the preparation and properties of calf liver ... · 2124 calf liver deozyribmucleoprotein vol. 238,...

THE JOURNAL cm BIOLOGICAL CHD~STRT Vol. 238, No. 6, June 1963

Printed in U.S.A.

The Preparation and Properties of Calf Liver Deoxyribonucleoprotein*

S. L. COMMERFORD,~ M. J. HUNTER,~ AND J. L. ONCLEY$

From the Department OJ Biological Chemistry, Harvard Medical School, Boston, Massachusetts

(Received for publication, December 27, 1962)

When deoxyribonucleic acid is estracted from mammalian tissues under mild conditions, it is always found to be in combination with an approximately equal amount of histone. This deoxyribonucleic acid-hi&one complex, or deoxyribonucleoprotein, was once thought to be an artifact, but more recent evidence has shown the deoxyribonucleic acid of the intact chromosome to exist as a complex with histone (l-3), suggesting that deosyribonucleoprotein may be a functional form of deoxyribonucleic acid.

Although many deoxyribonucleoprotein preparations have been reported in the literature, only a few of these have been sufficiently characterized by physical methods to permit any quantitative comparisons. These few preparations (4-l 1) have varied widely in their physical properties; for example, the reported intrinsic viscosities differ by over loo-fold. Since the method by which the DNA-histone complex was extracted from the cell also varied considerably in the above preparations much of the variation in physical properties may have been caused by the extraction procedure. Accordingly, in this study, the extraction was carried out under as mild conditions as possible and as rapidly as possible to obtain a preparation which had been subjected to minimal chemical and enzymatic stress and which would therefore closely reflect the DNA-histone interactions present in the chromosome. The tissue used as a source of deoxyribonucleoprotein (hereafter generally referred to as “the nucleoprotein”) was calf liver.

EXPERIMENTAL PROCEDURE

Hydraulic Press and Press Attachment-A Carver laboratory press (F. S. Carver, Inc., Summit, New Jersey) was used to obtain pressures of approximately 800 p.s.i. The press attachment was basically a stainless steel piston-cylinder combination with a disk perforated with many h-inch holes. The attachment was placed in the press and the tissue forced through the perforated disk.

Centl-ifugalion-A Sharples supercentrifuge (The Sharples

* This work has been supported by grants from the National Heart Institute (H-2127), the Milton Fund, Harvard University, and the American Cancer Society (through its institutional grant to Harvard University). Taken in part from a dissertation sub- mitted by Spencer L. Commerford in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Harvard University, Cambridge (1959).

t National Science Foundation, Pre-doctoral Fellow, 1955-1958. Present address, Brookhaven National Laboratories, Upton, Long Island, New York.

$ Present address, Biophysics Research Division, Institute of Science and Technology, University of Michigan, Ann Arbor, Michigan.

Corporation, Philadelphia) was used to centrifuge large volumes. The normal Sharples bowl with four blades at the bottom was satisfactory for all purposes except the sedimentation of nuclei. Since nuclei are quite susceptible to mechanical injury, a bowl without blades at the bottom was used in this special case. 9 special feed nozzle with a T-shaped channel long enough to ex- tend well into the bowl must be used with this bowl to prevent leakage.

Aborbancy-All measurements of absorbancy were performed on a Beckman model DU spectrophotometer.

pH Measurements-All pH measurements were made on a Cambridge pH meter at 25“ with a Maclnnes type electrode.

Dry weight determinations on the nucleoprotein preparations were made after dialyzing a solution containing approximately 0.1 g per dl against a large volume of distilled water for at least 18 hours at +l”. Aliquots of the dialyzed solution were placed in weighing bottles, freeze-dried, and heated in a vacuum oven at 110” to constant weight. The nucleoprotein dried in this manner remained snowy white and showed no evidence of char- ring. Exactly the same procedure was followed to obtain dry weights of aliquots of the dialysate. The dry weight of the dialysate was subtracted from the dry weight of the nucleoprotein solution.

Nitrogen Analysis-The nucleoprotein solution was digested either by the method of Kirk (12) or by the method of Grun- baum, Schaffer, and Kirk (13). Ammonia determinations were done either by the Conway diffusion technique (14) or by the use of Nessler’s reagent. Ammonium sulfate standards were run as controls with each determination. The recovery of ammonia by the Conway diffusion technique was always over 98 %. Before the nucleoprotein solutions were analyzed for nitrogen, they were dialyzed against a large volume of distilled water. The concentration of nitrogen in the dialysate was always found to be less than 0.1% of that present inside the dialysis bag.

Phosphorus determinations were done by the method of Lowry et al. (15). Disodium phosphate standards were run as controls. Before the nucleoprotein solutions were analyzed for phosphorus, they were dialyzed against distilled water. The concentration of phosphorus in the dialysate was never more than 0.1% of that inside the dialysis bag.

