohba, Y. (1974) Proc. Nut1 Acad. Sci. U.S.A. 71, 2419-2422. 24. Hayashi, K., Matsutera, E. & Ohba, Y. (1974) Biochim. Biophys. Acta, 342, 185-194.
293 25. Simpson, R. T. & Sober, H. A. (1970) Biochemistry, 0. 31033109. 26. Shih, T. & Fasman, G. D. (1970) J . Mo/. Biol. 52, 125-129. 27. Hanlon, S., Johnson, R. S., Wolf, B. & Chan, A . (1972) Proc. Nut1 Acad. Sci. U.S.A. 69, 3263-3267. 28. Johnson, R. S., Chan, A. & Hanlon, S. (1972) Biochemi~ylry,11, 4347-4358. 29. Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leigruber, W. Sr Weigele, M. (1972) Science (Wash. D.C.) 178,871 -872. 30. Van Lente, F. & Weintraub, H. (1975) Cell, 5, 45-SO. 31. Jackson, V. (1978) Cell, 15, 945-954.
Y. Ohba, Y. Morimitsu, and A. Watarai, Department of Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Takara-machi, Kanazawa-shi. Ishikawa-ken, Japan 920
Reaction of Formaldehyde with Calf-Thymus Nucleohistone Yoshiki OHBA, Yoshimi MORIMITSU, and Atsuko WATARAI Ilepartment of Biology, Faculty of Pharmaceutical Sciences, Kandzawa University (Received January 4, 1979)
The reactions of formaldehyde with calf thymus nucleohistone were analyzed in the following ways : measurement with fluorescamine of the decrease in primary amino groups resulting from hydroxymethylation and crosslinking reactions, measurement with dodecylsulphate-gel electrophoresis of formation of histone oligomers, measurement of fixation of histones to the DNA in nucleohistone, and measurement of changes in the circular dichroism spectrum in the region of 250 - 300 nm. In the presence of formaldehyde, the primary amino groups of histones decreased very rapidly, attaining an equilibrium within 60 min, and successively intermolecular crosslinks were also formed between histone molecules, the resulting dimers and oligomers being separable by dodecylsulfate-gel electrophoresis. Whereas the fixation reaction proceeded much more slowly. The extent of fixation could be measured more accurately by dodecylsulfate/sucrose centrifugation analysis than by sulfuric acid extraction. After removal of f-ormaldehyde from the reaction mixture, the fraction of masked amino groups decreased, perhaps due to the reverse reaction, but the extent of fixation of histones continued to increase with time. No specificity was observed among five molecular species of histones in the fixation reaction. With increase in formaldehyde concentration, the ellipticity of nucleohistone decreased to a minimum with about 0.4 % formaldehyde, and then increased.
Formaldehyde is known to bind to various groups of proteins, including primary amino and imidazole groups [l -31. There formaldehyde has both monofunctional and bifunctional actions. Many studies on the interactions of formaldehyde with nucleic acids have also shown that formaldehyde can react with both the exocyclic amino groups and endocyclic imino groups of DNA bases [4 - 61. Formaldehyde has recently been used in physical characterization of chromatin [7 - lo]. The mechanism of the reaction between formaldehyde and nucleohistone has been reported by several workers [ l l - 161. In this process formaldehyde reacts with both histones and DNA, acting as a fixative. The treatment of nucleohistone with formaldehyde results in complex in which protein-DNA interaction is no longer ionic as it is in native nucleohistone. The treated complexes are not dissociated either by 4 M CsCl or 0.1 M HzS04. In this work, we investigated the interactions of formaldehyde with calf thymus nucleohistone in following ways : namely by measuring decreases in primary amino groups of histone, fixation of histones to DNA, and changes of the circular dichroism spectrum in the Abbreviation. CD, circular dichroism
region of 250 - 300 nm, due to conformational changes of the DNA in chromatin.
