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Proteolytic contamination of calf thymus nucleohistone and its inhibition
252
SHORT COMMUNICATIONS
BBA 3308O
Proteolytic contamination of calf thymus nucleohistone and its inhibition During the past two decades, calf t h y m u s nuclei have often been used as a source of histones and of nucleohistonel, z. T h o u g h most workers have a t t e m p t e d to minimize degradation of extracted proteins and nucleic acids, the recent demonstration of extensive phagocytosis in the t h y m u s 3 and of extensive t h y m u s cell turnover 4 reemphasizes the danger of enzymic damage to macromolecules being isolated. We have detected proteolysis in all nucleohistone purified from calf t h y m u s tissue, though the extent of protease contamination depends on the isolation procedure and on the freshness of the t h y m u s tissue. Proteolysis is inhibited b y adding D F P or sodium bisulfite during nucleohistone isolation. Nucleohistone was prepared and analyzed as previously described 5. Free-zone electrophoresis employed the apparatus of OLIVERA, BAINE AND DAVIDSON6. Thermal denaturation profiles in 2.5" lO -4 M N a E D T A (pH 8) were obtained using a Gilford spectrophotometer (Model 2ooo), appropriately adapted. A convenient and sensitive method for monitoring proteolysis is to study the decrease in precipitability of nucleohistone in o.15 M NaC1. Such precipitation is dependent on the overall negative charge on the nucleohistone. The loss of a small IOC
2~
i
t
TIME
(HOURS)
Fig. I. Precipitation analysis of incubated nucleohistone. Samples of nucleohistone (A26om~ = IO) were precipitated as described previously s in o.15 M NaC1, o.oi M Tris-HCl (pH 8.o). V--E7, nucleohistone at 25°; ©--C), nucleohistone at o°; Kl--~, DFP-isolated nucleohistone at o% Fig. 2. Acrylamide gel electrophoresis of histones from calf thymus nueleohistone, a. Isolated in the absence of protease inhibitors, b. Isolated in the presence of DFP. a m o u n t of positively charged histone results in a significant increase in the solubility of nucleohistone in o. 15 M NaC1 s. Samples were assayed in this manner after incubation at either 0 ° or 25 °. The results in Fig. I show t h a t nucleohistone is degraded at both temperatures, more rapidly at the higher temperature. The degradation of histone also affects other physical properties of nucleohistone. Heat denaturation is accomplished at progressively lower temperatures (Table I) aad the electrophoretic mobility of the complex increases (Table II). Proteolytic degradation of calf t h y m u s nucleohistone is also shown b y a reduction in the yield of acid-soluble, trichloroacetic acid-precipitable histone. The fraction most Biochirn. Biophys. Acta, 16o (1968) 252 255
SHORT COMMUNICATIONS
253
TABLE I THE DEPENDENCE OF THERMAL DENATURATION PARAMETERS UPON PROTEOLYSIS %/ h is t h e p e r c e n t h y p e r c h r o m i c shift u p o n t h e r m a l d e n a t u r a t i o n . Tm is t h e t e m p e r a t u r e a t t h e m i d p o i n t of t h e r m a l d e n a t u r a t i o n (in 2.5" IO -a M E D T A , p H 8).
