[98] Determination of nucleic acids by phosphorus analysis

[98] D E T E R M I N A T I O N

OF N U C L E I C

ACIDS B Y P H O S P H O R U S

ANALYSIS

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[98] Determination of Nucleic Acids by Phosphorus Analysis By

GERHARD SCHMIDT

The methods for the determination of nucleic acids by phosphorus analysis were devised at a period preceding the development of the much more specific spectrophotometric and chromatographic procedures for nucleic acid analysis in tissues. The subsequent comparison of the results obtained by phosphorus analysis with the more recent techniques has shown that particularly the ribonucleic acid values obtained from some tissues include contaminating phosphorus compounds whose elimination is necessary, particularly in incorporation experiments with p32. Little reliable information regarding the nature or the origin of these contaminants is as yet available, apart from the observation that they are of low molecular weight. Consequently the only essential modifications of the original technique which have been developed for the purpose of avoiding this error consist in steps aiming at a preliminary isolation of the nucleic acids prior to the hydrolytic degradation procedures necessary for the analysis. The application of procedures including such steps is now generally considered as necessary for turnover experiments with p~2. The yield in isolation of the nucleic acid is at best 70%. These introductory remarks show that the procedures for nucleic acid determination in tissues by phosphorus analysis cannot be considered as routine techniques, but that the extent of contamination must be carefully evaluated in their application. A more detailed discussion of this problem will be given after the description of those procedures in which the isolation of the nucleic acid fraction is omitted. I. Determination of Total Nucleic Acids According to Berenblum, Chain, and Heatley 1

Principle. The phosphorus fraction of animal tissues remaining after complete extraction of the lipids and of the acid-soluble phosphorus compounds consists--according to present information--of the sum of nucleic acid phosphorus and phosphoprotein phosphorus. Since the amounts of the phosphoproteins in most tissues except eggs, mammary gland, and milk are very small in comparison to those of the nucleic acid phosphorus, the amounts of phosphorus obtained after extraction of the lipids and of the acid-soluble phosphorus compounds represent a good approximation 1 I. Berenblum, E. Chain, and N. G. Heatley, Biochem. J. 33, 68 (1939).

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of the concentration of nucleic acid phosphorus in m a n y animal tissues except for the materials listed above and for nervous tissue.

II. Partition of the Phosphorus Compounds Obtained According to the Berenblum-Chain-Heatley 1 Procedure into DNA, RNA, and Phosphoproteins According to Schmidt and Thannhauseff Principle. T h e mixture of the three fractions obtained b y the Berenblum-Chain-Heatley procedure can be quantitatively partitioned into its components b y selective alkaline hydrolysis. During a 16-hour incubation at room t e m p e r a t u r e in N sodium or potassium hydroxide, D N A remains still acid-insoluble, R N A is quantitatively transformed to acid-soluble nucleotides without a n y formation of inorganic phosphate, 3-6 and the phosphorus of the phosphoproteins is quantitatively converted to inorganic phosphate. 6

Removal of Phospholipids and of Acid-Soluble Phosphorus Compounds Modified Technique of Berenblum et al.1 Amounts of tissues, usually