MATERIALS AND METHODS Preparation of Nucleohistone Nucleohistone was prepared from 20 - 40 g of frozen calf thymus glands by a modification of the method of Zubay and Doty [17]. Thus 20-40g of frozen calf thymus glands were minced with scissors and then homogenized in 300 ml of standard solvent (0.14 M NaC1, 0.01 M EDTA pH 7.0) with a homogenizer (Nihonseiki) for 5 min. The mixture was filtered through double-layered gauze, and the filtrate was centrifuged at 3000 rev./min for 10min. The precipitate was again homogenized with the standard solvent and centrifuged as before. This cycle was repeated more than ten times. The final precipitate was then homogenized in 50-100 ml of doubledistilled water and the mixture was dialyzed against 0.7 mM phosphate buffer (pH 7.2) for 3 days. All procedures were carried out at 4 "C. The final preparation contained no detectable non-histone proteins
286
when examined by sodium dodecylsulfate electrophoresis [18]. An aliquot of the concentrated nucleohistone gel was weighed and diluted with water or phosphate buffer (0.7 mM, pH 7.2) by homogenization, until pipetting became feasible (generally diluted to 0.05% DNA). DNA was estimated from the absorbance at 260nm, being taken as 200, or by Burton's method [19]. Histones were estimated by the method of Lowry et al. [20], with isolated histones as a standard. The ratio of protein to DNA was 1.20-1.25.
Reaction with Formaldehyde Formaldehyde reagent grade (approximately 37 "/,) was obtained from Wako Chemicals. Contaminating formic acid present was removed by passing the cold solution through a small column of Dowex I in hydroxide form. The total formaldehyde content w7as calculated from the specific gravity measured with a hydrometer. For each experiment a sample of the stock solution was heated to 65 "C for 15 min and then diluted. The reaction was carried out at various concentrations of formaldehyde in 6 mM triethanolamine (pH 8.7), at a nucleohistone concentration of A260 1.0, unless otherwise mentioned. The reaction was allowed to proceed for various times at 4 " C ,or 20 "C, as indicated, and was usually stopped by adding either 0.1 vol. 10 % sodium dodecyl sulfate to remove the unfixed histones, or 0.5 vol. fluorescamine reagent, to estimate the amount of remaining primary amines of histones. Sometimes the reaction was stopped by rapid removal of excess formaldehyde. For this, 5 ml of the reaction mixture (A260 = 4.0) was applied to a Sephadex G-25 column (coarse, 2 x 30cm), and the column was eluted with 0.7 mM phosphate buffer (pH 6.7) at a flow rate of 5 ml/min at room temperature. Fractions of 5 ml were collected, and assayed first spectrophotometrically at 260 nm, and then by the Schiff test for formaldehyde. With the latter method 10 min was sufficient for complete separation of nucleohistone from formaldehyde.
Reaction with Fluorescarnine Fluorescamine (Fluram) was obtained from JapanRoche Diagonostics, and dissolved in anhydrous dioxane at a concentration of 0.1 mg/ml just before use. A sample of 1.0-20 pg protein in 2 ml of 0.2 M borate buffer (pH 9.5) was rapidly mixed with 1 ml of fluorescamine reagent, while holding the test tube on a Vortex mixer. The resulting fluorescence was measured at least 30 min later in a Hitachi spectrofluorometer, using an excitation wavelength of 340 nm and emission at a wavelength of 475 nm [21]. The extent of fluorescence remained unchanged for several hours at least. Under these conditions the amino
Reaction of Formaldehyde with Nucleohistone
groups of histones reacted quantitatively with fluoresamine, and the reaction v7as not influenced by the presence of DNA (Y. Ohba & K. Yoshii, unpublished results).
Separation oj Unfixed Histones After fixation of nucleohistone with formaldehyde, the unfixed histone fraction was separated by extraction with H 2 S 0 4 or centrifugation in dodecylsulfate/ sucrose. For extraction with HzS04, 2/3 vol. of 0.5 M HzS04 was added to the reaction mixture, containing partially fixed nucleohistone, and the mixture was stirred vigorously overnight at 4 "C, and then centrifuged at 10000 x g for 30 min and the supernatant was dialyzed against 0.7 mM phosphate. The volume of the dialyzed solution was calculated from the total weight, and then the total amount of dissociated protein was estimated. In the dodecylsulfate/sucrose method, 4-ml samples of the mixture of nucleohistone and formaldehyde were removed at intervals, mixed with 2 ml of 2 % sodium dodecylsulfate, and incubated at 37 "C for 30 min. Then the solution was layered on a discontinuous sucrose gradient consisting of layers of 2 ml of 60 % sucrose and 5 ml of 5 % sucrose, 0.1 % sodium dodecylsulfate. The gradient was centrifuged for 18 h at 35000 rev./min in the RPS 40 T rotor of a Hitachi P3 ultracentrifuge at 18 "C, and then 2-ml fractions were collected from the top of the tube with a microinfusion pump (LKB, Perpex). Their A260 was measured and the fractions in the peak position were combined and dialyzed against distilled water to estimate the ratio of protein to DNA. The DNA fraction obtained in this way from untreated nucleohistone contained less than 5 % of the total protein [23]. Sometimes the fractions of dissociated histones were lyophilized to analyze their amino acid composition or their dodecylsulfate-electrophoretic pattern.
