ANALYTICAL
BIOCHEMISTRY
Improvement
9,
167- 173 (1978)
of DNA Quantitation Proteolytic Extraction
following
C. A. BARTH’ AND B. S. WILLERSHAUSEN Forschrrgruppe
Erniihrung
un der Meditinischen Miinchen, Germany
Poliklinik,
Universitiit
Miinchen.
ReceivedOctober 14. 1977 A reliableand sensitive method for quantitation of deoxyribonucleic acid (DNA) is described herein. It is based on a proteinase K digestion step followed by a fiuorometric assay with 3,Sdiaminobenzoic acid (Kissane and Robins, 1958). The method proved to be applicable to fresh liver and to hepatocytes either in suspension or in culture when attached to a collagen substratum. A linear relationship between biological sample size and fluorometric response was observed only if the assay was preceded by proteolytic extraction. Moreover, the method resulted in DNA values per fresh liver weight that were about 100% higher than values reported earlier. The data show that proteolysis results in a more exhaustive extraction of DNA from tissues and a better separation of extracted DNA from other tissue constituents interfering with the assay.
Chemical and metabolic properties of cells or tissues are commonly expressed in terms of cell number, weight of specimen, or total protein content. Sometimes the DNA content is used as well because it is closely correlated to cell number. Furthermore it has the advantage of being very sensitive if suitable methods are applied. In the course of studies on the metabolic performance of hepatocytes attached to a collagen substratum we tried to determine DNA according to Kissane and Robins (1). In this procedure a fluorescent product is formed after reacting the deoxyribose moiety with 3,5-diaminobenzoic acid dihydrochloride (DABA).z We observed this sensitive method to give excellent results if applied to pure standards but to be of poor reliability in the case of biological samples. We thought that this might be mainly due to an incomplete separation of DNA from interfering cell constituents prior to reaction with DABA. In the following we report on an improvement of the assay by taking advantage of the proteinase K digestion method developed recently (2,3) to extract high molecular weight nucleic acids from biological specimens. I Postal address: Medizinische Poliklinik, Pettenkoferstr. Sa, D-8000 Germany. * Abbreviation used: DABA. 3,Sdiaminobenzoic acid dihydrochloride. 167
Miinchen,
0003-2697/78/0901-0167$02.00/O Copyright Q 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
168
BARTH
AND
WILLERSHAUSEN
Furthermore an inexpensive and easy procedure will be described yielding DABA of high purity suitable for fluorometry. MATERIALS
AND METHODS
Bovine serum albumin “reinst” (Catalog No. RHD 20) was bought from Behring-Hochst, Frankfurt-Main, West Germany. In some cases DABA from EGA Chemie, Steinheim/Albuch, West Germany, (Catalog No. 11,382-2) was used following purification according to Hinegardner (4). Generally, 3,5-diaminobenzoic acid (99%) (Catalog No. l-4943) was purchased from Roth, Karlsruhe, West Germany, and purified as described below. Calf thymus DNA from Sigma (Catalog No. D- 1501) dissolved in 1 N NHIOH was used as standard. Proteinase K was from Merck, Darmstadt, West Germany. All other chemicals were of reagent grade and bought from the same manufacturer. Male Sprague-Dawley rats of 150 g body weight were used throughout. 3,5Diaminobenzoic acid was purified by dissolving 4 g in 15 ml of 3.6 N HCl. The solution was stirred at about 60°C for 1 h after addition of 2.25 g of acid-washed charcoal. The charcoal was removed by filtration on a Buchner funnel (Paper No. 595, Schleicher and Schuell). The dihydrochloride derivative was precipitated by addition of an equal volume of concentrated HCl. The precipitate was separated from the solvent by filtration on a Buchner funnel. As the material generally had a yellowish color the charcoal, filtration, and precipitation steps were repeated after dissolving the material in water. The material then crystallized regularly as white needles which were dried over KOH in vucuo. Recovery was about 45%. Proton nmr of the purified material gave the expected signals (‘H nmr in water, r