of 1 to 2 g., are homogenized in 10 ml. of alcohol-ether mixture (3 : 1, v / v ) . The suspension is q u a n t i t a t i v e l y transferred to a 200-ml. Erlenmeyer flask with an additional 15 ml. of the alcohol-ether mixture and boiled on the water bath for a few minutes. After cooling, the suspension is filtered on a Bfichner funnel over W h a t m a n No. 1 paper which had been covered with a thin layer of Hyflo Filter-Aid (Johns-Manville) and washed several times with ether. T h e powdery filter cake is transferred to a small roundb o t t o m e d flask and refluxed with 30 ml. bf a mixture of equal volumes of chloroform and methanol. The lipid-free residue is filtered in a similar manner as described above. The a m o u n t of Filter-Aid should be kept at a minimum compatible with the quantitative removal of the filter cake from the paper. The dry precipitate is transferred quantitatively to a 50-ml. centrifuge tube preferably equipped with a ground-glass stopper and suspended in 15 ml. of approximately 0.1 N ice-cold glycine-perchloric acid buffer of p H 2.5. The suspension is mechanically shaken in a cold room for 20 minutes, centrifuged, 7 the s u p e r n a t a n t is discarded, and the residue is resus2 G. Schmidt and S. J. Thannhauser, J. Biol. Chem. 161, 83 (1945). 8 G. Schmidt, R. Cubiles, N. Z611ner, L. Heeht, N. Strickler, K. Seraidarian, M. Seraidarian, and S. J. Thannhauser, J. Biol. Chem. 192, 715 (1951). 4 K. C. Smith and F. W. Allen, J. Am. Chem. ,.%c. 75, 213 (1953). 5 G. de Lamirande, C. Allard, and A. Cantero, J. Biol. Chem. 214, 519 (1955). e A. M. Crestfield, K. C. Smith, and F. W. Allen, J. Biol. Chem. 216, 185 (1955). TThe supernatant solution occasionally contains floating particles resisting centrifugation. They are collected by filtration on a small Btichner funnel on Whatman No. 50 (hardened) filter paper and combined with the sediment.

[98] DETERMINATION OF NUCLEIC ACIDS BY PHOSPHORUS ANALYSIS 673 °

pended in 15 ml. of the acid buffer. Six such extractions are carried out. The last supernatant solution must be free of phosphorus after ashing. The last residue is suspended in 10 ml. of N potassium hydroxide and treated as described below. The extraction procedure just described has the advantage of permitting lipid-phosphorus determinations and nucleic acid determinations in the same sample. It also has the advantage of avoiding alterations of the solubility of phospholipids--a factor of some importance in experiments with nervous tissue. On the other hand, the protracted extraction with the acid buffer involves the danger of an appreciable degree of acid hydrolysis of the purine-deoxyriboside bonds of DNA. Technique of Schmidt and Thannhauser. ~ The danger just described is largely avoided in the method of Schmidt and Thannhauser, who extract the acid-soluble phosphorus compounds with trichloroacetic acid prior to the extraction of the phospholipids. Amounts of tissue up to 2 g. are homogenized under cooling in ice water and mixed immediately with several volumes of 10% trichloroacetic acid. After 10 minutes of standing in the cold, the suspension is filtered over a thin layer of Hyflo Filter-Aid (Johns-Manville Co.). If the presence of Filter-Aid interferes with the purposes of the experiment, the extractions and washings of the tissues may also be carried out on the centrifuge. The filtered material is washed extensively with at least twelve portions of 10% and later of 5% trichloroacetic acid. The last washing is free of acid-soluble phosphates. The residue is quantitatively transferred from the filter to a beaker and suspended in 95 % alcohol. (It is not advisable to leave the precipitate on the filter for the first alcohol washings, since the replacement of TCA by alcohol causes swelling of the particles of the residue and consequently clogging of the filter pores.) The suspension is filtered over a thin layer of Hyflo Filter-Aid and washed several times with alcohol and ether. The powder is refluxed for 10 minutes in 100 ml. of an ethanolether mixture (3 : 1, v/v), filtered, and refluxed for 2 hours with a mixture of chloroform and methanol (1:1, v/v). The extracted tissue powder is filtered, washed with ether, and dried in a vacuum desiccator until the ether is removed. The powder is suspended in 5 ml. of N potassium hydroxide 8 per gram of moist tissue (0.3 N potassium hydroxide, as 8 Although the concentration of N potassium hydroxide is sufficient to transform RNA-phosphorus completely to acid-soluble phosphorus compounds, de Lamirande et al. ~ found that 1.5 N potassium hydroxide is required for its complete conversion to mononucleotides, at least under the conditions of tissue analysis when the presence of considerable quantities of proteins exerts a strong buffer effect. Hydrolysis of the tissue nucleic acid fraction with 0.3 N potassium hydroxide yielded only