Amino Acid Analysis Amino acid analysis was carried out on fixed 10 7; histones or 20% histones without removal of DNA, and on 1 4 % histones remaining unfixed. When the fixation reaction was carried out in 0.2 %formaldehyde for 0.5 or 1 h, the partially fixed nucleohistones, including either 10% or 20% histones, were obtained by the dodecylsulfate/sucrose method described above. The 14 % histones remaining unfixed were obtained as the supernatant solution on dodecylsulfate centrifugation of reaction mixture treated with l % formaldehyde for 4 h at 4°C. When the reaction was carried out in 2 %, formaldehyde for 48 h, 100% fixation was achieved. These samples were hydrolysed in Nz-replaced sealed tubes with 6 M HC1 at 110 "C for 20 h, and analyzed in an amino acid analyzer (Joel 6SH). The
Y. Ohba, Y. Morimitsu, and A. Watarai
281
glycine content was not calculated, because glycine is produced during DNA degradation under these conditions of hydrolysis. Dodecylsuljate Electrophoresis
The unfixed histones in the fixation reaction were analyzed by dodecylsulfate electrophoresis. The samples were prepared by the same method as for dodecylsulfate/sucrose centrifugation method, and dissolved in the suitable amount of 2 sodium dodecylsulfate, 0.2 M phosphate buffer (pH 7.2), 50% glycerol to give a final concentration of 1 mg histone/ml. The samples were subjected to electrophoresis on 12.5 7; polyacrylamide gel in dodecylsulfate/phosphate buffer as previously described [24]. The gels were stained with Comassie brilliant blue, and scanned at 600 nm with a Gilford spectrophotometer model 2400s.
1 .o
0
"
f.
2.0
2 "D
610
do
1 o; Time (min)
I
18021h
Fig. 1 . Time course of formaldehyde reaction with primary amino groups of histones. Calf thymus nucleohistone ( A 2 6 0 = 1.0, final concentration) was treated with various concentrations of formaldehyde in 6 mM triethanolamine (pH 8.7) at 4°C as a function of time, and unchanged primary amino groups were measured by the fluorescamine method (see Materials and Methods). The relative fluorescence after normalization with native nucleohistone is plotted
Spectrophotometers
Ultraviolet-absorbance was measured with a Hitachi spectrometer, type 139, and spectra were obtained with a Hitachi recording spectrometer, type 323. Fluorescence was measured with a Hitachi spectrofluorometer, type MPF 4, and expressed in arbitrary units. Circular dichroism (CD) spectra were recorded in a Jeol spectropolarimeter model 5-20. Results were reported as molecular ellipticities. RESULTS Estimation of Free Amino groups of Histones after Reaction with Formaldehyde
Formaldehyde reacts in two ways with nucleoprotein : by masking primary amines with hydroxymethyl groups, and by crosslinking between two amino groups of histones, or between histones and DNA. The masking reaction is known to be reversible at equilibrium. In this work, the extent of the reaction was estimated by measuring the decrease in primary amino groups. A solution of calf thymus nucleohistone was mixed with various concentrations of formaldehyde at 4 ' C , and samples of the mixture were taken at intervals, and mixed with fluorescarnine solution. The amino groups free from hydroxymethylation with formaldehyde reacted immediately with fluorescamine to form a fluorophore, while unreacted fluorescamine was hydrolyzed to a non-fluorescent product. Fluorescence at 475 nm was measured after a 30-min incubation. As seen in Fig. 1, the amount of free amino groups decreased as a function of the formaldehyde concentration, attaining equilibrium within 30 min. On incubation for 21 h, however, the amount of free amino groups decreased further, possibly due to secondary crosslinking reactions. In Fig. 2 A, the