scale: 2.1, 2.4). In the DNA assay, reagent blanks with the freshly purified material gave a fluorescence which was lower than 5% of the fluorescence given by a standard containing 10 pg of DNA. Fluorometry was performed with an Eppendorf fluorometer Model 1130 attached to a photometer Model 1101. The primary filter was set at 436 nm, the secondary filter at 4703000 nm. Routine procedure. Hepatocyte cultures in petri dishes (60 mm) were washed three times with 4 ml of saline. Removal of dead cells and cell debris was enhanced by tapping the dish on the table. Four milliliters of a 500 mM Tris-HCl buffer, pH 8.0, containing 6 M urea, 10 mM EDTA and 10 mM NaCl was added to each dish. Proteinase K freshly dissolved in the above buffer was added to give a final concentration of 50 @/ml. After overnight incubation at 37°C the contents were transferred to a 14ml centrifuge tube and shaken vigorously on a Vortex-type mixer. It is critical that any cloudy material still present disappears at this moment. Total volume was measured and an aliquot of 0.5 ml was transferred to a 1.5-ml
DNA
ASSAY
FOLLOWING
PROTEOLYSIS
169
Eppendorf vial. Fifty microliters of bovine serum albumin (5 mg/ml) was added, and the DNA was precipitated by addition of 0.5 ml of 2 N HCl in 15% CCl,COOH. Albumin served to assure a complete precipitation of DNA at this and the following wash steps. The effect of albumin at this step will be discussed in detail later in this paper. Subsequently the original procedure given by Kissane and Robins (1) was followed essentially for washing the precipitate with CCl,COOH and ethanol. HCl, 1 N, was added before fluorometry according to Hinegardner (4). RESULTS
Figure 1 displays fluorometer readings after reacting different amounts of pure DNA or varying aliquots of a hepatocyte suspension with DABA, according to the original procedure of Kissane and Robins (1). There was an excellent linear response of fluorescence to rising amounts of pure DNA (Fig. 1A). But, deviation from linearity was observed in the case of biological samples (Fig. 1B) and could not be prevented even if samples were pretreated by such procedures as freezing and thawing or alkaline digestion in order to enhance DNA extraction from cells. Figure 2 shows that this problem was circumvented when proteolysis preceded the DNA assay; a linear fluorometer reading with sample size
0
0
5
10 [x10-4cekl
FIG. 1. Assay of DNA with purified calf thymus DNA (A) and isolated rat hepatocytes (B). (A) Fluorometer readings after reacting purified DNA with DABA, as outlined under Materials and Methods. Ordinate: Fluorescence in arbitrary units. Abscissa: Varying amounts of calf thymus DNA. (B) DNA determination in freshly isolated rat hepacytes (A, n ), hepatocytes suspended in 1 N NaOH (0). and hepatocytes frozen and thawed four times (0). For further details see (A).
170
BARTH
AND
WILLERSHAUSEN
1
0
2
[x lo-6cells]
FIG. 2. Assay of DNA in hepatocytes following proteolytic extraction. Freshly isolated hepatocytes were seeded on collagen gels, prepared according to Ref( 11) in petri dishes of60mm diameter. For proteolysis see Routine Procedure, except that no 0.5ml aliquot was taken out after proteolysis. Instead, the total material digested by proteinase was taken to DNA assay, the latter being scaled up correspondingly.
was observed. Table 1 shows results confirming this finding. Moreover, it is demonstrated that the results of four parallels agree perfectly well under the conditions chosen and that proteolysis by 200 pg of enzyme was sufficient to assure a satisfying extraction of DNA. It was observed that addition of 250 pg of albumin at the precipitation step, after proteolysis of a hepatocyte culture, raised the net fluorescence by 43%. A systematic examination revealed that DNA precipitation was TABLE INFLUENCE
OF DIFFERENT
1
AMOUNTS OF PROTEINASE IN HEPATOCYTE MONOLAYERS”
Net Proteinase
K
ON
DNA
EXTRACTION
fluorescence*
K added (f%)
0.2-ml
Sample
OS-ml
Sample
200
210 235
550 570
300
220 240
560 520
u Freshly isolated rat hepatocytes, 2.5 coated with a collagen gel (11). Subsequently Procedure with 200 or 300 pg of proteinase were analyzed for DNA. b Arbitrary units.
