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The effect of heparin on the porcine lymphocyte chromatin—I. The comparative study of DNA in different chromatin fractions
THE EFFECT OF HEPARIN ON THE PORCINE LYMPHOCYTE CHROMATIN-I. THE COMPARATIVE STUDY OF DNA IN DIFFERENT CHROMATIN FRACTIONS E. STRZ~LECKA.
D. SPI-IKOVSKY
and V. PAP~VOV
Department of Biophysics. Institute of Biochemistry and Biophysics. University of tcidi. Banacha 1?;16. t6di 9C-237. Poland and Laboratory of Molecular Biology. Institute of Medical Genetics. Academy of Medical Sciences of the U.S.S.R.. Kashirskoye shosse 6a. Mosco\l I Ii 478, U.S.S.R.
I. Porcine lymphocyte chromatin in the solution of 0.15 M NaCl + 0.01 M Tris. pH 7 treated Abstract with heparin liberated X”,, protein and 7.5”,, DNA to the supernatant. 2. DNA from the hupernatant and the pellet fractions as well as from control chromatin were isolated an identical conditions. 3. No slgnilicant changes were observed m spectral properties and melting points in SSC of compar.tble DNA specimens. 4. It was noted. howckcr. that DNA of the supernatant is subject to denaturatton in the process 01 Isolation. which. apart from the difference in protein composition of the supernatant and the pellet fractions. suggests dlflcrrnt chromatin structure of these fractions.
blood by the gelatin sedimentation method (Malec 1971). Lymphocyte pellets were frozen LO - I5 C. Polyanions such as heparin have been shown to produce certain structural and functional alterations in the eukaryotic chromatin. They cause loosening of the chromatin structure (decondensation) (Arnold et LI/.. 1971; Paponov ct (I/.. 1980) and produce chromatin destabilization to thermal denaturation (Ansevin er ul.. 1975). they also increase the DNA and RNA synthesis rates (Kracmer & Con‘ey, 1970; De Pomerai t’l al., 1974; Warnick & Lazarus, 1975; von der Decken. 19X1) and make possible the degradation of DNA of chromatin by DNases (CejkovB t,f crl.. 1978). The effect of heparin on chromatin consist mainly in the interaction of negatively charged polyanion molecules with positively charged histones. The specificity of this action was reported from several laboratories (cejkovi rt ctl.. 1971 ; Kitzis c’t trl., 1976: Hildebrand c’r al.. 1977; Paponov cf n/.. 1980). With the removal of hlstones from chromatin. consequent appearance of DNA was noted. It was referred to as slowly sedimenting DNA (Hildebrand (‘r trl.. 1977). solubilized DUA (Kraemer & Coffey. 1970). dispersed DNA or dccondensed DNA (Hildebrand & Tobey, 1975). In our previous work (Paponov t’~ (11.. 1980) we examined protein dissociation of calf thymus chromatin and its decompactization under the influence of heparin. This study was aimed at a comparison of certain physicochemical properties of DNA isolated from chromatin fraction under the influ&ce of heparin.
MATERIAI. .A%D \IETHODS L~wplwc rtl’s Lymphocytes
w’ere prepared
from
porcine
peripheral 793
(21 trl..
Chromatin was prepared according to the method of Zubay & Doty (1959). Frozen lymphocytes were homogcnIced in 0.075 M NaCl + 0.024 M EDTA. pH X In a Potter
Elvehjem homogenizer and washed 5 Iimcs in the same solution. The pellets were suspended in 0.1.5 M NaCl
+ 0.01 M Tris. pH 7.
Chromatin suspension was mcubated for 12 hr at 0 4 C with “Polfa” heparm in 0.15 M NaCl + 0.01 M Trls. pH 7 to obtain the foollowing (chromatin DNA:heparin) conccntration ratios: 1:0.5, l:l. 1:2, 1:5, 1:lO. I:50 and I:lOO. The final concentration of DNA was 200 /lg,,rnl and that of heparin 0.1-20 mg/ml. Corresponding volume of 0.15 M NaCl + 0.01 M Tris was added to the control chromatin. The samples were centrifuged at 6O.OOOy for 1.5 hr In a VAC-601 centrifuge. After centrifugation. supernatants and pellets were removed for further analyui\.