Temp. o °
Temp. 25 °
Time (h)
% Precipiration in o.I 5 M NaC1
Tm
% h
Time (h)
% Precipitation in o.z5 M NaCl
Tm
% h
o 26 48 96
89 78 42 36
75 74.8 71.4 71-°
35.0 32.6 34 .0 33.3
o 2 4 6 14
89 72 45 42 39
75 74.2 73 .o
35 .0 32.2 32.1 33.7 32.7
72.2 7 o-2
sensitive to degradation is the lysine-rich (histone I) fraction as shown by acrylamidegel electrophoresis in Fig. 2. REID AND COLE7 have reported a similar observation following incubation of thymus nuclei at 37 °. Apparently, deoxyribonuclease is not present in large amounts. The sedimentation velocity of DNA isolated from nucleohistones (s~0,w -----14) did not change significantly over the time period examined. The NaCl-soluble material which is produced by degradation consists of partially TABLE I[ THE DEPENDENCE OF ELECTROPHORETIC MOBILITY OF THE NUCLEOHISTONE COMPLEX UPON PROTEOLYSIS
Time (h)
% Precipitation
Mobility (cm 2. V -1. sec -t)
0 24 48 72
85 77 74 65
1.28 1.33 1.33 1.42
deproteinized nucleohistone mixed with fragments of histone which dissociated at the elevated (o.i5) ionic strength. This was ascertained by sedimenting the NaCl-soluble material at 35 ooo rev./min for I2 h. The pellet which contained > 95% of the DNA present was dissolved in 2.5. Io-4 M NaEDTA (pH 8) and dialyzed exhaustively against this buffer. A small quantity of DNA (50/~g/ml) was added to the supernatant fraction which was then dialyzed against the same low ionic strength buffer. Fig. 3 shows the melting profiles of each of these samples as compared to undegraded nucleohistone and to deproteinized DNA. The dissolved nucleohistone pellet has a much reduced Tm and the material in the supernatant has complexed with, and stabilized, the added DNA against melting. Proteolysis was substantially inhibited by D F P (o.ooI M) and sodium bisulfite (o.oo5 M) and to a lesser extent by iodoacetamide and fl-mercaptoethanol (Fig. 4). Biochim. Biophys. Acta, 16o (I968) 252-255
254
SHORT COMMUNICATIONS
1.40
/O/Q.o..o- o.'o-o- o
/
1.30
/
L20
~" I.lO
o t
J
/
/z
/
4Q 44
1,00 I
/'C7::: /./j-/
I
t
o ~ 1.3G
1.20
1.10
t
42 40 38
E" 32
30
./i/, "/"
././
22 l.O0
~O
~o
~'o
TEMPERATURE
18
go
do "riME
"3do
( rnln )
Fig. 3. a. Thermal denaturation profiles of D N A and nucleohistone in 2.5 • IO -~ M E D T A (pH 8). O - - O , D N A ; [ ~ - - D , n u c l e o h i s t o n e , b. Thermal denaturation profiles of the pellet from o.15 M NaCl-soluble fraction (El--B) ; a n d D N A + supernatant f r o m 35 ooo rev,/min supernatant (O---OL All samples dissolved in 2.5" lO -4 M E D T A (pH 8). Fig. 4. Inhibition of proteolysis in nucleohistone. Nucleohistone w a s incubated at 37 ° in the presence of (a) o.ooi M /~-mercaptoethanol ( X - X); (b) o.ooI M i o d o a e e t a m i d e ( ( 9 - - 0 ) ; (c) o.ooi M D F P ( A - - A ) ; (d) o.oo 5 M sodium bisulfite ( V - - V ) ; (e) no addition ( [ ~ - - D ) ; e x t e n t of proteolysis measured b y precipitation in o.i 5 M NaC1.
Calf thymus nucleohistone prepared in the presence of D F P or sodium bisulfite did not lose acid-extractable, trichloroacetic acid-precipitable histone as a function of time. Also, the o.15 M NaC1 precipitability is at a high level and essentially constant over a long time, as shown in Fig. I. We would stress the need to define the concentration of nucleohistone in a precipitation assay as the extent of precipitation is strongly dependent upon this variable s. The lysine-rich histone fraction of DFP-treated nucleohistone is clearly visible upon acrylamide-gel electrophoresis (Fig. 2). Much research is currently directed towards understanding genetic control. Isolated chromosomal material particularly from calf thymus is frequently used as a standard system in vitrog, 1°. As the role of proteins in regulation of genetic activity is thought to be prime, the utmost caution should be used in analysis of data obtained in the presence of proteolytic enzymes. However, the use of our precipitation assay provides a convenient and rapid monitor of proteolytic contamination* and the use of D F P or sodium bisulfite during the isolation promises, at least for calf thymus * W e do not k n o w how m a n y histone peptide bonds m u s t be broken to change solubility characteristics in NaC1; clearly the sensitivity of the assay to initial proteolysis remains to be determined.