RNA Analysis-RNA-containing samples were heated at 90” with an equal volume of 10% trichloroacetic acid for 20 minutes, cooled, and centrifuged. RNA determinations on the clear trichloroacetic acid extract were done by the method of Ceriotti (16). Adenosine (Schwarz Laboratories, Inc.), m.p. 222-225”, was used as a primary standard.

DNA ,4naZysis-DNA-containing samples were heated at 90”

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2124 Calf Liver Deozyribmucleoprotein Vol. 238, NTo. 6

with an equal volume of 10% trichloroacetic acid for 20 minutes, cooled, and centrifuged. The supernatant was analyzed for DNA either by the method of Keck (17) or by the method of Dische (18). A commercial sample of DNA (Schwarz Labora- tories, Inc.) was used as a standard for both methods.

The nucleotide base composition of the nucleoprotein was determined by the method of Wyatt (19).

Determination of Nucleoprotein Concentration-The concentration of the nucleoprotein solutions was routinely determined by measurements of the ultraviolet absorption at 260 rnp. Ultra- violet absorption and dry weight measurements gave a value of 89 for the E:?m of the nucleoprotein. Since the absorption spectrum of the nucleoprotein is irreversibly changed when the nucleoprotein is exposed to high or low pH values, this method was not used to determine the concentrations of nucleoprotein solutions which had been exposed to pH values lower than 5 or higher than 9.

The electrophoresis of the nucleoprotein solutions was performed in a Spinco model H electrophoresis instrument at + lo in either pH 8.0, P/2 0.01 Tris-HCl buffer or in pH 8.0, I’/2 1.01 buffer (1 M NaCl-0.01 M Tris-HCl.)

Equilibrium Dialysis-The binding of ribonucleosides by the nucleoprotein was determined by equilibrium dialysis at +I”. All experiments were done in duplicate and included three controls, one with buffer in place of the nucleoprotein, one with buffer in place of the ribonucleoside, and one with buffer in place of both ribonucleoside and the nucleoprotein. When the dialysis bags were well washed and the nucleoprotein solutions were fresh, the amount of ultraviolet-absorbing material in the dialysates of the last two controls was negligible. The distribution of ribonucleoside in the first control always verified that equilibrium had been attained.

Viscosity measurements of the nucleoprotein solutions were made by means of an Ubbelohde-type viscometer (20) or by means of an Ostwald-Fenske No. 50 viscometer (21). The Ubbelohde viscometer, made available through the courtesy of Professor Paul Doty, Harvard University, was equipped with four bulbs, which made it possible to determine the viscosity at four different flow gradients. Only the three lower bulbs, which corresponded to average flow gradients of approximately 100, 60, and 25 set-l, were used. The Ostwald-Fenske No. 50 viscometer had an average flow gradient of approximately 200 se@. Duplicate or triplicate flow times agreed to within 0.3 second. Since the flow times were all greater than 300 seconds, the relative viscosities should be accurate to approximately 2 parts in 1000. The nucleoprotein solutions were filtered through sintered glass before being placed in the viscometer. All measurements were made at 25 f 0.01”.

Sedimentation measurements were made by means of a Spinco model E ultracentrifuge equipped with an ultraviolet optical system. The sedimentation patterns were recorded on Kodak commercial film when this optical system was used. The films were developed under carefully standardized conditions and read in a recording densitometer (Spinco Analytrol) which plots film density as a function of distance along the cell axis. The readings were converted to a plot of nucleoprotein concentration versus distance from the center of rotation by means of a cali- bration curve relating film density to nucleoprotein concentration. Corrections for effects due to uneven illumination along the centrifuge cell or to dirt in the optical system were made with the use of the zero time sedimentation pattern as a base-line.

The distribution of sedimentation coefficients in the nucleoprotein solutions was calculated by the method of Schumaker and Schachman (22). It is often convenient, however, to char- acterize the sedimentation of the nucleoprotein by means of a single sedimentation coefficient rather than by a distribution of sedimentation coefficients. Either the sedimentation coefficient at the maximal value of t.he concentration gradient or the sedimentation coefficient at the median concentration would be suitable. The latter was chosen because the position of the median concentration can be determined much more accurately than the position at which the concentration gradient is largest, especially at low concentrations. The value of the median sedimentation coefficient was determined in most instances from the slope of a plot of log z versus time, in which z is the distance from the center of rotation to a point in the centrifuge cell at which the nucleoprotein concentration is one-half the initial value. This procedure is not strictly correct.’ The rigorous method for determining the median sedimentation coefficient is to calculate the distribution of sedimentation coefficients and then to integrate numerically over this distribution. This was done in a few instances but is very laborious, and use of the simpler method seemed justified since the plots of log x/x0 versus time appeared to be linear and the median sedimentation coefficients obtained in this manner varied only slightly from those determined by the rigorous method. Good agreement between the two methods will always be obtained if the distribution function is sharply peaked in the region of the median sedimentation coefficient.