DNA was isolated from the control chromatln and from the supernatant and pellet of the chromatin under the influence of heparin (chromatin DNA: heparin ratlo wah l:5). The chromatin was added with a solution of NaCl and SDS up to final concentrations of 2 M and I”,,. resnectively. heated at 60 C for 30mm. cooled to 0 C’ and siorcd overnight. The aliquots were centrifuged at 40.000 9 for I hr in a Beckman ultracentrifuge (a SW 27 rotor). Supernatants were shaken with equal amounts of chloroform: isoamyl alcohol (1:24) for 30 min at 0 C and fhen centrifuged at 5000 9 for 30 min (Janetzki K-23 centrifuge). DNA was precipitated from the top layer with cooled ethanol. The pellets were dissolved m small volumes of water with the addition of equal volumes of phosphate buffer
(2.5 M K2HP04 + 3i”,, H,PO,. 20: I I and equal volumes of 2.tncthou~ethanol. The extraction was carried out for I5 min at 0 C and ccntrifugcd at 3000 y for I5 men. DNA was prcclpltatcd from supernatants with cooled 2-etoxyethanal. The dried DNA pellets wcrc soluhili/ed ,n SSC (0.15 M NaCI + 0.015 M sodium citrate) and dialyxd against this solvent. Before T, readings the preparations wcrc diluted to 3 concentration of 70 beg ml.
(Al Protein 30
20 s
IO
Measurement:, were made m a self-recording Unicam SP 1700 spcctrophotometer with thermostated chamber hold~ng four cubcttcs. The average rate of temp. increase was 0.5 C. min. The rate of registering of melting curves was I cm’5 min. The ahsorhance was read at X0 nm. The initial absorbance of the sample ranged from 0.35 to 0.5. The hqperchromic ctl’cct. whxh is percentage of increase of ahsorhance 1n relation to initial temperature after the thermal denaturation of DNA was determined a< AT The temperaturc of meltmg uf DNA 7; value and the helix -coil transitlon range ~A’r”, value. were determined grnphicnlly from the melting cur\c. The 7;,, point for which AT= 50”,, of map. value and the point for which A7’ is c-fold smaller (1X”,,) as well a\ the agmmctrlcal point to the latter in relation to 7;,, (AT = X2” (,I were chtabllshcd. When dctcrmining 76,. correction fbr solution thermal expansion aas takw 1nt0 account
The protcln ua determined according to the method of Lowry vr u/ (1951). DNA was dctermincd according to the method of Spirin (195X). RNA by the orcinol method (C‘olowlck & Kaplan. 1959) and heparln xxordmg to the method of Hlttcr cY: Muir (1962).
The effect of polyanions on the chromatin structure is beyond any doubt today. but some molecular mechanisms of this activity still remain unexplained. Most of the previous experiments have dealt with the effect of polyanions on chromatin in some solutions which differ in their ionic strength from the intracellular environment. In our previous work we presented the possibility of dissociation of calf thymus chromatin histones in 0.15 M NaCl under the influence of heparin (Paponov (‘I trl.. 19X0). Our present investigations confirm these early suggestions. In our recent work chromatin was isolated from the preparation of porcine lymphocytes in which lymphocytes constituted about 75”,, of all the cells. The remainder con&cd of neutrophil leukocytes (ZS’,,) and monocytes (3.5”,,). The resulting chromatin had protein/DNA ratio of I .3 I .4 and RNA/DNA ratio of 0.03 0.08. After the addition of hcparin to chromatin. some protein and DNA were noted in supernatants at physiological ionic strength employed. The quantities of protein and DNA increased with the increase of polqanion concentration (Fig. I). The protein,DNA ratio in the supernatants increased and was, e.g. 4.6 at DNA:heparin cont. ratio of I :0.5. or 6.7 at the ratio of I: IO. which indicates increasing protein dissociation from chromatin DNA. The most effective heparin concentrations seem to be upto 200 gg;ml (at the DNA: heparin ratio of I : I ). Such concentrations of polyanion cause the release of 10.6 & 3.1”,, protein and 2.0 f O.Y,, DNA (for loo”,, we assumed protein and DNA concentrations in chromatin suspension of
Heparln
,
mg /ml
Fi_e. 1. Removal of protein and DNA from porclnc lymphocyte chromatin after heparin treatment. Abscissa is heparln concentration in mg/ml. Ordinate is percentage of protein (* l ) and DNA (0 01 in wpernatanta. IOO”,, final concentration of DNA ( :0.2 mgml) and protcln ( :0.26 mg/rnl) in chromatm juspcnsion (see Materials and Method\).