Biochim. Biophys. Acta, 16o (1968) 252-255
255
SHORT COMMUNICATIONS
nucleohistone, to reduce this problem substantially. There remains the possibility that these protease inhibitors may introduce disadvantageous chemical or biological effects. This matter is being investigated but no deleterious effects have yet been found. The authors are grateful to Professor J. DONNER, in whose laboratory much of this work was performed, and to Miss M. THOMASand Mrs. J. EARNHARDTfor technical assistance.
Department of Biochemistry, University of Iowa, Iowa City, Iowa, and Division of Biology, California Institute of Technology, Pasadena, Calif. (U.S.A.)
SAKOL PANYIM RONALD H . J E N S E N * R O G E R CHALKLEY**
I J. DONNER AND P. O. P. TS'O, The Nucleohistones, H o l d e n - D a y , S a n Francisco, Calif., 1964 . 2 H. B u s c H , Histories and Other Nuclear Proteins, A c a d e m i c Press, N e w York, N.Y., 1965 3 D. METCALF, in R. A. GOOD AND A. E. GABRIELSON, The Thymus in Immunobiology, H a r p e r Row, L o n d o n , 1965, p. 156. 4 D. ~{ETCALF,in R. A. GOOD AND A. E. GABRIELSON, The Thymus in Immunbiology, H a r p e r Row, L o n d o n , 1965, p. 16o. 5 R. CHALKLEY AND R. H. JENSEN, J. Mol. Biol., s u b m i t t e d . 6 B. M. OLIVERA, P. BAINE AND N. DAVIDSON, Biopolymers, 2 (1964) 245. 7 B. R. REID AND R. D. COLE, Proc. Natl. Acad. Sci. U.S., 51 (lO64) lO44. 8 R. H. JENSEN AND R. CltALKLEY, J. Mol. Biol., s u b m i t t e d . 9 Y- OHBA, Biochim. Biophys. Acta, 123 (1966) 84. IO P. H. LLOYD, B. H. NICHOLSON AND A. R. PEACOCKE, Biochem. J., lO 4 (1967) 999.
Received December 22nd, 1967 Revised manuscript received February 8th, 1968 P r e s e n t a d d r e s s : I n t e r n a t i o n a l Minerals a n d Chemical Corp., Libertyville, Ill., U.S.A. ** P r e s e n t a d d r e s s : D e p a r t m e n t of B i o c h e m i s t r y , U n i v e r s i t y of Iowa, I o w a City, Iowa, U.S.A.
Biochim. Biophys. Acta, 16o (1968) 252-255
BBA 33O86
Nuclear magnetic investigation of pre-denaturational conformational transition of metmyoglobin in aqueous solution* Because of the great importance of the haemoproteins, they have been the object of investigation by a considerable variety of methods. In the last few years the method of nuclear magnetic resonance 1-7 has also been applied in these studies. Aqueous solutions of haemoproteins are of special interest with regard to NMR studies. These substances can be considered as macromolecular complexes of iron 8. The unpaired electrons of the iron atom in the haemin group give the whole molecule the character of a paramagnetic particle, of which the magnetic moment interacts with the magnetic moments of the protons of the solvent and shortens their relaxation * T h e m a i n p a r t o f this p a p e r was p r e s e n t e d at t h e I n t e r n a t i o n a l S y m p o s i u m on t h e Struct u r e a n d R e a c t i v i t y of Organic C o m p o u n d s , Sofia, J u n e 21-24, 1966.