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suggested b y D a v i d s o n et al., 9,~° is p r o b a b l y preferable b u t has not been tested so far b y the author) and is slowly shaken a t room t e m p e r a t u r e for 16 hours. Under these conditions, the tissue powder is practically completely dissolved, and the small deposit at the end of the hydrolysis consists only of the Filter-Aid. 11 T h e s u p e r n a t a n t liquid is centrifuged off, and an aliquot is set aside for the determination of the total phosphorus (sample A). Another aliquot is used for the determination of the ultraviolet absorption at 260 mu. F o r the partition of the phosphorus into D N A , R N A , and phosphoprotein phosphorus, a 5-ml. aliquot is acidified to p H 1 b y the addition of a measured volume of a 15% solution of perchloric acid. T h e copious precipitate is centrifuged off and contains q u a n t i t a t i v e l y the deoxyribonucleic acid and a large a m o u n t of protein degradation products. T h e s u p e r n a t a n t , which contains q u a n t i t a t i v e l y the ribonucleotides and the inorganic p h o s p h a t e formed from the phosphoproteins, is set aside. T h e precipitate is suspended in a small a m o u n t of w a t e r and dissolved b y the addition of the necessary a m o u n t of N p o t a s s i u m hydroxide. I t is reprecipitated b y adding a sufficient volume of a mixture of trichloroacetic acid and hydrochloric acid to bring the final concentration of free hydrochloric acid to a p p r o x i m a t e l y 0.2 N. T h e precipitate is centrifuged and washed two times with a mixture of 5 % trichloroacetic acid and 0.2 N hydrochloric acid. I n a suitable aliquot of the s u p e r n a t a n t solution (without the washings) the total phosphorus is determined according to the m e t h o d of Fiske and S u b b a R o w (sample B). A n o t h e r aliquot of the s u p e r n a t a n t solution (exclusive of the wash70% of the amount of mononucleotides as compared to the yield observed with 1.5 N potassium hydroxide. Smith and Allen 4,8 similarly reported that, after hydrolysis of 200 mg. of yeast RNA with 5 ml. of N sodium hydroxide at room temperature for 24 hours, approximately 3% of the total nucleotides were present in the form of oligonucleotides. Although the presence of acid-soluble oligonucleotides does not affect the results of RNA-phosphorus determinations, complete conversion to mononucleotides is, of course, essential for experiments designed to check the result of phosphorus determination by chromatographic separation of the nucleotides. Incomplete conversion of tissue RNA to mononucleotides might be partially responsible for some discrepancies 8,9 observed by several authors between the RNA-phosphorus values and the yield of mononucleotides. J. N. Davidson and R. N. Smellie, Biochem. J. 52, 594 (1952). l0 I. Leslie, in "The Nucleic Acids" (E. Chargaff and J. N. Davidson, eds.), Vol. 2, p. 1. Academic Press, New York, 1955. 11 When the procedure is carried out as described in this chapter, the silicates of the Filter-Aid do not interfere with the colorimetrie phosphorus determinations, although small amounts of inert silicates are extracted by the potassium hydroxide and appear as insoluble granules during the ashing. Contact of alkali-treated FilterAid with acid, however, must be carefully avoided. For this reason, the alkaline hydrolyzate must be centrifuged prior to acidification.

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ings) is neutralized with potassium hydroxide and stirred with 1 to 3 g. of Amberlite cation exchanger IR-100H (analytical grade). By this treatment, peptones are adsorbed which would interfere with the phosphorus determination by the formation of precipitates on addition of ammonium molybdate. The ion exchanger is centrifuged, washed with water, the combined filtrate and washings are made up to a measured volume with water, and the inorganic phosphorus is determined in the combined filtrate and washings according to Fiske and SubbaRow (sample C). When samples A, B, and C represent the values per 100 g. of tissue, the difference A - B represents the concentration of DNA-phosphorus, B - C that of RNA-phosphorus, and C that of the phosphoprotein phosphorus.