260 and 200/lg;ml. respectively). The next IO-fold increase of heparin concentration up to 2 mg;ml DNA: heparin ratio is then I: 10 caused the release of IX.1 I 2.1”,, protein and 3.2 k, 0.4”,, DNA) and l000-fold increase of concentration upto 30 mg:ml (DNA:hcparin ratio of I: 100) caused the release of 30.8 k 5.X”,, protein and 7.5 + l.O”,, DNA. At high heparin concentrations (above DNA: heparin ratio of 1:50) the release of protein and DNA reached the saturation point. For such high heparin concentrations (IO-20 mg/ml). the determination of protein quantity in supernatants exhibited additional error reaching S”,,. which was a result of heparin inlluence on protein determination according to the method of Lowry c’f trl. (1951). This error does not influence, however. the character of the curve A (Fig. I). The release of protein and DNA from porcine lymphocyte chromatin under the influence of heparin was lower than in the case of calf thymus chromatin studied in our previous work (Paponov or ctl.. 1980). In supernatants there wcrc 2-fold smaller quantities of proteins and 4-fold smaller quantities of DNA. Most likely it IS connected with higher lymphocyte chromatin and heparin concentrations in suspension, which causes a certain increase in sedimentation of dificrent types of complexes containing protein and DNA. On the basis of the above-mentioned results. it can be deduced that at a given physiological ionic strength. certain heterogenous. protein-enriched chromatin fraction remains in supcrnatants after high-speed centrlfugation. It is supposed that chromatin structure of this fraction is loosened. The diffcrences in chromatin fibre condensation of supernatant and pellet fractions are likely to be related to differences in quantitative and qualitative protein compositions of these fractions. In accordance with that. it seemed interesting to ask if the differences in chromatin fibre condensation and in the protein composition of supernatant and pellet fractions are related to different DNA structure and DNA nucleotidc composition of these fractions. In our recent work we compare spectra and melting curves of DNA of lymphocyte chromatin separated after heparin treatment with the supcrnatant and the
The effect of heparin
on the porcme
r\
I i
06
t
i
I
\
i
\ \ :, ::““*-...\\ *.:.\ ‘.*.. \
;/ /
;.
‘:.\ \ \
...*
A f
, 220
,
,
,
240
260
280
Wave
length,
k ...
L_
300
320
nm
FI:. 2. Lltra\lolet ahsorption spectra of supernatant DNA -----. pellet DNA and control chromatin DNA .,,,_ DNA was isolated from porcine lymphocyte chromatin trc,lted and,‘or nontreated with heparm as describd in Materials and Methods. Solvent was SSC. with DNA of chromatin preparation not subjected to this polyanion activity. While isolating DNA it was difficult to separate DNA from heparin, especially in the case of supernatants. Separating this polysaccharide from DNA with 2-metoxyethanol we obtained some supernatant DNA preparations in pellet
Table
I. Comparative
thermal
3
4
I
295
which the DNA: heparin ratio ranged from 5: 1 to 6: I while in DNA preparations from pellet it ranged from 50: 1 to 60: 1. In our quadruplicate experiments we isolated DNA from control porcine lymphocyte chromatin and heparin-treated chromatin divided into pellet and supernatant at a chromatin DNA: heparin ratio of 1:5. In the case of control chromatm. a treatment with 2-metoxyethanol was also employed. The efficiency of particular preparations ranged from 30 to 507/,,. Figure 2 shows spectra of some individual preparations in SSC (0.15 M NaCl + 0.015 M sodium citrate). A280/A260 ratio of given preparations of control chromatin DNA was 0.54-0.64 and 0.55$0.5X for the pellet and supernatant DNA. AZjO:A2h0 ratio for all the three preparations of DNA ranged from 0.85 to 0.88. DNA preparations isolated from supernatant and from pellet exhibited less protein impurities than in the case with control chromatin DNA preparations. Maximum of absorbance for all the preparations obtained ranged from 259 to 260 nm (Fig. 2). Comparing DNA solution concentrations determined by the absorption coefficient-~ C, (it was assumed that 1 mg DNA/ml in SSC corresponds to 20 optlcal units) and according to the method of Spirin we observed that for the supernatant (1958&C, DNA C, > C, approx. 