amount of free amino groups is plotted against the formaldehyde concentration. The extent of reaction decreased with increase in the nucleohistone concentration : on treatment with 1 % formaldehyde for 30 min, 15 % of the total free amino groups remained unmasked with nucleohistone at ,4260 = 1.0, while 35 % of the free amino grops remained unmasked with nucleohistone at A 2 6 0 = 4.0. The hydroxymethylation reaction of primary amines is known to be reversible. To confirm the reversibility of this reaction, reaction mixtures countaining various concentrations of formaldehyde were dialyzed to eliminate unreacted formaldehyde, and then the amount of free amino groups in nucleohistone was measured again. The results are also shown in Fig.2A. On treatment with 1 % formaldehyde most of the free amino groups of histones became masked, but on dialysis of the solution for 48 h, nearly 50% of these groups became unmasked again. Thus the reverse reaction must be slower than the forward reaction. The fact that the reverse reaction was not complete was partly due to the formation of crosslinking methylene bridges, which is known not to be reversible. In most of these experiments free amino groups were estimated with fluorescarnine in the presence of formaldehyde. The observed reaction might be due to the reaction of fluorescamine with formaldehyde. Therefore, to test whether formaldehyde affects the reaction of fluorescamine, we measured the amount of free amino groups in the presence of formaldehyde using two different concentrations of fluorescamine (0.1 mg/ml and 0.2 mgiml). As shown in Fig. 2 B, there was only a slight difference between the value with these two concentrations of fluorescamine. This slight difference is attributable to the competition between fluorescamine and formaldehyde
Reaction of Formaldehyde with Nucleohistone
288
1
.Ot
A
7-
loo
Ii C H O j
80 -
-
- - _ _- - 0 c '
"
0
1
2
3
4
Time (h) Formaldehyde (%)
Fig. 3 . Fixation reaction qfformaldehyde between proteins and DNA. The fixation reaction was measured with the indicated formaldehyde concentrations in nucleohistone solution of A260 = 10.0 in 6 mM triethanolamine (pH 8.7). The reaction was stopped by adding either 2/3 vol. 0.5 M HzSO4 or 1 vol. 2 % sodium dodecyl sulfate. Unfixed histone was separated by the dodecylsulfate/sucrose method (0 -0) or the sulfuric acid method (0-0) (see Materials and Methods)
3
1 .o
2.0
Formaldehyde
3 .O
(%)
Fig. 2. Reaction of primary nniino groups qf nucleohistone with increasing fijr?nddehj,de concentrations. (A) Effects of nucleohistone concentration, and reaction time. (a) ,4260 = 4.0. reaction time = 30 min; (b) i l ~=~1.0, ~ !reaction time = 30 min; (c) A260 = 2.0, reaction time = 66 h: (c') 3 ml of reaction mixture of (c) were dialyzed against I 1 of 6 mM triethanolamine buffer (pH 8.5) for 48 h at 4'C:. (d) A260 = 1.0, reaction time = 22 h. (B) Effect of fluorescamine concentration on the formaldehyde reaction. Nucleohistone final concentration of A260 = 2.0; reaction time, 60 min. Two con0.1 mg/ml centrations of fluorescamine were used: (00) dioxane; (O----o) 0.2 mg!ml dioxane. Other conditions were as described in the legend to Fig. 1
for the amino groups of histones, and thus the results indicate that fluorescamine does not seem to react directly with formaldehyde. Fimtion of Histonrs to D N A In the presence of formaldehyde the ionic bonds between histones and DNA are converted so that the treated complexes are not dissociable either by salt or acid. The extent of this fixation was estimated by separating unfixed histones from DNA by two methods. Dodecylsulfate treatment results in complete dis-
sociation of unfixed histones from DNA, and the D NA and fixed histone fraction can then be separated by centrifugation. Alternatively, after the fixation reaction unfixed histones can be extracted with sulfuric acid. The results in Fig. 3 show that in 1.OX formaldehyde more than SO:/;; of the histones were fixed to DNA within 4 h, whereas after treatment with 0.2% formaldehyde for 4 h, 60 % of the histones could still be dissociated by dodecylsulfate treatment. Using 0.2 M HzS04 instead of dodecylsulfate, a great difference was observed in values for the unfixed fraction of histones. From these results, it is concluded that histones became less soluble in sulfuric acid on reaction with formaldehyde not only by fixation, but also by hydroxymethylation of the amino groups, and the dodecylsulfate method is preferable to the sulfuric acid method for estimating the extent