Limits of Application of the Determination of Nucleic Acids by Phosphorus Analysis The procedure described gave very satisfactory recoveries of DNA, RNA, casein, or ovovitellin, when mixtures of these substances were added to tissue homogenates. Obviously, however, the accuracy of the figures greatly depends on the mutual proportions, particularly between DNA and RNA. (Since the phosphoprotein phosphorus is based on determinations of inorganic P, this fraction can be determined with a fair degree of accuracy even in the presence of large amounts of nucleic acids.) The proportion between DNA and RNA varies within very wide ranges from very small values (for example, in the unfertilized arbacia egg, where this ratio is extremely small, and in pancreas, where it is approximately 0.1) to the high values in thymus or in mature sperm cells or in isolated chromosomes. Since the procedure is essentially a differential method, the accuracy of the values of the two components is of similar degrees only when both their concentrations are within similar ranges. A more serious shortcoming of the procedure is the occurrence in some tissues of unknown phosphorus-containing substances of nonnucleotide nature which are not completely removed by the extraction of the acid-soluble and the lipid-phosphorus compounds. These phosphorus compounds appear in the R N A fraction, and their presence may result in RNA-phosphorus figures which are too high. Davidson and Smellie 9.1° tested this possibility by paper electrophoresis of the ribonucleotides of the alkaline hydrolyzate of liver after injection of P32-phosphate. Comparison of the spots visualized by ultraviolet absorption with radioautographs of the same hydrolyzate revealed spots showing radioactivity without ultraviolet absorption. These authors estimate that the RNA-

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phosphorus values obtained with the Schmidt-Thannhauser procedure on rat liver are approximately 25% too high. On the basis of a quantitative comparison between the RNA-phosphorus figures and the ultraviolet absorption of brain hydrolyzates, Folch,12 as well as Rossiter and collaborators, 13.14came to the conclusion that the discrepancies are much higher in analyses of nervous tissues. It is difficult to decide at present to what extent the presence of these contaminations is due to incomplete removal of acid-soluble and lipidbound phosphorus from the nucleic acid fraction or to the actual occurrence of unknown phosphorus compounds linked to protein. The difficulty created by the incomplete extraction--even of inorganic phosphate-from tissue is known to many investigators studying the incorporation of labeled--inorganic or organic--phosphorus into the protein fraction of homogenates. Davidson and Smellie 9,1° have convincingly demonstrated contamination of the R N A fraction with inorganic p32 added in the form of phosphate simultaneously with trichloroacetic acid to liver homogenates. The author has experienced a similar difficulty in incorporation studies with the protein fraction of mammary gland homogenates. On the other hand, the almost negligible amounts of inorganic phosphorus found in the R N A fraction of all tissues except those containing appreciable amounts of phosphoproteins render it unlikely that incompleteness of the extraction of acid-soluble phosphorus compounds could cause appreciable errors in the chemical determination of RNA, although it interferes with measurements of the specific radioactivity. Of course, the possibility is not excluded that certain acid-soluble phosphoric acid esters might remain adsorbed to the protein fraction to a higher degree than does inorganic phosphate. For the serious discrepancies between ultraviolet extinction and phosphorus~Concentration encountered in the analysis of nerve tissue, Folch and Le Baron 1~ advanced the explanation that certain phospholipids, in particular phosphoinositides, are not completely extracted by the lipid solvents used. The presence of unknown phosphorus components of proteins must likewise be considered as a source of error in nucleic acid determinations. So far, however, no phosphorus-containing constituents of proteins except phosphoric acid esters of hydroxyamino acids have been reported. In regard to Davidson and Smellie's experiments in which the phosphorus figures obtained by direct determination were compared with those calculated from the ultraviolet extinctions of the nucleotide spots 1~ j. Folch and F. N. Le Baron, Federation Proc. 10, 185 (1951). 13 j . E. Logan, W. A. Nannell, and R. J. Rossiter, Biochem. J. §1, 470 (1953). ~ H. A. Deluca, R. J. Rossiter, and K. P. Strickland, Biochem. J. 55, 193 (1953).