4.5 /cg/ml. while for the pellet DNA and control chromatin DNA--results of determinations of DNA concentrations by the two above methods are consistent. After thermal denaturation of the supernatant and pellet DNA and the control chromatin DNA in standard conditions. no significant changes in melting temperature of the preparations were found in SSC (Table I). For the control chromatin DNA the average melting temperature was 85.7 C (X5~~ X6 C). for the from different
Pellet DNA
chroma-
Supernatant DNA
AT
T,
AT,
(“,,I
( C)
( Cl
40.0
86.5
6.0
40.5 4 1.o 41.2 3x.7
86.0 86.0 85.0 85.0
7.0 7.0 8.0 7.0
42.3 43.7 39.9 39.2
86.0 85.0 85.5 85.5
37.0 39.0 41.2
85.5 86.0 86.0
38.1 42.1 43.8 37.3 37.1 40.0 38.9
85.0 87.5 89.0 88.0 85.0 85.0 87.0
7.0 7.0 6.5 5.0 8.0 6.5 8.0
26.3 32.7 30.3
X6.0 89.0 88.5
7.5 6.0 7.5
18.9 18.6 15.1
x7.0 89.0 86.0
6.5 7.0 6.0
7.0 6.0 7.0 6.0
40.0 37.7 41.9 43.4
85.0 84.5 86.0 85.0
7.0 6.5 8.5 9.0
12.1 14.9 16.2 13.6 14.7 14.5
87.0 85.0 84.0 84.5 86.0 X6.0
6.5 7.0 7.5 7.5 9.0 9.0
5.5 6.5 7.5
37.9 42.2 40.2
85.0 84.5 86.5
5.5 8.5 7.5
22.9 20.3 21.6 30.0
86.5 85.7 X6.5 86.5
7.5 8.5 7.0
I
2
chromatin-
denaturation of DNA isolation tin fraction
Control DNA
Nr
lymphocyte
Nr -preparation
IO.0
number. Porcine lymphocyte chromatin and DNA were isolated
four times in identical conditions (see Materials and Methods). AT-- hyperchromic effect. T,, melting temperature for which AT = 50% of max. value. AT, helix + coil transitlon range for which AT = 18.~82”,,.
:* 1 .,..........+a..............."' 0.4
t
I25
30
70
75 T,
80
85
90
95
100
“C
Fig. 3. ,Absorhance temperature profiles fat- aupernatant and control chromatin DNA DNA ----I. pellet DNA DNA was lsolatcd from porcine lymphocqtc chromatln treated a&or nontreatcd with hcpar~n i\\ described in Materials and Method,. Denaturation solvent
DNA it was X5.9 C (85 XY C) and for the superDNA it was Xh.5’C (X4- 89 C). It was also found that the helix + random cniii transition range (AT,)was very similar in all the three types of prrparations. However, the supernatant and pellet DNA preparations are characterized by low reproducibility of results. The supernatant DNA is also charactertzed by a decreased hyperchromic effect (upprox. W,,) and decreased slope of the melting curve in the helix+ coil transition area (Table 1. Fig. 3). &hi& evidences den~~~L~rat~onof the DNA ~rep~~riiti~~t~isolated from supernatant. Also the difference in DNA concentrations determined according to absorption cocfficient and according to the method of Spirin (1958) demonstrates denaturation of the supcrnatant DNA preparation. Higher absorbance of the supernatant DNA than the pellet and control chromatin DNA may be due to nitrogen bases uncovcteci in DNA after denat~iration. The fact that uphen isolating DNA m an identical way from diKerent chromatin preparations only the supernatant DNA is subject to denaturntion suggests different structure of the fractions compared. Most likely. the supernatant DNA partially devoid of proteins. of loosened and more cxtendrd structure than the pellet fraction DNA is prcatly susceptible to denaturation under the influence of the operations applied. pellet
natant
The pot&is
results that
of
our
polyanions
investi~~~t~ol~s such
contirm
as heparin
con
the
hy-
be used
for detection of structural changes in cucaryotic chromatin and can also be useful for chromatin fractionation. Further experiments are planned to analyse the structure of different chromatin fractions and the mechanism of chromatin fractionation with heparin. The elucidation of these questions could contribute to a better ilnderstaildin~ of the role of po~yanions in cellular regulatory processes.