Biochim. Biophys. Acta, 16o (1968) 255-258
SHORT COMMUNICATIONS
BBA 3308O
Proteolytic contamination of calf thymus nucleohistone and its inhibition During the past two decades, calf t h y m u s nuclei have often been used as a source of histones and of nucleohistonel, z. T h o u g h most workers have a t t e m p t e d to minimize degradation of extracted proteins and nucleic acids, the recent demonstration of extensive phagocytosis in the t h y m u s 3 and of extensive t h y m u s cell turnover 4 reemphasizes the danger of enzymic damage to macromolecules being isolated. We have detected proteolysis in all nucleohistone purified from calf t h y m u s tissue, though the extent of protease contamination depends on the isolation procedure and on the freshness of the t h y m u s tissue. Proteolysis is inhibited b y adding D F P or sodium bisulfite during nucleohistone isolation. Nucleohistone was prepared and analyzed as previously described 5. Free-zone electrophoresis employed the apparatus of OLIVERA, BAINE AND DAVIDSON6. Thermal denaturation profiles in 2.5" lO -4 M N a E D T A (pH 8) were obtained using a Gilford spectrophotometer (Model 2ooo), appropriately adapted. A convenient and sensitive method for monitoring proteolysis is to study the decrease in precipitability of nucleohistone in o.15 M NaC1. Such precipitation is dependent on the overall negative charge on the nucleohistone. The loss of a small IOC
2~
i
t
TIME
(HOURS)
Fig. I. Precipitation analysis of incubated nucleohistone. Samples of nucleohistone (A26om~ = IO) were precipitated as described previously s in o.15 M NaC1, o.oi M Tris-HCl (pH 8.o). V--E7, nucleohistone at 25°; ©--C), nucleohistone at o°; Kl--~, DFP-isolated nucleohistone at o% Fig. 2. Acrylamide gel electrophoresis of histones from calf thymus nueleohistone, a. Isolated in the absence of protease inhibitors, b. Isolated in the presence of DFP. a m o u n t of positively charged histone results in a significant increase in the solubility of nucleohistone in o. 15 M NaC1 s. Samples were assayed in this manner after incubation at either 0 ° or 25 °. The results in Fig. I show t h a t nucleohistone is degraded at both temperatures, more rapidly at the higher temperature. The degradation of histone also affects other physical properties of nucleohistone. Heat denaturation is accomplished at progressively lower temperatures (Table I) aad the electrophoretic mobility of the complex increases (Table II). Proteolytic degradation of calf t h y m u s nucleohistone is also shown b y a reduction in the yield of acid-soluble, trichloroacetic acid-precipitable histone. The fraction most Biochirn. Biophys. Acta, 16o (1968) 252 255
SHORT COMMUNICATIONS
253
TABLE I THE DEPENDENCE OF THERMAL DENATURATION PARAMETERS UPON PROTEOLYSIS %/ h is t h e p e r c e n t h y p e r c h r o m i c shift u p o n t h e r m a l d e n a t u r a t i o n . Tm is t h e t e m p e r a t u r e a t t h e m i d p o i n t of t h e r m a l d e n a t u r a t i o n (in 2.5" IO -a M E D T A , p H 8).