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on paper electrophoresis strips, the question might also be raised whether some nucleotide losses might not have occurred during the 18-hour electrophoresis at pH 3.5, and whether the conversion of R N A to mononucleotides was complete, s These comments show that the interpretation of the phosphorus values should always be checked by determinations of the ultraviolet absorption (see Vol. III [106]) of the final filtrate containing the R N A nucleotides. For incorporation studies with p32, this precaution is not sufficient, however, and a preliminary isolation of the nucleic acid fraction must precede its partition by alkaline hydrolysis. The yield of the several isolation techniques with gram amounts of tissue is 70 % at best, and the conclusions regarding turnover rates reached on the basis of these procedures rest on the assumption that the behavior of the isolated portions is representative of that of the fractions lost during the procedure. III. Procedures for Incorporation Studies of p3~ in DNA and RNA of Tissues Method of E. Hammarsten, 15 as Applied by Deluca, Rossiter and Strickland 14

Extraction of Acid-Soluble Phosphorus Compounds. The tissue (amounts of 500 mg. or more) is suspended in approximately 20 vol. of 10% trichloroacetic acid solution at 0 ° and homogenized in a PotterElvehjem type of apparatus. After centrifugation, the residue is extracted seven times with 10-vol. portions of a solution of 0.04 M of acid potassium phosphate in 10% trichloroacetic acid and two times with 20 vol. of a 10 % trichloroacetic acid solution. The radioactivity of the final extracts is negligible. Extraction of Phospholipids. The residue is extracted two times in the cold with 10-vol. portions of 95% ethanol and four times with 10-vol. portions of Bloor's alcohol-ether mixture, at boiling temperature. The final residue is washed with 10 vol. of ether. Extraction and Isolation of the Nucleic Acid Fraction. The residue is suspended in 2 ml. of a 10 M aqueous solution of urea per 500 rag. of fresh tissue, and the suspension is kept at room temperature for 5 minutes. After addition of 6 vol. (of the original tissue sample) of a saturated aqueous solution of sodium chloride and ammonium sulfate, the suspension is boiled for 1 minute. The suspension is centrifuged, the supernatant liquid is set aside, and the residue is extracted twice with 4 vol. of the salt-urea mixture (150 g. of urea dissolved to 1 1. with the salt mixture) 1~ E. H a m m a r s t e n , Acta Med. ~cand. 128, Suppl. 196, 634 (1947).

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at boiling temperature. The nucleic acids are precipitated from the combined extracts by the addition of approximately 1 ml. of a saturated aqueous copper sulfate solution to 7 ml. of the extract. After standing overnight, the suspension is centrifuged and the precipitate is washed twice with a 1.8% solution of copper chloride. The copper nucleates are decomposed in the following manner: The precipitate is suspended in 0.8 ml. of a concentrated potassium acetate buffer of pH 6.4 (100 g. of potassium acetate dissolved in 100 ml. of water and titrated to pH 6.4 with glacial acetic acid). The suspension is centrifuged, and the residues are extracted three times with portions of 0.2 ml. of a 5 M urea solution. The nucleates are precipitated at - 1 5 ° with 10 ml. of 95% ethanol and a drop of a saturated solution of sodium chloride. The precipitate is redissolved in 1 ml. of water, and the solution is reprecipitated at 0 ° by the addition of 0.11 ml. of hydrochloric acid followed by addition of 10 ml. of a mixture of 9 vol.-parts of 95% ethanol and 1 vol.-part of aqueous N hydrochloric acid at - 1 5 °. After 2 hours of standing, the centrifuged precipitate is dissolved by neutralizing with 0.01 N sodium hydroxide, and the solution is repreclpitated with acid and acid alcohol as described before. This precipitate is used for the partition into DNA and RNA according to Schmidt and Thannhauser 2 (see below). M e t h o d of D a v i d s o n and S m e l l i e 9

