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Differences in the amino acid composition of deoxyribonucleoproteins from white blood cells of normal and leukemic human beings
pp. 541-548, 1970.
Life Sciences Vol. 9, Part II, Printed in Great Britain
Pergamon Press
DIFFERENCES IN THE AMINO ACID COMPOSITION OF OBOXYRIBONUCLEOPROTEINS FROM WHITE BLOOD CELLS OF NORMAL AND LEUKEMIC HUMAN BEINGS L . Messineo and J . R . Kongsvik Biochemistry Research Laboratory, Veterans Administration Center 45428 4100 West Third Street, Dayton, Ohio
(Received 22 January 1970; in final form 20 March 1970)
It has been reported that deoxyribonucleoproteins (DNP) from the white blood cells of normal and leukemic human beings elicit in rabbits an immunological response which is specific to the extent of distinguishing between DNP's from normal and leukemic subjects (1,2) .
Furthermore, antibodies against peripheral
leukocyte DNP from normal subjects cross-react with ONP from bone marrow cells of the same subjects, but not with DNP from marrow cells of leukemic subjects . No cross reaction was found between antisera to DNP from white blood cells and bone marrow cells of leukemic individuals and the DNP of leukocytes and bone marrow cells from normal subjects (2) . On the basis of these immunological distinctions we have compared the amino acid composition of DNP from the peripheral leukocytes of normal subjects with that from leukemic peripheral leukocytes .
We wish to report differences
in the amino acid composition of the respective DNP's and would like to suggest that these differences could explain the immunological differences noted earlier . Materials and Methods In this study human beings are considered normal or leukemic on the basis of clinical diagnostic tests .
In no instance was the white blood cell count of
the chronic lymphocytic leukemic patients less than 80,000 sn 3 . Deoxyribonucleoproteins were extracted from normal and leukemic leukocytes as reported earlier (1,2) .
Previous to amino acid analyses the clarified gly-
cine extracts from both sources were characterised by ultracentrifugal, electro-
541
542
Vol. 9, No . 10
AMINO ACID COMPOSITION
phoretic, chromatographic,, immunologic and chemical procedures (3,4) . were found to be intact, homogeneous DNA-basic protein complexes .
They
Chemical
analyses of the DNP's by the modified biuret test (5) and spectrophotometric analyses at 260 mr indicated a protein and DNA content of 71 and 29% respectively .
The DNP preparations were highly polymerized as evidenced by a sedi-
mentation coefficient (S20 ,w ) of 60 .
The DNA moiety of the respective leuko-
cyte DNP's was undegraded as evidenced by an alkaline induced hyperchromicity at 260 mp of 33% (6) and highly fibrous DNA is recovered upon deproteinization and subsequent ethanol precipitation (7) .
The molar base composition of both
normal and leukemic DNA were similar (A = 27 .4, T = 27 .8, G = 22 .2, C = 22 .5), thereby corresponding to previous reports (8) .
Elution of the DNP's from
Sephadex G-200 columns results in the recovery of both protein and DNA under a single peak . Immunological assays in test tubes with rabbit antibodies, obtained as previously described (2), result in the complete precipitation of the DNP's, while in agar gel plates one single band is formed when the diffusing DNP antigen and antibodies meet (1,2) . Cellulose acetate electrophoresis and column chromatography on Sephadex were performed as reported elsewhere (3) . Solubility studies, which have been reported elsewhere (9) show that glycine extraction recovers more intact DNP particles without the loss of fractions which accompanies other methods of extraction (10) . The amino acid analyses were done on a model 034-0030 Hitachi (PerkinElmer) amino acid analyzer by the ligand exchange method .
DNP preparations
were separated from the glycine of the medium by filtration through Sephadex G-25, eluted with double distilled water at 50 C . and recovered by lyophilization .
The hydrolysis was carribd out on the whole DNP particles in a sealed,
evacuated tube with 6N HCl under reflux for 20 hours at 101 0 C . eight milligrams of whole DNP were used in each analysis .
From five to
Whole DNP was
analyzed instead of the extracted protein moieties to avoid the loss of
Vol. 9, No. 10
AMINO ACID COMPOSITION
543
fractions and to maintain the quantitative relationship of the protein to DNA . Results and Discussion Aliquots of purified normal and leukemic DNP preparations were routinely tested by cellulose acetate electrophoresis and column chromatography before acid hydrolysis .
Figure 1 shows the electrophoretic migration of normal (A)
FIG . lA Blectrophoretic migration of leukemic leukocyte DNP at 150 volts for 35 minutes .
The CLL-DNP was applied at
2 .6 (A) and at 1 .3 (B) mg/ml . point of application .