of fixation. Besides the fixation reaction of histone to DNA, intermolecular crosslinking reactions must occur between histone molecules in nucleohistone, which should be detectable as formation of dimers or oligomers of unfixed histones by dodecylsulfate electrophoresis. As shown in Fig.4, after treatment of nucleohistone with 2 % formaldehyde for 30 min, which resulted in 40% fixation, five peaks were observed in the region of higher molecular weight on dodecylsulfate electrophoresis. The molecular weight of each fraction was calculated from its mobility, although it is uncertain whether the molecular weights of formaldehyde-treated proteins are correlated with their mobilities on dodecylsulfate/gel electrophoresis. The fraction of M , = 29000, which is presumably
Y. Ohba. Y. Morimitsu, and A. Watarai 2% HCHO
289 0.2 n/o
HCHO
Table 1. Amino acidanalysis ojfised OP unfixed histones after fi)rmaIdehyde treatment No corrections were made for hydrolytic loss. Values for glycine are omitted Amino acid
Amount in histones 10%
fixed
l h
A
JV
I\
I
8888 8'L P%Xd R
100"" fixed
isolated H1
6.1 6.0 3.9 9.7 5.2 15.9 7.9
2.7 6.0 6.0 4.0 9.9 26.2 5.8
mol/lO0 mol Aspartic acid Threonine Serine Glutamic acid Proline Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Arginine Lysine
0 0 00
Migration
16% unfixed ~~
n 0.5 h
20"/, fixed
-
Fig. 4. Dodecylsulfate electrophoresis of foemaldehyde-treated histones. Nucleohistone solution (AZ6"= 7.0, final concentration) was treated with either 2 % o r 0.2% formaldehyde for 0.5, 1, 2 and 4 h at 4" C. After the reaction the unfixed histone was extracted by the dodecylsulfate/sucrose method. The released histone was subjected to electrophoresis, and the gels were stained and scanned at 650 nm. Molecular weights were calculated from the mobilities with reference to marker proteins
histone dimer, has a slightly higher mobility than H I histones. The mobility of this supposed dimer in 2 % formaldehyde was higher than that of the reaction product in 0.2 "/, formaldehyde, which had a mobility corresponding to Mr = 32000. This finding suggests that increase in intramolecular crosslinks may affect the interaction of the proteins with dodecylsulfate. After treatment with 2 % formaldehyde for 4 h, more than 90 % the histones had become fixed to DNA, and the remaining unfixed fraction gave nearly the same electrophoretic pattern as that observed after 30-min treatment, but with more of the dimer fraction. Even after treatment with 0.2 % formaldehyde for 30 min, when only 5 % of the histones were fixed to DNA, these oligomer peaks were detectable in the unfixed histone fraction, and after treatment for 4 h, which resulted in 45% fixation, the amount of the dimer fraction was slightly h o r e than after 30-min treatment. Thus some, but not all, crosslinking reactions between histones themselves seem to proceed much faster than the fixation reactions. Therefore, the decrease of primary amino groups shown in Fig. 1 must be due to intra-crosslinking and inter-crosslinking of
6.1 5.1 3.9 9.5 6.2 16.7 8.0 0.7 4.1 8.2 1.5 2.1 2.0 7.5 16.2
1.9 5.6 3.1 9.4 6.3 18.3 7.3
7.1 5.6 3.0 10 7 4.1 15.2 8.2
-
-
0.1
4.5 8.5 0.2 2.1 3.4 7.3 16.3
5.5 9.8 0.4 2.1 2.5 9.9 14.8
5.3 9.6 -
2.1 1.8 10.1 16.3
-
1.0 4.8 1.0 1 .0 -
1.9 28.9
histone molecules themselves, as well as to hydroxymethylation. The electrophoretic pattern of these oligomer fractions remains unchanged during the fixation reaction, and the dimer is not converted to higher oligomer, but the amount of dimer fraction gradually increases with time. Therefore, the rates of fixation of each of these oligomers to DNA must be nearly the same as the rate of fixation of histone monomer. Is there any difference in the rates of fixation of the five histone components to DNA? To answer this question, we prepared three samples of histones : the 10 % and 20 % histone fractions fixed first, and the final 14 % histone fraction remaining unfixed. These fractions were subjected to amino acid analysis. As shown in Table 1, no difference was found in the amino acid compositions of these three fractions, which are identical to that of