A somewhat simpler isolation procedure was described by Davidson and Smellie. The tissue powder obtained after extraction with trichloroacetic acid and lipid solvents is extracted three times for 1 hour each time with a solution containing 10 g. of sodium chloride in 100 ml. of solution, at 100 °. The combined extracts are precipitated with 2 vol. of 95% alcohol, and the precipitate is washed with alcohol and ether and dried. Partition of the Nucleic Acid into Ribo- and Deoxyribonucleic Acids According to Schmidt and Thannhauser. The nucleic acid precipitate is dissolved in 0.3 N potassium hydroxide (Davidson and Smellie's modification of the method of Schmidt and Thannhauser, who used N alkali). One milliliter of alkali is recommended for 5 mg. of nucleic acid. The amount of alkali must be sufficient to prevent a substantial lowering of the pH by the hydrolytic liberation of acidic groups from RNA; on the other hand, an undue excess of alkali should be avoided, since it would cause unnecessary dilution of the nucleate solution. The alkaline solution is incubated for 16 hours at 30 °. During this time the degradation of ribonucleic acid to acid-soluble nucleotide is quantitative, whereas that of deoxyribonucleic acid results in the formation of products which can still be precipitated quantitatively by acidification with perchloric acid at pH 1. If the temperature does not exceed 30 °, no appreciable destruc-

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tion of the purine or pyrimidine moieties occurs, whereas at higher temperatures (37 °, as originally recommended by Schmidt and Thannhauser) considerable deamination of cytidylic acid results. After the completion of the hydrolysis, the largest part of the potassium ions is removed by dropwise addition of 10% perchloric acid until a pH between 7 and 8 is reached. The suspension of potassium perchlorate is kept for several hours in the refrigerator for the purpose of maximal precipitation and is diluted to a measured volume with a minimal amount of water. After centrifugation, an aliquot of the supernatant is brought to pH 1 by further addition of a measured volume of 1% perchloric acid. The precipitate which contains the deoxyribonucleic acid is centrifuged off after a few minutes standing. Deoxyribonucleic Acid Fraction. The precipitated deoxyribonucleic acid is dissolved in a small volume of dilute sodium hydroxide and reprecipitated with perchloric acid. For p3~ determinations, it is obviously advisable to dissolve the precipitates in solutions of nonlabelled ribonucleotides and inorganic phosphates. This purification is repeated several times. The final solution is used for phosphorus and radioactivity determinations. The absence of contaminating ribonucleotides can be checked by ionophoresis and radioautography. Remarks. The extent of purification required for specific activity determinations depends on the ratio between ribonucleic and deoxyribonucleic acids in the isolated total nucleic acid fraction. This ratio varies over a very wide range according to the analyzed tissue. It is easy to obtain sufficiently pure deoxyribonucleic acid samples from tissues like thymus or lymph glands or from sperm cells; but extensive purification is necessary in work with liver, pancreas, intestinal mucosa, and unfertilized eggs. For this reason, a detailed description of a generally valid purification procedure of the deoxyribonucleic acid fraction cannot be given. The general principle descril~ed in the preceding paragraph must be adapted to the specific requirements of the investigation. Ribonucleotide Fraction. The supernatant contains the ribomononucleotides but might still contain traces of inorganic phosphate. These can be removed by treatment of the neutralized hydrolyzate with magnesium oxide, preferably after addition of amounts of unlabeled phosphate containing 5 to 10 mg. of phosphorus. The purity of the ribonucleotide fraction can be checked by ionophoresis and radioautographyaccording to Davidson and Smellie. 9