Temp. o °
Temp. 25 °
Time (h)
% Precipiration in o.I 5 M NaC1
Tm
% h
Time (h)
% Precipitation in o.z5 M NaCl
Tm
% h
o 26 48 96
89 78 42 36
75 74.8 71.4 71-°
35.0 32.6 34 .0 33.3
o 2 4 6 14
89 72 45 42 39
75 74.2 73 .o
35 .0 32.2 32.1 33.7 32.7
72.2 7 o-2
sensitive to degradation is the lysine-rich (histone I) fraction as shown by acrylamidegel electrophoresis in Fig. 2. REID AND COLE7 have reported a similar observation following incubation of thymus nuclei at 37 °. Apparently, deoxyribonuclease is not present in large amounts. The sedimentation velocity of DNA isolated from nucleohistones (s~0,w -----14) did not change significantly over the time period examined. The NaCl-soluble material which is produced by degradation consists of partially TABLE I[ THE DEPENDENCE OF ELECTROPHORETIC MOBILITY OF THE NUCLEOHISTONE COMPLEX UPON PROTEOLYSIS
Time (h)
% Precipitation
Mobility (cm 2. V -1. sec -t)
0 24 48 72
85 77 74 65
1.28 1.33 1.33 1.42
deproteinized nucleohistone mixed with fragments of histone which dissociated at the elevated (o.i5) ionic strength. This was ascertained by sedimenting the NaCl-soluble material at 35 ooo rev./min for I2 h. The pellet which contained > 95% of the DNA present was dissolved in 2.5. Io-4 M NaEDTA (pH 8) and dialyzed exhaustively against this buffer. A small quantity of DNA (50/~g/ml) was added to the supernatant fraction which was then dialyzed against the same low ionic strength buffer. Fig. 3 shows the melting profiles of each of these samples as compared to undegraded nucleohistone and to deproteinized DNA. The dissolved nucleohistone pellet has a much reduced Tm and the material in the supernatant has complexed with, and stabilized, the added DNA against melting. Proteolysis was substantially inhibited by D F P (o.ooI M) and sodium bisulfite (o.oo5 M) and to a lesser extent by iodoacetamide and fl-mercaptoethanol (Fig. 4). Biochim. Biophys. Acta, 16o (I968) 252-255
254
SHORT COMMUNICATIONS
1.40
/O/Q.o..o- o.'o-o- o
/
1.30
/
L20
~" I.lO
o t
J
/
/z
/
4Q 44
1,00 I
/'C7::: /./j-/
I
t
o ~ 1.3G
1.20
1.10
t
42 40 38
E" 32
30
./i/, "/"
././
22 l.O0
~O
~o
~'o
TEMPERATURE
18
go
do "riME
"3do
( rnln )
Fig. 3. a. Thermal denaturation profiles of D N A and nucleohistone in 2.5 • IO -~ M E D T A (pH 8). O - - O , D N A ; [ ~ - - D , n u c l e o h i s t o n e , b. Thermal denaturation profiles of the pellet from o.15 M NaCl-soluble fraction (El--B) ; a n d D N A + supernatant f r o m 35 ooo rev,/min supernatant (O---OL All samples dissolved in 2.5" lO -4 M E D T A (pH 8). Fig. 4. Inhibition of proteolysis in nucleohistone. Nucleohistone w a s incubated at 37 ° in the presence of (a) o.ooi M /~-mercaptoethanol ( X - X); (b) o.ooI M i o d o a e e t a m i d e ( ( 9 - - 0 ) ; (c) o.ooi M D F P ( A - - A ) ; (d) o.oo 5 M sodium bisulfite ( V - - V ) ; (e) no addition ( [ ~ - - D ) ; e x t e n t of proteolysis measured b y precipitation in o.i 5 M NaC1.
Calf thymus nucleohistone prepared in the presence of D F P or sodium bisulfite did not lose acid-extractable, trichloroacetic acid-precipitable histone as a function of time. Also, the o.15 M NaC1 precipitability is at a high level and essentially constant over a long time, as shown in Fig. I. We would stress the need to define the concentration of nucleohistone in a precipitation assay as the extent of precipitation is strongly dependent upon this variable s. The lysine-rich histone fraction of DFP-treated nucleohistone is clearly visible upon acrylamide-gel electrophoresis (Fig. 2). Much research is currently directed towards understanding genetic control. Isolated chromosomal material particularly from calf thymus is frequently used as a standard system in vitrog, 1°. As the role of proteins in regulation of genetic activity is thought to be prime, the utmost caution should be used in analysis of data obtained in the presence of proteolytic enzymes. However, the use of our precipitation assay provides a convenient and rapid monitor of proteolytic contamination* and the use of D F P or sodium bisulfite during the isolation promises, at least for calf thymus * W e do not k n o w how m a n y histone peptide bonds m u s t be broken to change solubility characteristics in NaC1; clearly the sensitivity of the assay to initial proteolysis remains to be determined.