x
106. were seeded in each of four petri dishes they were treated as outlined under Routine K and two different aliquots of 0.2 and 0.5 ml
DNA
ASSAY
FOLLOWING
171
PROTEOLYSIS
optimal if 200-300 ,ug of albumin was added (Fig. 3); therefore 250 pg of albumin was added routinely. The data show furthermore that too high concentrations of protein interfere with the assay; fluorescence was lower by 23% in the presence of 1000 pg of albumin if compared to optimal protein concentrations. It was observed that DNA added as internal standards to the DNA assay of a liver homogenate gave more than 90% of the expected fluorescence (Table 2). From this finding we concluded that the amount of albumin added routinely assured complete precipitation of extracted DNA without interfering with the fluorescence assay. Fifty sequential DNA measurements were performed on hepatocyte cultures, as outlined under Materials and Methods. The assays were run in duplicate. The deviation from the mean of duplicates averaged 5.8% in this series, the DNA content ranging from 4 to 86 /Lg of DNA per dish. DNA content was found to amount to 76.2 2 6.6 pg per 2.5 x IO6 rat hepatocytes (X & SEM; n = 6) isolated according to Bischoff et al. (5) with collagenase perfusion of the liver, if proteinase digestion preceded DNA determination. As, on the other hand, we estimated 1 g of wet isolated hepatocytes to contain 1.15 x lOa cells in agreement with others (6) alevelof(76.2pg x 1.15 x 10*)/(2.5 x 106) = 3.51mgofDN~Vgwetweight of liver may be calculated from our data. This is considerably higher than values reported by earlier investigators who reported DNA to range from 2.05 (7) to 2.75 (8) mg/g wet weight. Even if one takes into account that about 15% of the intact rat liver volume consists of cells other than hepatocytes (9). which makes comparison between
3ooq
01
’ 0
200
1 400
8 800
600 Albumin
I 1000
[pg]
Ftc. 3. Influence of varying amounts of albumin added at the precipitation step after proteolytic extraction. Hepatocyte suspension, 2.5 ml, containing IO6 cells/ml, was seeded in petri dishes as outlined in Fig. 2. For proteolysis see Routine Procedure in Materials and Methods. Duplicates of 0.2- or 0.5-ml aliquots were then transferred to Eppendorf vials and DNA was precipitated and assayed (as outlined under Materials and Methods) after addition of varying amounts of albumin. Albumin. by itself, proved not to give any fluorescence above that of blanks in the DNA assay.
172
BARTH AND WILLERSHAUSEN TABLE RECOVERY
OF FLUORESCENCE ADDED
2
CAUSED
BY DNA
STANDARD
TO A LIVER HOMOGENATE”
Net fluorescenceb Liver
extract +
Assay
Liver extract (4
DNA standard (B)
DNA standard (Cl
Difference (B - A)
1 2 3 4 5 6
125 100 180 125 115 -
240 195 285 225 225 -
115
115 95 105 100 110 -
x
Recovery (%) (B - A)/C 100 83 91 87 96 91.4
u Rat livers were homogenized so that the wet weight of tissue was diluted 500-fold. The homogenates were treated with proteinase K as outlined under Materials and Methods. Aliquots of 0.5 ml were then taken to DNA assay with (B) or without (A) addition of 5 pg of calf thymus DNA. The routine procedure was followed. (r Arbitrary units.