D. SPI-IKOVSKY
and V. PAP~VOV
Department of Biophysics. Institute of Biochemistry and Biophysics. University of tcidi. Banacha 1?;16. t6di 9C-237. Poland and Laboratory of Molecular Biology. Institute of Medical Genetics. Academy of Medical Sciences of the U.S.S.R.. Kashirskoye shosse 6a. Mosco\l I Ii 478, U.S.S.R.
I. Porcine lymphocyte chromatin in the solution of 0.15 M NaCl + 0.01 M Tris. pH 7 treated Abstract with heparin liberated X”,, protein and 7.5”,, DNA to the supernatant. 2. DNA from the hupernatant and the pellet fractions as well as from control chromatin were isolated an identical conditions. 3. No slgnilicant changes were observed m spectral properties and melting points in SSC of compar.tble DNA specimens. 4. It was noted. howckcr. that DNA of the supernatant is subject to denaturatton in the process 01 Isolation. which. apart from the difference in protein composition of the supernatant and the pellet fractions. suggests dlflcrrnt chromatin structure of these fractions.
blood by the gelatin sedimentation method (Malec 1971). Lymphocyte pellets were frozen LO - I5 C. Polyanions such as heparin have been shown to produce certain structural and functional alterations in the eukaryotic chromatin. They cause loosening of the chromatin structure (decondensation) (Arnold et LI/.. 1971; Paponov ct (I/.. 1980) and produce chromatin destabilization to thermal denaturation (Ansevin er ul.. 1975). they also increase the DNA and RNA synthesis rates (Kracmer & Con‘ey, 1970; De Pomerai t’l al., 1974; Warnick & Lazarus, 1975; von der Decken. 19X1) and make possible the degradation of DNA of chromatin by DNases (CejkovB t,f crl.. 1978). The effect of heparin on chromatin consist mainly in the interaction of negatively charged polyanion molecules with positively charged histones. The specificity of this action was reported from several laboratories (cejkovi rt ctl.. 1971 ; Kitzis c’t trl., 1976: Hildebrand c’r al.. 1977; Paponov cf n/.. 1980). With the removal of hlstones from chromatin. consequent appearance of DNA was noted. It was referred to as slowly sedimenting DNA (Hildebrand (‘r trl.. 1977). solubilized DUA (Kraemer & Coffey. 1970). dispersed DNA or dccondensed DNA (Hildebrand & Tobey, 1975). In our previous work (Paponov t’~ (11.. 1980) we examined protein dissociation of calf thymus chromatin and its decompactization under the influence of heparin. This study was aimed at a comparison of certain physicochemical properties of DNA isolated from chromatin fraction under the influ&ce of heparin.
MATERIAI. .A%D \IETHODS L~wplwc rtl’s Lymphocytes
w’ere prepared
from
porcine
peripheral 793
(21 trl..
Chromatin was prepared according to the method of Zubay & Doty (1959). Frozen lymphocytes were homogcnIced in 0.075 M NaCl + 0.024 M EDTA. pH X In a Potter
Elvehjem homogenizer and washed 5 Iimcs in the same solution. The pellets were suspended in 0.1.5 M NaCl
+ 0.01 M Tris. pH 7.
Chromatin suspension was mcubated for 12 hr at 0 4 C with “Polfa” heparm in 0.15 M NaCl + 0.01 M Trls. pH 7 to obtain the foollowing (chromatin DNA:heparin) conccntration ratios: 1:0.5, l:l. 1:2, 1:5, 1:lO. I:50 and I:lOO. The final concentration of DNA was 200 /lg,,rnl and that of heparin 0.1-20 mg/ml. Corresponding volume of 0.15 M NaCl + 0.01 M Tris was added to the control chromatin. The samples were centrifuged at 6O.OOOy for 1.5 hr In a VAC-601 centrifuge. After centrifugation. supernatants and pellets were removed for further analyui\.