Biochim. Biophys. Acta, 16o (1968) 252-255
255
SHORT COMMUNICATIONS
nucleohistone, to reduce this problem substantially. There remains the possibility that these protease inhibitors may introduce disadvantageous chemical or biological effects. This matter is being investigated but no deleterious effects have yet been found. The authors are grateful to Professor J. DONNER, in whose laboratory much of this work was performed, and to Miss M. THOMASand Mrs. J. EARNHARDTfor technical assistance.
Department of Biochemistry, University of Iowa, Iowa City, Iowa, and Division of Biology, California Institute of Technology, Pasadena, Calif. (U.S.A.)
SAKOL PANYIM RONALD H . J E N S E N * R O G E R CHALKLEY**
I J. DONNER AND P. O. P. TS'O, The Nucleohistones, H o l d e n - D a y , S a n Francisco, Calif., 1964 . 2 H. B u s c H , Histories and Other Nuclear Proteins, A c a d e m i c Press, N e w York, N.Y., 1965 3 D. METCALF, in R. A. GOOD AND A. E. GABRIELSON, The Thymus in Immunobiology, H a r p e r Row, L o n d o n , 1965, p. 156. 4 D. ~{ETCALF,in R. A. GOOD AND A. E. GABRIELSON, The Thymus in Immunbiology, H a r p e r Row, L o n d o n , 1965, p. 16o. 5 R. CHALKLEY AND R. H. JENSEN, J. Mol. Biol., s u b m i t t e d . 6 B. M. OLIVERA, P. BAINE AND N. DAVIDSON, Biopolymers, 2 (1964) 245. 7 B. R. REID AND R. D. COLE, Proc. Natl. Acad. Sci. U.S., 51 (lO64) lO44. 8 R. H. JENSEN AND R. CltALKLEY, J. Mol. Biol., s u b m i t t e d . 9 Y- OHBA, Biochim. Biophys. Acta, 123 (1966) 84. IO P. H. LLOYD, B. H. NICHOLSON AND A. R. PEACOCKE, Biochem. J., lO 4 (1967) 999.
Received December 22nd, 1967 Revised manuscript received February 8th, 1968 P r e s e n t a d d r e s s : I n t e r n a t i o n a l Minerals a n d Chemical Corp., Libertyville, Ill., U.S.A. ** P r e s e n t a d d r e s s : D e p a r t m e n t of B i o c h e m i s t r y , U n i v e r s i t y of Iowa, I o w a City, Iowa, U.S.A.
Biochim. Biophys. Acta, 16o (1968) 252-255
BBA 33O86
Nuclear magnetic investigation of pre-denaturational conformational transition of metmyoglobin in aqueous solution* Because of the great importance of the haemoproteins, they have been the object of investigation by a considerable variety of methods. In the last few years the method of nuclear magnetic resonance 1-7 has also been applied in these studies. Aqueous solutions of haemoproteins are of special interest with regard to NMR studies. These substances can be considered as macromolecular complexes of iron 8. The unpaired electrons of the iron atom in the haemin group give the whole molecule the character of a paramagnetic particle, of which the magnetic moment interacts with the magnetic moments of the protons of the solvent and shortens their relaxation * T h e m a i n p a r t o f this p a p e r was p r e s e n t e d at t h e I n t e r n a t i o n a l S y m p o s i u m on t h e Struct u r e a n d R e a c t i v i t y of Organic C o m p o u n d s , Sofia, J u n e 21-24, 1966.
Biochim. Biophys. Acta, 16o (1968) 255-258