isolated parenchymal cells and intact liver questionable, this difference between results of earlier investigators and our results is most easily explained by a better extraction of DNA after proteolysis. In fact, direct determination on total liver tissue resulted in a DNA content of 4.7 + 0.27 mg/g wet weight of rat liver in our hands (X + SD; n = 6). The finding that DNA content of total liver is 34% higher than that of isolated hepatocytes (4.7 vs 3.51 mg) can be explained by the fact that Kupffer and other sinusoidal cells contain about 10 times more DNA per gram wet weight than hepatocytes (13). For the purpose of comparison we attempted to determine DNA content in the total liver without prior proteolysis as well. However no reliable results could be calculated because of differing parallels; values obtained from the same sample ranged between 3.3 and 4.06 mg of DNA/g wet weight of liver. DISCUSSION
The data presented herein strongly suggest that a more complete extraction of DNA from tissue and/or a better separation from interfering tissue constituents is achieved if proteinase K digestion is used. This leads to definitively higher values of DNA content in rat liver tissue. Higher contents of DNA in different tissues due to proteolytic extraction have been observed recently by other investigators as well (10). More-
DNA
ASSAY
FOLLOWING
PROTEOLYSIS
173
over, more consistent results are obtained as judged from conforming parallels and the increase in fluorescence with increasing sample size. The higher content of DNA observed cannot be due to the nonspecificity of the method used because in accordance with earlier investigators (12) we have found that a 60-min incubation of liver homogenates with 100 pg of DNase I results in blank fluorescence. For a complete precipitation of extracted DNA, addition of albumin is recommended; 250 pg of albumin added before precipitation proved to be optimal under our conditions. One has to keep in mind that too high amounts of albumin lower the fluorescence as shown in Fig. 3. This points to the importance of removing protein by the proposed extraction procedure for the improvement of the assay. Finally, a modified procedure to purify DABA to a satisfying degree based on simple charcoal treatment and pH change is proposed. The complicated and expensive filtration through 0.65~pm pores proposed in an earlier paper (4) proved to be unnecessary in this procedure. Moreover, the method described is considerably less expensive because the less pure starting material can be obtained at a lower price. ACKNOWLEDGMENTS The expert technical assistance of Mrs. 1. Lend1 is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft through the Forschergruppe Ernahrung (Zo 7121). We would like to thank Dr. Igo-Kemenes. Institut fur Physiologische Chemie. University of Munich, for helpful suggestions and Dr. Afting. Biochemisches Institut. Universitat Freiburg, who acquainted us with the fluorometric assay of Kissane and Robins.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13.
Kissane, J. M.. and Robins, E. (1958) J. Bid/. Chem. 233, 184- 188. Wiegers, U., and Hilz, H. (1971) Biochem. Biophys. Res. Commun. 44, 513-519. Gross-Belard, M., Oudet, P.. and Chambon. P. (1973) Eur. J. B&hem. 36, 32-38. Hinegardner. R. T. (1971)Anal. Biwhrm. 39, 197-201. Bischoff, E.. Wilkening, J.. Tran-Thi. T., and Decker, K. (1976) Eur. J. Biochem. 62, 279-283. Zahlten, R. N., and Stratman, F. W. (1974) Arch. Biwhem. Biuphys. 163, 600-608. Leslie, I. (1955) in The Nucleic Acids (Chargaff. E., and Davidson, J. N.. eds.). Vol. 2, p. 8, Academic Press. New York. Ceriotti, G. (1955) J. Bid. Chem. 214, 59-70. Wilson, M. E.. Stowell. R. E., Yokoyama. H. 0.. and Tsuboi, K. K. (1953) Cancer Res. 13, 86-92. Kasche, V.. Amnens, H.. Naslund, L.. and Zollner, R., (1977) Z. Physiol. Chrm. 358, 256 (abstract). Elsdale. T., and Bard, J. (1972) J. Ceil Bid. 54, 626-637. Klevecz. R. R., Kapp, L. N.. and Remington, J. A. (1975) in Control of Proliferation in Animal Cells (Clarkson B.. and Baserga. R., eds.) pp. 817-831, Cold Spring Harbor, New York. Decker. K.. Hofmann. F.. Kreusch, J., Maier, K. P.. Wunder. P. G.. and Wagle. S. R. (1970) in Kupffer Cells and Other Sinusoidal Cells of Liver (Wisse. E.. and Knook. D. L.. eds.) pp. 315-324, Elsevier. North Holland Biomedical Press, Amsterdam.