,411 sedimentation studies were performed at 25’. The sedimentation coefficients reported herein have been corrected to 2OO.2

The apparent molecular weight and partial specific volume of DNA in a cesium chloride density gradient were calculated according to the method introduced by Meselson (23).

Electron photumicrographs of a calf liver nucleoprotein preparation were made through the courtesy of Professor C. E. Hall, Massachusetts Institute of Technology, by a method which he has recently described (24). According to this technique, the nucleoprotein solution was sprayed onto freshly cleaved mica. The dimensions of the nucleoprotein molecules were revealed by shadow casting. Polystyrene latex was added to the nucleoprotein solution to provide an internal standard.

RESULTS

Preparation of Deoxyribonucleoprotn

Isolation of Nuclei-Fresh calf livers were obtained approximately 60 minutes after the death of the animal. The large

1 Due to the radial dilution effect, x refers to the position of the median sedimentation coefficient only at zero time; at finite times, z refers to the position of populations of nucleoprotein molecules with successively greater sedimentation coefficients. The log z versus time plot measured in this way will therefore not necessarily be linear and its slope will not give the true median sedimentation coefficient.

2The sedimentation coefficients at 20”, SZO, were calculated from the sedimentation coefficients measured at 25”, ~26, by use of the equation

s25 ?I% 8.20 = - = 0.89 s25

120

in which $25 is the viscosity of water at 25” and 720 is the viscosity of water at 20”.


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June 1963 S. L. Commerjowi, n/l. J. Hunter, and J. L. Oncleg 2125

blood vessels were cut away, and the liver was extruded at +l” through the perforated disk of the press attachment by use of the hydraulic press. The coarse mince was then extruded successively through 16-, 30-, and 80-mesh stainless steel screens at pressures up to 800 psi. The final mince was suspended in 40 volumes of ice-cold phosphate buffer (0.12 M NaCl-0.008 M

K&IPOd-0.002 M KHzP04-0.0002 M CaCb,, pH 7.4) with thorough stirring and allowed to stand 30 minutes while the coarse connective tissue fragments settled out. The supernatant was siphoned off. On microscopic examination, this supernatant was seen to contain many isolated nuclei and to be almost free of whole cells and large connective tissue fragments.

The nuclei were sedimented from the above supernatant by centrifugation at +l“ in the Sharp& centrifuge (with the special feed nozzle with the T-shaped channel) at approximately 8000 r.p.m. and a flow rate of 160 to 200 ml per minute. By this procedure, 500 g (wet weight) of liver could be processed in 3 to 4 hours. The yield of nuclei was approximately 70 to 80% of those present in the tissue mince. The nuclei so obtained appeared to be intact and almost completely free from gross contamination by connective tissue or adhering cytoplasmic remnants when examined microscopically. Some details of yield in a typical nuclear preparation are shown in Table I.

&traction of Deoxyribonucleoprotin from Isolated Nuclei- Calf liver nuclei which had been freshly isolated from 500 g of calf liver were suspended in 10 liters of sodium-EDTA buffer (0.025 M EDTA-0.075 M NaCl), pH 8.0, to extract soluble nuclear components which would otherwise contaminate the nucleoprotein. The suspension was stirred for 30 minutes, then centrifuged in the Sharples centrifuge at 30,000 r.p.m. and a flow rate of 200 ml per minute. The sediment was resuspended in 500 ml of the same buffer. Sufficient EDTA buffer was then added to give a final volume of 10 liters, and the suspension was stirred for 30 minutes and centrifuged as before. This operation was repeated twice with 10 liters of bicarbonate buffer (0.05 nf NaHCOa-0.10 nt NaCl), also at pH 8.0 and ionic strength 0.15. The sediment collected after the final wash still contained the nucleoprotein. This residue was blended in a Waring Blendor for 2 minutes with 500 ml of distilled water. Sufficient distilled water (approximately 1 liter) was then added to extract the nuclear nucleoprotein and give a nucleoprotein concentration of approximately 0.1 g per dl. After being stirred for 20 minutes,

the suspension was centrifuged in a Servall centrifuge at 15,060 r.p.m. for 30 minutes. The supernatant was withdrawn with a sterile pipette and stored in sterile flasks at +l”. All opera- tions were performed at O-5”.

Although the nuclei could be frozen for over 24 hours without any noticeable change in the extractability or physical properties of the resultant nucleoprotein, freezing the nucleoprotein solutions caused an irreversible aggregation of the nucleoprotein into a gel. This gel remained stable for many days. It could be dispersed in 8 M urea but not in 2 nc NaCl nor by stirring in a Waring Blendor.