The arrows indicate the
The anode is on the right .
544
AMINO ACID COMPOSITION
Vol. 9, No . 10
FIG . 1B Electrophoretic migration of normal leukocyte DNP at 210 volts for 25 minutes .
The DNP applied was 971 pg/ml .
The arrow indicates the point of application .
The anode
is on the right . and leukemic DNP's (B) . as a single band . on Sephadex G-200 .
In either case, the DNP's migrated towards the anode
Figure 2 shows the elution profile of leukemic leukocyte DNP Filtration of the DNP through G-200 consistently resulted
in the recovery of one peak containing both DNA and protein .
(See Fig . 2)
Superimposable profiles were obtained when normal leukocyte DNP's were chromatographed .
AMINO ACID COMPOSITION
Vol. 9, No. 10
545
2.5-
2 .0-
LO-
0 .5 --~
0.0 1
5
le
i l§ 11 1 1118 1 191 i p mol l i 20 25 30 10 15
FRA C TION NUMBER FIG . 2
Gel exclusion chromatography of leukemic leukocyte DNP on Sephadex G-200 .
The sample (1 ml at 4 .3 mg/ml) was
eluted from the column (90 x 1 .5 cm) with 0 .0007 M KH2 P04' pH 7 .1 .
The fraction size was 7 ml .
35
548
AMINO ACID COMPOSITION
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Vol. 9, No. 10
AMINO ACED COMPOSMON
547
Table I shows the amino acid composition of five leukemic and four normal leukocyte DNP preparations .
The data are expressed as the mole percentage of
total moles of amino acids recovered .
It is clear that differences exist in
the amino acid composition of the respective DNP's .
The sum of basic amino
acids (lysine, arginine, and histidine) in leukemic DKP is 21%, while it is 15% in the DNP from normal leukocytes .
This is an absolute difference of 29% .
Conversely, the sum of aspartic and glutamic acids, proline, serine, threonine and valine in normal DNP is 29% greater than that of leukemic DIO? . Since the extraction and analyses were carried out under the same conditions, it is difficult to envision these differences to be due to loss of fractions, oxidation of basic amino acids or contamination .
The consistency of
the analyses of separate preparations enhances the validity of the data . The presence of unbound contaminants in the DNP preparations are excluded by the results of the physico-chemical characterizations mentioned under methods (above) . On the basis of these physico-chemical characterizations of normal and leukemic white blood cell DNP's, we think that the results of the amino acid analyses is a better reflection of the composition of the protein moiety bound to DNA in vivo .
This concept is enhanced also because the loss of innate
fractions has been avoided by analyzing the whole DNP .
Thus, these results
would suggest that a real difference exists in the protein moieties of normal and leukemic DNP . Since the molar base composition of the DNA from normal and leukemic DIP is similar, the differences in the amino acid composition of the protein moieties could prove to be of significant value with respect to the expression of the genetic code in health and neoplasia .
Furthermore, these results may
imply the existence of structural differences between normal and leukemic DNA .
Sammy The basic amino acid content of the protein moieties of the glycineextracted deoxyribonucleoproteins (DBP) from normal and leukemic peripheral
548
ALTO ACID COMPOSITION
white blood cells was found to be different .
Vol . 9, No. 10
The absolute sum of the basic
amino acids in leukemic DNP was 29% greater than that in the normal DNP, while in the normal DNP an equal magnitude of difference was found in the acidicneutral amino acid content .
These differences are discussed in relation to the
known immunological distinctions between normal and leukemic DNP's and also as a consequence of the etiological action of the leukemic agent(s) . References 1.
L . MESSINEO, Nature 180,
1122 (1961) .
2.
L. MESSINEO, Arch . Biochem . Biophys . 108, 471 (1964) .
3.
L . MESSINEO, J . R . KONGSVIK, Arch Biochem. Biophys . 135,
4.
L . MESSINEO, J. R . KONGSVIK, Intnl . J . Biochem. in press (1970) .
5.
G . L . ELLMAN, Anal . Biochem. 3,
6.
R . D . HOTCHKISS, in S . P . Colowick and N. 0. Kaplan (Editors), Methods in Enzymology , vol . 3, p . 708, Academic Press Inc ., New York (1957) .
7.
E . R . M. KAY, N. W. SIMMONS, and A. L . DODNCE, J . Am . Chem . Soc . 74, 1724 (1952) .
8.
E .E . POLLI, M . ROSOFF, G . Di MAYORCA, and L. F. CAVALIERI, Cancer Research 19, 159 (1959) .
9.
L. MESSINEO, Biophysical Society Abstracts (1970) .
10 .
R . VENDRELY, and C . VENDRELY, Protoplasmolocia 5 (3Ç), 1 (1966) .