BIOCHEMISTRY
Improvement
9,
167- 173 (1978)
of DNA Quantitation Proteolytic Extraction
following
C. A. BARTH’ AND B. S. WILLERSHAUSEN Forschrrgruppe
Erniihrung
un der Meditinischen Miinchen, Germany
Poliklinik,
Universitiit
Miinchen.
ReceivedOctober 14. 1977 A reliableand sensitive method for quantitation of deoxyribonucleic acid (DNA) is described herein. It is based on a proteinase K digestion step followed by a fiuorometric assay with 3,Sdiaminobenzoic acid (Kissane and Robins, 1958). The method proved to be applicable to fresh liver and to hepatocytes either in suspension or in culture when attached to a collagen substratum. A linear relationship between biological sample size and fluorometric response was observed only if the assay was preceded by proteolytic extraction. Moreover, the method resulted in DNA values per fresh liver weight that were about 100% higher than values reported earlier. The data show that proteolysis results in a more exhaustive extraction of DNA from tissues and a better separation of extracted DNA from other tissue constituents interfering with the assay.
Chemical and metabolic properties of cells or tissues are commonly expressed in terms of cell number, weight of specimen, or total protein content. Sometimes the DNA content is used as well because it is closely correlated to cell number. Furthermore it has the advantage of being very sensitive if suitable methods are applied. In the course of studies on the metabolic performance of hepatocytes attached to a collagen substratum we tried to determine DNA according to Kissane and Robins (1). In this procedure a fluorescent product is formed after reacting the deoxyribose moiety with 3,5-diaminobenzoic acid dihydrochloride (DABA).z We observed this sensitive method to give excellent results if applied to pure standards but to be of poor reliability in the case of biological samples. We thought that this might be mainly due to an incomplete separation of DNA from interfering cell constituents prior to reaction with DABA. In the following we report on an improvement of the assay by taking advantage of the proteinase K digestion method developed recently (2,3) to extract high molecular weight nucleic acids from biological specimens. I Postal address: Medizinische Poliklinik, Pettenkoferstr. Sa, D-8000 Germany. * Abbreviation used: DABA. 3,Sdiaminobenzoic acid dihydrochloride. 167
Miinchen,
0003-2697/78/0901-0167$02.00/O Copyright Q 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
168
BARTH
AND
WILLERSHAUSEN
Furthermore an inexpensive and easy procedure will be described yielding DABA of high purity suitable for fluorometry. MATERIALS
AND METHODS
Bovine serum albumin “reinst” (Catalog No. RHD 20) was bought from Behring-Hochst, Frankfurt-Main, West Germany. In some cases DABA from EGA Chemie, Steinheim/Albuch, West Germany, (Catalog No. 11,382-2) was used following purification according to Hinegardner (4). Generally, 3,5-diaminobenzoic acid (99%) (Catalog No. l-4943) was purchased from Roth, Karlsruhe, West Germany, and purified as described below. Calf thymus DNA from Sigma (Catalog No. D- 1501) dissolved in 1 N NHIOH was used as standard. Proteinase K was from Merck, Darmstadt, West Germany. All other chemicals were of reagent grade and bought from the same manufacturer. Male Sprague-Dawley rats of 150 g body weight were used throughout. 3,5Diaminobenzoic acid was purified by dissolving 4 g in 15 ml of 3.6 N HCl. The solution was stirred at about 60°C for 1 h after addition of 2.25 g of acid-washed charcoal. The charcoal was removed by filtration on a Buchner funnel (Paper No. 595, Schleicher and Schuell). The dihydrochloride derivative was precipitated by addition of an equal volume of concentrated HCl. The precipitate was separated from the solvent by filtration on a Buchner funnel. As the material generally had a yellowish color the charcoal, filtration, and precipitation steps were repeated after dissolving the material in water. The material then crystallized regularly as white needles which were dried over KOH in vucuo. Recovery was about 45%. Proton nmr of the purified material gave the expected signals (‘H nmr in water, r scale: 