DNA was isolated from the control chromatln and from the supernatant and pellet of the chromatin under the influence of heparin (chromatin DNA: heparin ratlo wah l:5). The chromatin was added with a solution of NaCl and SDS up to final concentrations of 2 M and I”,,. resnectively. heated at 60 C for 30mm. cooled to 0 C’ and siorcd overnight. The aliquots were centrifuged at 40.000 9 for I hr in a Beckman ultracentrifuge (a SW 27 rotor). Supernatants were shaken with equal amounts of chloroform: isoamyl alcohol (1:24) for 30 min at 0 C and fhen centrifuged at 5000 9 for 30 min (Janetzki K-23 centrifuge). DNA was precipitated from the top layer with cooled ethanol. The pellets were dissolved m small volumes of water with the addition of equal volumes of phosphate buffer
(2.5 M K2HP04 + 3i”,, H,PO,. 20: I I and equal volumes of 2.tncthou~ethanol. The extraction was carried out for I5 min at 0 C and ccntrifugcd at 3000 y for I5 men. DNA was prcclpltatcd from supernatants with cooled 2-etoxyethanal. The dried DNA pellets wcrc soluhili/ed ,n SSC (0.15 M NaCI + 0.015 M sodium citrate) and dialyxd against this solvent. Before T, readings the preparations wcrc diluted to 3 concentration of 70 beg ml.
(Al Protein 30
20 s
IO
Measurement:, were made m a self-recording Unicam SP 1700 spcctrophotometer with thermostated chamber hold~ng four cubcttcs. The average rate of temp. increase was 0.5 C. min. The rate of registering of melting curves was I cm’5 min. The ahsorhance was read at X0 nm. The initial absorbance of the sample ranged from 0.35 to 0.5. The hqperchromic ctl’cct. whxh is percentage of increase of ahsorhance 1n relation to initial temperature after the thermal denaturation of DNA was determined a< AT The temperaturc of meltmg uf DNA 7; value and the helix -coil transitlon range ~A’r”, value. were determined grnphicnlly from the melting cur\c. The 7;,, point for which AT= 50”,, of map. value and the point for which A7’ is c-fold smaller (1X”,,) as well a\ the agmmctrlcal point to the latter in relation to 7;,, (AT = X2” (,I were chtabllshcd. When dctcrmining 76,. correction fbr solution thermal expansion aas takw 1nt0 account
The protcln ua determined according to the method of Lowry vr u/ (1951). DNA was dctermincd according to the method of Spirin (195X). RNA by the orcinol method (C‘olowlck & Kaplan. 1959) and heparln xxordmg to the method of Hlttcr cY: Muir (1962).
The effect of polyanions on the chromatin structure is beyond any doubt today. but some molecular mechanisms of this activity still remain unexplained. Most of the previous experiments have dealt with the effect of polyanions on chromatin in some solutions which differ in their ionic strength from the intracellular environment. In our previous work we presented the possibility of dissociation of calf thymus chromatin histones in 0.15 M NaCl under the influence of heparin (Paponov (‘I trl.. 19X0). Our present investigations confirm these early suggestions. In our recent work chromatin was isolated from the preparation of porcine lymphocytes in which lymphocytes constituted about 75”,, of all the cells. The remainder con&cd of neutrophil leukocytes (ZS’,,) and monocytes (3.5”,,). The resulting chromatin had protein/DNA ratio of I .3 I .4 and RNA/DNA ratio of 0.03 0.08. After the addition of hcparin to chromatin. some protein and DNA were noted in supernatants at physiological ionic strength employed. The quantities of protein and DNA increased with the increase of polqanion concentration (Fig. I). The protein,DNA ratio in the supernatants increased and was, e.g. 4.6 at DNA:heparin cont. ratio of I :0.5. or 6.7 at the ratio of I: IO. which indicates increasing protein dissociation from chromatin DNA. The most effective heparin concentrations seem to be upto 200 gg;ml (at the DNA: heparin ratio of I : I ). Such concentrations of polyanion cause the release of 10.6 & 3.1”,, protein and 2.0 f O.Y,, DNA (for loo”,, we assumed protein and DNA concentrations in chromatin suspension of
Heparln
,
mg /ml
Fi_e. 1. Removal of protein and DNA from porclnc lymphocyte chromatin after heparin treatment. Abscissa is heparln concentration in mg/ml. Ordinate is percentage of protein (* l ) and DNA (0 01 in wpernatanta. IOO”,, final concentration of DNA ( :0.2 mgml) and protcln ( :0.26 mg/rnl) in chromatm juspcnsion (see Materials and Method\).