378 (1969) .
40 (1962) .
Life Sciences Vol. 9, Part II, Printed in Great Britain
Pergamon Press
DIFFERENCES IN THE AMINO ACID COMPOSITION OF OBOXYRIBONUCLEOPROTEINS FROM WHITE BLOOD CELLS OF NORMAL AND LEUKEMIC HUMAN BEINGS L . Messineo and J . R . Kongsvik Biochemistry Research Laboratory, Veterans Administration Center 45428 4100 West Third Street, Dayton, Ohio
(Received 22 January 1970; in final form 20 March 1970)
It has been reported that deoxyribonucleoproteins (DNP) from the white blood cells of normal and leukemic human beings elicit in rabbits an immunological response which is specific to the extent of distinguishing between DNP's from normal and leukemic subjects (1,2) .
Furthermore, antibodies against peripheral
leukocyte DNP from normal subjects cross-react with ONP from bone marrow cells of the same subjects, but not with DNP from marrow cells of leukemic subjects . No cross reaction was found between antisera to DNP from white blood cells and bone marrow cells of leukemic individuals and the DNP of leukocytes and bone marrow cells from normal subjects (2) . On the basis of these immunological distinctions we have compared the amino acid composition of DNP from the peripheral leukocytes of normal subjects with that from leukemic peripheral leukocytes .
We wish to report differences
in the amino acid composition of the respective DNP's and would like to suggest that these differences could explain the immunological differences noted earlier . Materials and Methods In this study human beings are considered normal or leukemic on the basis of clinical diagnostic tests .
In no instance was the white blood cell count of
the chronic lymphocytic leukemic patients less than 80,000 sn 3 . Deoxyribonucleoproteins were extracted from normal and leukemic leukocytes as reported earlier (1,2) .
Previous to amino acid analyses the clarified gly-
cine extracts from both sources were characterised by ultracentrifugal, electro-
541
542
Vol. 9, No . 10
AMINO ACID COMPOSITION
phoretic, chromatographic,, immunologic and chemical procedures (3,4) . were found to be intact, homogeneous DNA-basic protein complexes .
They
Chemical
analyses of the DNP's by the modified biuret test (5) and spectrophotometric analyses at 260 mr indicated a protein and DNA content of 71 and 29% respectively .
The DNP preparations were highly polymerized as evidenced by a sedi-
mentation coefficient (S20 ,w ) of 60 .
The DNA moiety of the respective leuko-
cyte DNP's was undegraded as evidenced by an alkaline induced hyperchromicity at 260 mp of 33% (6) and highly fibrous DNA is recovered upon deproteinization and subsequent ethanol precipitation (7) .
The molar base composition of both
normal and leukemic DNA were similar (A = 27 .4, T = 27 .8, G = 22 .2, C = 22 .5), thereby corresponding to previous reports (8) .
Elution of the DNP's from
Sephadex G-200 columns results in the recovery of both protein and DNA under a single peak . Immunological assays in test tubes with rabbit antibodies, obtained as previously described (2), result in the complete precipitation of the DNP's, while in agar gel plates one single band is formed when the diffusing DNP antigen and antibodies meet (1,2) . Cellulose acetate electrophoresis and column chromatography on Sephadex were performed as reported elsewhere (3) . Solubility studies, which have been reported elsewhere (9) show that glycine extraction recovers more intact DNP particles without the loss of fractions which accompanies other methods of extraction (10) . The amino acid analyses were done on a model 034-0030 Hitachi (PerkinElmer) amino acid analyzer by the ligand exchange method .
DNP preparations
were separated from the glycine of the medium by filtration through Sephadex G-25, eluted with double distilled water at 50 C . and recovered by lyophilization .
The hydrolysis was carribd out on the whole DNP particles in a sealed,
evacuated tube with 6N HCl under reflux for 20 hours at 101 0 C . eight milligrams of whole DNP were used in each analysis .
From five to
Whole DNP was
analyzed instead of the extracted protein moieties to avoid the loss of
Vol. 9, No. 10
AMINO ACID COMPOSITION
543
fractions and to maintain the quantitative relationship of the protein to DNA . Results and Discussion Aliquots of purified normal and leukemic DNP preparations were routinely tested by cellulose acetate electrophoresis and column chromatography before acid hydrolysis .
Figure 1 shows the electrophoretic migration of normal (A)
FIG . lA Blectrophoretic migration of leukemic leukocyte DNP at 150 volts for 35 minutes .
The CLL-DNP was applied at
2 .6 (A) and at 1 .3 (B) mg/ml . point of application .
The arrows indicate the
The anode is on the right .