2.1, 2.4). In the DNA assay, reagent blanks with the freshly purified material gave a fluorescence which was lower than 5% of the fluorescence given by a standard containing 10 pg of DNA. Fluorometry was performed with an Eppendorf fluorometer Model 1130 attached to a photometer Model 1101. The primary filter was set at 436 nm, the secondary filter at 4703000 nm. Routine procedure. Hepatocyte cultures in petri dishes (60 mm) were washed three times with 4 ml of saline. Removal of dead cells and cell debris was enhanced by tapping the dish on the table. Four milliliters of a 500 mM Tris-HCl buffer, pH 8.0, containing 6 M urea, 10 mM EDTA and 10 mM NaCl was added to each dish. Proteinase K freshly dissolved in the above buffer was added to give a final concentration of 50 @/ml. After overnight incubation at 37°C the contents were transferred to a 14ml centrifuge tube and shaken vigorously on a Vortex-type mixer. It is critical that any cloudy material still present disappears at this moment. Total volume was measured and an aliquot of 0.5 ml was transferred to a 1.5-ml
DNA
ASSAY
FOLLOWING
PROTEOLYSIS
169
Eppendorf vial. Fifty microliters of bovine serum albumin (5 mg/ml) was added, and the DNA was precipitated by addition of 0.5 ml of 2 N HCl in 15% CCl,COOH. Albumin served to assure a complete precipitation of DNA at this and the following wash steps. The effect of albumin at this step will be discussed in detail later in this paper. Subsequently the original procedure given by Kissane and Robins (1) was followed essentially for washing the precipitate with CCl,COOH and ethanol. HCl, 1 N, was added before fluorometry according to Hinegardner (4). RESULTS
Figure 1 displays fluorometer readings after reacting different amounts of pure DNA or varying aliquots of a hepatocyte suspension with DABA, according to the original procedure of Kissane and Robins (1). There was an excellent linear response of fluorescence to rising amounts of pure DNA (Fig. 1A). But, deviation from linearity was observed in the case of biological samples (Fig. 1B) and could not be prevented even if samples were pretreated by such procedures as freezing and thawing or alkaline digestion in order to enhance DNA extraction from cells. Figure 2 shows that this problem was circumvented when proteolysis preceded the DNA assay; a linear fluorometer reading with sample size
0
0
5
10 [x10-4cekl
FIG. 1. Assay of DNA with purified calf thymus DNA (A) and isolated rat hepatocytes (B). (A) Fluorometer readings after reacting purified DNA with DABA, as outlined under Materials and Methods. Ordinate: Fluorescence in arbitrary units. Abscissa: Varying amounts of calf thymus DNA. (B) DNA determination in freshly isolated rat hepacytes (A, n ), hepatocytes suspended in 1 N NaOH (0). and hepatocytes frozen and thawed four times (0). For further details see (A).
170
BARTH
AND
WILLERSHAUSEN
1
0
2
[x lo-6cells]
FIG. 2. Assay of DNA in hepatocytes following proteolytic extraction. Freshly isolated hepatocytes were seeded on collagen gels, prepared according to Ref( 11) in petri dishes of60mm diameter. For proteolysis see Routine Procedure, except that no 0.5ml aliquot was taken out after proteolysis. Instead, the total material digested by proteinase was taken to DNA assay, the latter being scaled up correspondingly.
was observed. Table 1 shows results confirming this finding. Moreover, it is demonstrated that the results of four parallels agree perfectly well under the conditions chosen and that proteolysis by 200 pg of enzyme was sufficient to assure a satisfying extraction of DNA. It was observed that addition of 250 pg of albumin at the precipitation step, after proteolysis of a hepatocyte culture, raised the net fluorescence by 43%. A systematic examination revealed that DNA precipitation was TABLE INFLUENCE
OF DIFFERENT
1
AMOUNTS OF PROTEINASE IN HEPATOCYTE MONOLAYERS”
Net Proteinase
K
ON
DNA
EXTRACTION
fluorescence*
K added (f%)
0.2-ml
Sample
OS-ml
Sample
200
210 235
550 570
300
220 240
560 520
u Freshly isolated rat hepatocytes, 2.5 coated with a collagen gel (11). Subsequently Procedure with 200 or 300 pg of proteinase were analyzed for DNA. b Arbitrary units.