the hydrolyzate of completely fixed nucleohistone. Thus we tentatively conclude that there is no specificity in the fixation of different molecular species of histone to DNA. In the fixation reaction, which is much slower than the hydroxymethylation of primary amino groups, the hydroxymethyl group bound to amino groups of histone seems further to react with DNA. The following experiment was carried out to confirm that hydroxymethylation and fixation were successive reactions. A solution of nucleohistone (A260 = 4.0) was treated with 5 % formaldehyde for 15 min. During this treatment free amino groups decreased to 5 % of the initial value. Then unchanged formaldehyde was removed by passing the mixture through a column of
Reaction of Formaldehyde with Nucleohistone
290 Table 2. Relation hetueen hydroxymethylution and the fixation reaction
-~
-
Sample
Primary ammo group
Fixation
[8]275x
-
% Control nucleohistone
100
deg.cm2dmol
0
3.9
after elimination of formaldehyde: immediately 4 h later 35 h later
011
0.5
' 2' 1
I
1
5
10
100
Triethanolamine (mM)
7.5 20 40
46 60 63
0 2.6 2.2
Fig. 5. Influence of ionic strength on the ,formaldehyde reaction. Nucleohistone (A*,," = 10.0, final concentration) was allowed to react with 1 7; formaldehyde at various triethanolamine concentrations (pH 8.7). After 30 min, changes primary amino groups were and after 1 h the extent of estimated with fluorescamine (-0) fixation was measured by the dodecylsulfate/sucrose method (O-.)
Sephadex G-25 (see Materials and Methods). In this way nucleohistone was completely separated from formaldehyde in 10 min. The excluded fraction was collected to measure the changes in the amount of free amino groups and in the extent of fixation with time (Table 2). Just after passing through the column, the nucleohistone preparation contained 7.5 "/, free amino groups, presumably formed by the reverse reaction after removal of formaldehyde. The unchanged amount of free amino group after removal of excess formaldehyde also supports the conclusion that formaldehyde does not react directly with fluorescamine. After 4 h, the amount of free amino group increased to 20% and after 35 h to 40%, due to the slow reverse reaction. In spite of the decrease of hydroxymethyl groups, the extent of fixation increased from 46 (I/, to 60 % during the first 4 h after removal of formaldehyde. This increase was probably due to a further reaction of hydroxymethyl groups bound to amino groups of histone to make crosslinks with DNA. The above experiments were carried out in 6 mM triethanolamine (pH 8.7). How is the formaldehyde reaction affected by ionic strength? To examine this question, we measured the extents of hydroxymethylation and fixation with 1.O % formaldehyde in various concentrations of triethanolamine, measuring the amount of free amino groups after 30min, and the extent of fixation after 60 min. As shown in Fig. 5 , the amount of free amino groups decreased with increase in ionic strength, showing a critical point with about 7 mM triethanolamine, while the fixation reaction was not influenced by ionic strength over the range tested. Clmizge of Circulur Dichroism Ellipticity The conformational characteristics of DNA can be estimated by measuring the positive ellipticity in the range of 250-300 nm in the C D spectrum, The positive ellipticity is known to decrease when DNA
0
% -10 E
D N
5 m
g
-20
b
30
-1 Wavelength (nrn)
Fig. 6. Circular dichroic. specrru of calf thymus nucleohistone, after 66 / I of reaction at 4 ' C with various concentrations of'formaldehj&. Formaldehyde concentrations: (......) 0 % ; (- - ) O.lYo; (.-.-.) 1 (----) 5 "4. CD measurements were made at 20 'C on the reaction mixture (AX,,) = 1.0) in a cuvette of 1.0-cm light path
x;
interacts with histones [25 - 281. On reaction of nucleohistone with formaldehyde the C D ellipticity decreased further. The CD spectra of nucleohistone with various concentrations of formaldehyde are shown in Fig. 6. The ellipticity values at 275 nm, [ H I 2 7 5 were 7.5 x lo3 deg . cm2 . (dmol nuc1eotide)-' for calf thymus DNA and 3.5 x lo3 deg . cm2 . dmol-I for the nucleohistone.