260 and 200/lg;ml. respectively). The next IO-fold increase of heparin concentration up to 2 mg;ml DNA: heparin ratio is then I: 10 caused the release of IX.1 I 2.1”,, protein and 3.2 k, 0.4”,, DNA) and l000-fold increase of concentration upto 30 mg:ml (DNA:hcparin ratio of I: 100) caused the release of 30.8 k 5.X”,, protein and 7.5 + l.O”,, DNA. At high heparin concentrations (above DNA: heparin ratio of 1:50) the release of protein and DNA reached the saturation point. For such high heparin concentrations (IO-20 mg/ml). the determination of protein quantity in supernatants exhibited additional error reaching S”,,. which was a result of heparin inlluence on protein determination according to the method of Lowry c’f trl. (1951). This error does not influence, however. the character of the curve A (Fig. I). The release of protein and DNA from porcine lymphocyte chromatin under the influence of heparin was lower than in the case of calf thymus chromatin studied in our previous work (Paponov or ctl.. 1980). In supernatants there wcrc 2-fold smaller quantities of proteins and 4-fold smaller quantities of DNA. Most likely it IS connected with higher lymphocyte chromatin and heparin concentrations in suspension, which causes a certain increase in sedimentation of dificrent types of complexes containing protein and DNA. On the basis of the above-mentioned results. it can be deduced that at a given physiological ionic strength. certain heterogenous. protein-enriched chromatin fraction remains in supcrnatants after high-speed centrlfugation. It is supposed that chromatin structure of this fraction is loosened. The diffcrences in chromatin fibre condensation of supernatant and pellet fractions are likely to be related to differences in quantitative and qualitative protein compositions of these fractions. In accordance with that. it seemed interesting to ask if the differences in chromatin fibre condensation and in the protein composition of supernatant and pellet fractions are related to different DNA structure and DNA nucleotidc composition of these fractions. In our recent work we compare spectra and melting curves of DNA of lymphocyte chromatin separated after heparin treatment with the supcrnatant and the
The effect of heparin
on the porcme
r\
I i
06
t
i
I
\
i
\ \ :, ::““*-...\\ *.:.\ ‘.*.. \
;/ /
;.
‘:.\ \ \
...*
A f
, 220
,
,
,
240
260
280
Wave
length,
k ...
L_
300
320
nm
FI:. 2. Lltra\lolet ahsorption spectra of supernatant DNA -----. pellet DNA and control chromatin DNA .,,,_ DNA was isolated from porcine lymphocyte chromatin trc,lted and,‘or nontreated with heparm as describd in Materials and Methods. Solvent was SSC. with DNA of chromatin preparation not subjected to this polyanion activity. While isolating DNA it was difficult to separate DNA from heparin, especially in the case of supernatants. Separating this polysaccharide from DNA with 2-metoxyethanol we obtained some supernatant DNA preparations in pellet
Table
I. Comparative
thermal
3
4
I
295
which the DNA: heparin ratio ranged from 5: 1 to 6: I while in DNA preparations from pellet it ranged from 50: 1 to 60: 1. In our quadruplicate experiments we isolated DNA from control porcine lymphocyte chromatin and heparin-treated chromatin divided into pellet and supernatant at a chromatin DNA: heparin ratio of 1:5. In the case of control chromatm. a treatment with 2-metoxyethanol was also employed. The efficiency of particular preparations ranged from 30 to 507/,,. Figure 2 shows spectra of some individual preparations in SSC (0.15 M NaCl + 0.015 M sodium citrate). A280/A260 ratio of given preparations of control chromatin DNA was 0.54-0.64 and 0.55$0.5X for the pellet and supernatant DNA. AZjO:A2h0 ratio for all the three preparations of DNA ranged from 0.85 to 0.88. DNA preparations isolated from supernatant and from pellet exhibited less protein impurities than in the case with control chromatin DNA preparations. Maximum of absorbance for all the preparations obtained ranged from 259 to 260 nm (Fig. 2). Comparing DNA solution concentrations determined by the absorption coefficient-~ C, (it was assumed that 1 mg DNA/ml in SSC corresponds to 20 optlcal units) and according to the method of Spirin we observed that for the supernatant (1958&C, DNA C, > C, approx. 