x
106. were seeded in each of four petri dishes they were treated as outlined under Routine K and two different aliquots of 0.2 and 0.5 ml
DNA
ASSAY
FOLLOWING
171
PROTEOLYSIS
optimal if 200-300 ,ug of albumin was added (Fig. 3); therefore 250 pg of albumin was added routinely. The data show furthermore that too high concentrations of protein interfere with the assay; fluorescence was lower by 23% in the presence of 1000 pg of albumin if compared to optimal protein concentrations. It was observed that DNA added as internal standards to the DNA assay of a liver homogenate gave more than 90% of the expected fluorescence (Table 2). From this finding we concluded that the amount of albumin added routinely assured complete precipitation of extracted DNA without interfering with the fluorescence assay. Fifty sequential DNA measurements were performed on hepatocyte cultures, as outlined under Materials and Methods. The assays were run in duplicate. The deviation from the mean of duplicates averaged 5.8% in this series, the DNA content ranging from 4 to 86 /Lg of DNA per dish. DNA content was found to amount to 76.2 2 6.6 pg per 2.5 x IO6 rat hepatocytes (X & SEM; n = 6) isolated according to Bischoff et al. (5) with collagenase perfusion of the liver, if proteinase digestion preceded DNA determination. As, on the other hand, we estimated 1 g of wet isolated hepatocytes to contain 1.15 x lOa cells in agreement with others (6) alevelof(76.2pg x 1.15 x 10*)/(2.5 x 106) = 3.51mgofDN~Vgwetweight of liver may be calculated from our data. This is considerably higher than values reported by earlier investigators who reported DNA to range from 2.05 (7) to 2.75 (8) mg/g wet weight. Even if one takes into account that about 15% of the intact rat liver volume consists of cells other than hepatocytes (9). which makes comparison between
3ooq
01
’ 0
200
1 400
8 800
600 Albumin
I 1000
[pg]
Ftc. 3. Influence of varying amounts of albumin added at the precipitation step after proteolytic extraction. Hepatocyte suspension, 2.5 ml, containing IO6 cells/ml, was seeded in petri dishes as outlined in Fig. 2. For proteolysis see Routine Procedure in Materials and Methods. Duplicates of 0.2- or 0.5-ml aliquots were then transferred to Eppendorf vials and DNA was precipitated and assayed (as outlined under Materials and Methods) after addition of varying amounts of albumin. Albumin. by itself, proved not to give any fluorescence above that of blanks in the DNA assay.
172
BARTH AND WILLERSHAUSEN TABLE RECOVERY
OF FLUORESCENCE ADDED
2
CAUSED
BY DNA
STANDARD
TO A LIVER HOMOGENATE”
Net fluorescenceb Liver
extract +
Assay
Liver extract (4
DNA standard (B)
DNA standard (Cl
Difference (B - A)
1 2 3 4 5 6
125 100 180 125 115 -
240 195 285 225 225 -
115
115 95 105 100 110 -
x
Recovery (%) (B - A)/C 100 83 91 87 96 91.4
u Rat livers were homogenized so that the wet weight of tissue was diluted 500-fold. The homogenates were treated with proteinase K as outlined under Materials and Methods. Aliquots of 0.5 ml were then taken to DNA assay with (B) or without (A) addition of 5 pg of calf thymus DNA. The routine procedure was followed. (r Arbitrary units.