544
AMINO ACID COMPOSITION
Vol. 9, No . 10
FIG . 1B Electrophoretic migration of normal leukocyte DNP at 210 volts for 25 minutes .
The DNP applied was 971 pg/ml .
The arrow indicates the point of application .
The anode
is on the right . and leukemic DNP's (B) . as a single band . on Sephadex G-200 .
In either case, the DNP's migrated towards the anode
Figure 2 shows the elution profile of leukemic leukocyte DNP Filtration of the DNP through G-200 consistently resulted
in the recovery of one peak containing both DNA and protein .
(See Fig . 2)
Superimposable profiles were obtained when normal leukocyte DNP's were chromatographed .
AMINO ACID COMPOSITION
Vol. 9, No. 10
545
2.5-
2 .0-
LO-
0 .5 --~
0.0 1
5
le
i l§ 11 1 1118 1 191 i p mol l i 20 25 30 10 15
FRA C TION NUMBER FIG . 2
Gel exclusion chromatography of leukemic leukocyte DNP on Sephadex G-200 .
The sample (1 ml at 4 .3 mg/ml) was
eluted from the column (90 x 1 .5 cm) with 0 .0007 M KH2 P04' pH 7 .1 .
The fraction size was 7 ml .
35
548
AMINO ACID COMPOSITION
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Vol. 9, No. 10
AMINO ACED COMPOSMON
547
Table I shows the amino acid composition of five leukemic and four normal leukocyte DNP preparations .
The data are expressed as the mole percentage of
total moles of amino acids recovered .
It is clear that differences exist in
the amino acid composition of the respective DNP's .
The sum of basic amino
acids (lysine, arginine, and histidine) in leukemic DKP is 21%, while it is 15% in the DNP from normal leukocytes .
This is an absolute difference of 29% .
Conversely, the sum of aspartic and glutamic acids, proline, serine, threonine and valine in normal DNP is 29% greater than that of leukemic DIO? . Since the extraction and analyses were carried out under the same conditions, it is difficult to envision these differences to be due to loss of fractions, oxidation of basic amino acids or contamination .
The consistency of
the analyses of separate preparations enhances the validity of the data . The presence of unbound contaminants in the DNP preparations are excluded by the results of the physico-chemical characterizations mentioned under methods (above) . On the basis of these physico-chemical characterizations of normal and leukemic white blood cell DNP's, we think that the results of the amino acid analyses is a better reflection of the composition of the protein moiety bound to DNA in vivo .
This concept is enhanced also because the loss of innate
fractions has been avoided by analyzing the whole DNP .
Thus, these results
would suggest that a real difference exists in the protein moieties of normal and leukemic DNP . Since the molar base composition of the DNA from normal and leukemic DIP is similar, the differences in the amino acid composition of the protein moieties could prove to be of significant value with respect to the expression of the genetic code in health and neoplasia .
Furthermore, these results may
imply the existence of structural differences between normal and leukemic DNA .
Sammy The basic amino acid content of the protein moieties of the glycineextracted deoxyribonucleoproteins (DBP) from normal and leukemic peripheral
548
ALTO ACID COMPOSITION
white blood cells was found to be different .
Vol . 9, No. 10
The absolute sum of the basic
amino acids in leukemic DNP was 29% greater than that in the normal DNP, while in the normal DNP an equal magnitude of difference was found in the acidicneutral amino acid content .
These differences are discussed in relation to the
known immunological distinctions between normal and leukemic DNP's and also as a consequence of the etiological action of the leukemic agent(s) . References 1.
L . MESSINEO, Nature 180,
1122 (1961) .
2.
L. MESSINEO, Arch . Biochem . Biophys . 108, 471 (1964) .
3.
L . MESSINEO, J . R . KONGSVIK, Arch Biochem. Biophys . 135,
4.
L . MESSINEO, J. R . KONGSVIK, Intnl . J . Biochem. in press (1970) .
5.
G . L . ELLMAN, Anal . Biochem. 3,
6.
R . D . HOTCHKISS, in S . P . Colowick and N. 0. Kaplan (Editors), Methods in Enzymology , vol . 3, p . 708, Academic Press Inc ., New York (1957) .
7.
E . R . M. KAY, N. W. SIMMONS, and A. L . DODNCE, J . Am . Chem . Soc . 74, 1724 (1952) .
8.
E .E . POLLI, M . ROSOFF, G . Di MAYORCA, and L. F. CAVALIERI, Cancer Research 19, 159 (1959) .
9.
L. MESSINEO, Biophysical Society Abstracts (1970) .
10 .
R . VENDRELY, and C . VENDRELY, Protoplasmolocia 5 (3Ç), 1 (1966) .
378 (1969) .
40 (1962) .