Y. Ohba, Y. Morimitsu, and A. Watarai
I
I
I
,
0.01
0.1
1
10
Formaldehyde 10
('lo)
I
I
0
9
P
29 1
'r
100
-
X'
60 .x LL
O
0
0.01
0.1
1
10
Formaldehyde (%)
Fig. 7. Variation of' ellepticity with formaldehyde concentration. (A) [HI275 as a function of formaldehyde concentration in a solution of nucleohistone ( A 2 6 0 = 1.0) after 2 h ( x), 8 h (0)and 20 h (0)at 4 "C. Nucleohistone concentration in the reaction mixture; (-) ,4260 = 1.0; (----) A260 = 5.0. (B) Comparison of [0]275 (-) with the fixation reaction (----), as a function of the formaldehyde concentration for nuclehistone (0) and for DNA (O), treated for 48 h at 4°C. The concentrations of nucleohistone or DNA were ,4260 = 1.0 for ellipticity measurements, and A260 = 10.0 for the fixation reaction. Conditions for the fixation reaction were as described in the legend to Fig. 3
In a solution of 0.1 % formaldehyde, the positive ellipticity at 275 nm decreases to 1.5x l o 3 deg.cm2 .dmol-l, the maximum shifts slightly to 280 nm, and the crossover point shifts from 260 nm to 263 nm. Furthermore a negative ellipticity appears in the region of 287300 nm. Surprisingly the positive ellipticity increases with increases in the concentration of formaldehyde. In Fig.7, is plotted as a function of formaldehyde concentration. The ellipticity begins to decrease with above 0.01 % formaldehyde, reaches a minimum with about 0.4% formaldehyde, and then increases again to a constant value above 2 formaldehyde. This change of ellipticity can be explained as follows. As seen in Fig. 2A, more than 80 % of the primary amino groups are masked with formaldehyde at concentrations above 0.4%, and -NH2 groups are converted to -NH2CH20H.The extent of this reaction depended on the nucleohistone concentration, but the decrease of with increase in formaldehyde concentration up to 0.5 % did not depend on the nucleo-
histone concentration. Thus the decrease of ellipticity is probably due to other reactions of formaldehyde than hydroxymethylation of histone moiety : that is presumably crosslinks or fixation reaction. It is not likely that the DNA double-helix structure in nucleohistone is unwound in the presence of formaldehyde, because there was no hyperchromism at 260 nm. As shown in Fig. 7B there is a good correspondence between the fixation reaction and decrease of [O]275. If the decrease of [fl]275 is caused by the fixation reaction, the change must occur slowly and gradually. As shown in Fig.7A, even 2 h after the reaction started, there was only a slight change in ellipticity. After 8 h the minimum point began to appear with about 0.4% formaldehyde, when more than 60% of the total histones were fixed to DNA. This decrease of ellipticity with formaldehyde is a unique character of nucleohistone: with isolated DNA, [6]2,5 did not change with increase in concentration of formaldehyde until 1 and then decreased by 10 % (Fig. 7 B). Thus we conclude tentatively that the decrease of C D ellipticity at 275 nm observed with 0.01 -0.4:< formaldehyde is due to the fixation reaction. It is uncertain why the ellipticity increased with formaldehyde concentrations of more than 0.4 %. With formaldehyde concentration of about 1 %, the changes in values of nucleohistone and isolated DNA both showed a critical point. This finding suggests that formaldehyde at concentrations of above 1 % may react directly with DNA bases of nucleohistone.