isolated parenchymal cells and intact liver questionable, this difference between results of earlier investigators and our results is most easily explained by a better extraction of DNA after proteolysis. In fact, direct determination on total liver tissue resulted in a DNA content of 4.7 + 0.27 mg/g wet weight of rat liver in our hands (X + SD; n = 6). The finding that DNA content of total liver is 34% higher than that of isolated hepatocytes (4.7 vs 3.51 mg) can be explained by the fact that Kupffer and other sinusoidal cells contain about 10 times more DNA per gram wet weight than hepatocytes (13). For the purpose of comparison we attempted to determine DNA content in the total liver without prior proteolysis as well. However no reliable results could be calculated because of differing parallels; values obtained from the same sample ranged between 3.3 and 4.06 mg of DNA/g wet weight of liver. DISCUSSION
The data presented herein strongly suggest that a more complete extraction of DNA from tissue and/or a better separation from interfering tissue constituents is achieved if proteinase K digestion is used. This leads to definitively higher values of DNA content in rat liver tissue. Higher contents of DNA in different tissues due to proteolytic extraction have been observed recently by other investigators as well (10). More-
DNA
ASSAY
FOLLOWING
PROTEOLYSIS
173
over, more consistent results are obtained as judged from conforming parallels and the increase in fluorescence with increasing sample size. The higher content of DNA observed cannot be due to the nonspecificity of the method used because in accordance with earlier investigators (12) we have found that a 60-min incubation of liver homogenates with 100 pg of DNase I results in blank fluorescence. For a complete precipitation of extracted DNA, addition of albumin is recommended; 250 pg of albumin added before precipitation proved to be optimal under our conditions. One has to keep in mind that too high amounts of albumin lower the fluorescence as shown in Fig. 3. This points to the importance of removing protein by the proposed extraction procedure for the improvement of the assay. Finally, a modified procedure to purify DABA to a satisfying degree based on simple charcoal treatment and pH change is proposed. The complicated and expensive filtration through 0.65~pm pores proposed in an earlier paper (4) proved to be unnecessary in this procedure. Moreover, the method described is considerably less expensive because the less pure starting material can be obtained at a lower price. ACKNOWLEDGMENTS The expert technical assistance of Mrs. 1. Lend1 is gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft through the Forschergruppe Ernahrung (Zo 7121). We would like to thank Dr. Igo-Kemenes. Institut fur Physiologische Chemie. University of Munich, for helpful suggestions and Dr. Afting. Biochemisches Institut. Universitat Freiburg, who acquainted us with the fluorometric assay of Kissane and Robins.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13.
Kissane, J. M.. and Robins, E. (1958) J. Bid/. Chem. 233, 184- 188. Wiegers, U., and Hilz, H. (1971) Biochem. Biophys. Res. Commun. 44, 513-519. Gross-Belard, M., Oudet, P.. and Chambon. P. (1973) Eur. J. B&hem. 36, 32-38. Hinegardner. R. T. (1971)Anal. Biwhrm. 39, 197-201. Bischoff, E.. Wilkening, J.. Tran-Thi. T., and Decker, K. (1976) Eur. J. Biochem. 62, 279-283. Zahlten, R. N., and Stratman, F. W. (1974) Arch. Biwhem. Biuphys. 163, 600-608. Leslie, I. (1955) in The Nucleic Acids (Chargaff. E., and Davidson, J. N.. eds.). Vol. 2, p. 8, Academic Press. New York. Ceriotti, G. (1955) J. Bid. Chem. 214, 59-70. Wilson, M. E.. Stowell. R. E., Yokoyama. H. 0.. and Tsuboi, K. K. (1953) Cancer Res. 13, 86-92. Kasche, V.. Amnens, H.. Naslund, L.. and Zollner, R., (1977) Z. Physiol. Chrm. 358, 256 (abstract). Elsdale. T., and Bard, J. (1972) J. Ceil Bid. 54, 626-637. Klevecz. R. R., Kapp, L. N.. and Remington, J. A. (1975) in Control of Proliferation in Animal Cells (Clarkson B.. and Baserga. R., eds.) pp. 817-831, Cold Spring Harbor, New York. Decker. K.. Hofmann. F.. Kreusch, J., Maier, K. P.. Wunder. P. G.. and Wagle. S. R. (1970) in Kupffer Cells and Other Sinusoidal Cells of Liver (Wisse. E.. and Knook. D. L.. eds.) pp. 315-324, Elsevier. North Holland Biomedical Press, Amsterdam.