4.5 /cg/ml. while for the pellet DNA and control chromatin DNA--results of determinations of DNA concentrations by the two above methods are consistent. After thermal denaturation of the supernatant and pellet DNA and the control chromatin DNA in standard conditions. no significant changes in melting temperature of the preparations were found in SSC (Table I). For the control chromatin DNA the average melting temperature was 85.7 C (X5~~ X6 C). for the from different
Pellet DNA
chroma-
Supernatant DNA
AT
T,
AT,
(“,,I
( C)
( Cl
40.0
86.5
6.0
40.5 4 1.o 41.2 3x.7
86.0 86.0 85.0 85.0
7.0 7.0 8.0 7.0
42.3 43.7 39.9 39.2
86.0 85.0 85.5 85.5
37.0 39.0 41.2
85.5 86.0 86.0
38.1 42.1 43.8 37.3 37.1 40.0 38.9
85.0 87.5 89.0 88.0 85.0 85.0 87.0
7.0 7.0 6.5 5.0 8.0 6.5 8.0
26.3 32.7 30.3
X6.0 89.0 88.5
7.5 6.0 7.5
18.9 18.6 15.1
x7.0 89.0 86.0
6.5 7.0 6.0
7.0 6.0 7.0 6.0
40.0 37.7 41.9 43.4
85.0 84.5 86.0 85.0
7.0 6.5 8.5 9.0
12.1 14.9 16.2 13.6 14.7 14.5
87.0 85.0 84.0 84.5 86.0 X6.0
6.5 7.0 7.5 7.5 9.0 9.0
5.5 6.5 7.5
37.9 42.2 40.2
85.0 84.5 86.5
5.5 8.5 7.5
22.9 20.3 21.6 30.0
86.5 85.7 X6.5 86.5
7.5 8.5 7.0
I
2
chromatin-
denaturation of DNA isolation tin fraction
Control DNA
Nr
lymphocyte
Nr -preparation
IO.0
number. Porcine lymphocyte chromatin and DNA were isolated
four times in identical conditions (see Materials and Methods). AT-- hyperchromic effect. T,, melting temperature for which AT = 50% of max. value. AT, helix + coil transitlon range for which AT = 18.~82”,,.
:* 1 .,..........+a..............."' 0.4
t
I25
30
70
75 T,
80
85
90
95
100
“C
Fig. 3. ,Absorhance temperature profiles fat- aupernatant and control chromatin DNA DNA ----I. pellet DNA DNA was lsolatcd from porcine lymphocqtc chromatln treated a&or nontreatcd with hcpar~n i\\ described in Materials and Method,. Denaturation solvent
DNA it was X5.9 C (85 XY C) and for the superDNA it was Xh.5’C (X4- 89 C). It was also found that the helix + random cniii transition range (AT,)was very similar in all the three types of prrparations. However, the supernatant and pellet DNA preparations are characterized by low reproducibility of results. The supernatant DNA is also charactertzed by a decreased hyperchromic effect (upprox. W,,) and decreased slope of the melting curve in the helix+ coil transition area (Table 1. Fig. 3). &hi& evidences den~~~L~rat~onof the DNA ~rep~~riiti~~t~isolated from supernatant. Also the difference in DNA concentrations determined according to absorption cocfficient and according to the method of Spirin (1958) demonstrates denaturation of the supcrnatant DNA preparation. Higher absorbance of the supernatant DNA than the pellet and control chromatin DNA may be due to nitrogen bases uncovcteci in DNA after denat~iration. The fact that uphen isolating DNA m an identical way from diKerent chromatin preparations only the supernatant DNA is subject to denaturntion suggests different structure of the fractions compared. Most likely. the supernatant DNA partially devoid of proteins. of loosened and more cxtendrd structure than the pellet fraction DNA is prcatly susceptible to denaturation under the influence of the operations applied. pellet
natant
The pot&is
results that
of
our
polyanions
investi~~~t~ol~s such
contirm
as heparin
con
the
hy-
be used
for detection of structural changes in cucaryotic chromatin and can also be useful for chromatin fractionation. Further experiments are planned to analyse the structure of different chromatin fractions and the mechanism of chromatin fractionation with heparin. The elucidation of these questions could contribute to a better ilnderstaildin~ of the role of po~yanions in cellular regulatory processes.