(x,
DISCUSSION Formaldehyde has been extensively used in studies on protein and protein/nucleic acid complex. It has long been known to react with the amino groups of amino acids and proteins. The reaction of formaldehyde with primary amines can be decribed by following two steps [4]. R-NH2
+ HCHO
R-NH-CH20H
+ R'NH2 -+ R-NH-CHz-NH-R' + H2O. R-NH-CH20H
The first step is rapid and reversible, and the second step is slow and irreversible. We estimated the extent of hydroxylation with fluorescarnine, which reacts very rapidly with primary amines only ( t l , z = 0.1 s). Fluorescamine can be used to estimate the amount of free amines in the presence of formaldehyde, because the rate of the reverse reaction to hydroxymethylation is slow and remaining fluorescamine decomposes rapidly ( f l p = 5 s) [29]. Moreover this fluorescent dye does not react with native DNA. Immediately after the nucleohistone is exposed to formaldehyde, a rapid reaction will occur between the
292
formaldehyde and the amino groups of histones. The hydroxymethyl group formed can react further with other binding sites, if present, to form methylene crosslinks between two neighbouring amino groups of the same or different molecules of histones. The crosslinked products are detectable as dimers or oligomers of histones on dedecylsulfate-gel electrophoresis. Histone oligomers formed by treatment of chromatin with formaldehyde was first reported by Van Lente et al. [30]. They observed five sorts of specific histone-crosslinked products, and determined two major fractions as a dimer of histone 2 A and 2B, and that of histone 2B and 3. In this report also, five sorts of histone oligomers have been detected (Fig. 4), and these crosslinks must be formed much faster than the fixation reaction. Regardless of the reaction time and concentration of formaldehyde, approximatively two-thirds of histones remained as monomers. Most of the amino groups of histones are hydroxymethylated immediately after the reaction starts, as shown in Fig. 1, and these masked forms of all reactive sites may interfere the formation of crosslinks. Chalkley and Hunter [15] reported that formaldehyde cannot induce intermolecular crosslinking of histones. Recently Jackson has succeeded in the selective reversal of formaldehyde crosslinkings in both histone-histone and histone-DNA [31]. He analyzed ten possible dimers of the four sorts of histones after formaldehyde treatment and HzS04 extraction, though he could not detect oligomer complex. These discrepancies are probably because we used the dodecylsulfate method but they used sulfuric acid for extraction of unfixed histones, and presumably intermolecular and intramolecular crosslinkings reduce the solubility of histones in sulfuric acid. The fixation reaction seems to occur much more slowly than the reaction of hydroxymethylation or crosslinks of histones themselves. Brutlag et al. [ l l ] first reported that formaldehyde-treated chromatin contained covalently linked histone-DNA complexes, which could not be dissociated by treatment with acid or a high concentration of cesium chloride. They represented protein-DNA crosslinks by formaldehyde which remains attached to the complex after pronase treatment. Pronase treatment of the fixed chromatin removes 96 of the protein. The formatin of crosslinks between histones and DNA results in a conformational change of DNA, which is detectable as a change of positive ellipticity in the region of 250-300 nm. Decrease of positive ellipticity of nucleohistone on reaction with formaldehyde has been reported by Senior and Olins [16], but their results do not agree with our results. They reported that the positive ellipticity above 240 nm progressively decreased with increase in the formaldehyde concentration, finally attaining a negative value with 3 % formaldehyde. They did not observe
Reaction of Formaldehyde with Nucleohistone
a minimum point on increasing the formaldehyde concentration. In an attempt to find out why they obtained different results, we exanlined calf thymus nucleohistone under their reaction conditions, but we found that in either water of 5 mM NaC1,0.035 mM phosphate buffer (pH 6.8) it gave the same pattern as that in Fig. 7. Thus this discrepancy may be attributed to the different chromatins used: that is, a difference between calf thymus and chicken erythrocyte chromatin. In fact, they reported that the [0]z75 value of chicken erythrocyte chromatin is more than 5000 deg . cm2 . dmol-' in water, while that of our calf thymus chromatin was 3500 deg . cm2 . dmol-', as indicated in this paper. The reason for the increase of ellipticity at 275 nm with concentrations of over 0.4 7; formaldehyde is unkown. Two possible explanations for this change may be considered. One is the direct reaction of formaldehyde with DNA bases in this concentration range, causing a conformational change in the secondary structure of DNA. The results in Fig.7 support this consideration. The other possibility is that the fraction of formaldehyde diadduct in histones may increase with over 0.4 % formaldehyde, and this increase in the amount of diadduct may reduce the interaction of histones with DNA. This work was supported in part by Scientific Research Grants from the Ministry of Education, Science and Culture of Japan.
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Y. Ohba, Y. Morimitsu, and A. Watarai, Department of Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, Takara-machi, Kanazawa-shi. Ishikawa-ken, Japan 920