Thorough washing of the nuclei (as described above) with iso- tonic solutions before the extraction of the nucleoprotein removed a large amount of protein and RNA which would otherwise have been extracted along with the nucleoprotein. In the first wash, approximately 60% of the RNA, 60% of the dry weight, and 70% of one protein (adenosine deaminase) were extracted from the nuclear fragments. The loss of over half the nuclear sub- stance was paralleled by a marked change in the nature of the sediment obtained after centrifugation of the nuclear suspension. In contrast to intact nuclei which, when sedimcnted, formed a light brown sticky paste, the sediment of the washed nuclear fragments was medium brown, granular, and compact.

Progressively smaller amounts of material were removed from the nuclei in the second and third washes. After the third wash, the nuclear remnants contained all their original nucleoprotein, approximately 20% of the original RNA and 30% of the original dry weight. The nuclei were routinely washed three times since it had been found that further extractions at the same ionic strength (0.15) did not remove significant amounts of the re- maining RNA or protein. In the final two washes, the EDTA buffer was replaced by a bicarbonate buffer to simplify the nitrogen analyses that were subsequently made on the extracted nucleoprotein.

When the washed nuclear fragments were extracted in a sufli- cient volume of distilled water, over 90% of the nucleoprotein dissolved within 20 minutes. Although nucleoprotein concentrations up to 0.2 to 0.25 g per dl could be obtained by the addition of smaller volumes of water, it was more convenient to extract with a volume of water sufficient to give a final nucleoprotein concentration of 0.10 g per dl. At higher concentrations, the solutions of nucleoprotein were so viscous that it was difli-

TABLE I

Yield of nuclei jronb &pica1 nuclear preparation

Liver mince 120 g, dry weight (500 g, wet weight) 7.6 g of RNA, 1.3 g of DNA

Suspend in 200 dl of buffer, pH 7.4, r/2 0.15 Allow to settle for 30 minutes Decant

Connective tissue 9.6 g, dry weight

Centrifuge 3000 X g

Nuclei 8.1 g, dry weight 0.30 g of RNA, 0.89 g of DNA

Microsomes Mitochondris Cytoplasmic supcrnstant


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2126 Calf Liver Deoxyribonucleoprotein

TABLE II

Vol. 238, No. 6

Comparison of properties of nucleoprotein extract with properties reported for other deoxyribonucleoprotein preparations*

Nitrogen ............................. Phosphorus .......................... N:P. ................................ Nucleic acidt. ....................... Et:,,, (259 mp) ........................ c(P) (259 mp) ........................ RNA

Orcinol ............................ Dialyzable P .......................

Base composition Adenine (A) ....................... Thymine (T). ...................... Cytosine (C). ...................... Guanine (G). ...................... (A + T)/(G + C) .................

.-

-

7--

Nucleoprotein extract

Doty (11) LEk Carter c”4;d Hall

-

_

Iavison Id Butle

(25)

15.7 f 0.3 16.8 4.5 f 0.2 4.5 3.5 f 0.2 3.7 f 0.2 45 f 2 46 f 2 89 f 2 106 f 5

6100 i 150 7200

16.6 i 0.2 15.7 16.73 f 0.02 15.7 4.2 f 0.1 4.4 4.6 f 0.1 3.7 3.9 f 0.1 3.6 3.6 4.2 42 44 46 37

5 4

‘a 2

27.0 27.4 27.7 28.2 22.6 21.9 22.8 22.5 1.21 1.25

* All analytical measurements are given as percentage of composition. t These values were calculated from the reported phosphorus contents and are not necessarily those listed by the authors. $ This figure was obtained by Dounce and O’Connell (27) for nucleoprotein prepared from calf thymus by the method of Doty and

Zubay.

cult to remove the nuclear remnants from the nucleoprotein by centrifugation. Approsimately two-thirds of the RNA still present in the washed nuclear fragments was estracted along with the nucleoprotein. The rest of the RNA remained in the dark brown nuclear sediment.

When washing the nuclei, it was convenient, but not necessary, to resuspend the nuclear sediments by means of a Waring Blendor. The nuclei could be efficiently washed and the nucleoprotein almost quantitatively extracted when only gentle stirring (e.g. by means of a magnetic stirring bar) was used throughout the procedure.

Chemical Composition of Deozyribonucleoprotein Preparation

Data on the chemical composition of deoxyribonucleoprotein prepared as indicated above are given in Table II. This nucleoprotein preparation will be designated “nucleoprotein estract” to distinguish it from other nucleoprotein preparations. For comparison, the chemical compositions reported in the literature for five different nucleoprotein preparations and one DNA preparation are also listed. Four of the nucleoprotein preparations (those of &bay and Doty (11)) Steiner (6)) Carter (4)) a.nd Davison and 13utler (25)) were obtained from calf thymus. The other nucleoprotein preparation (7) and the DNA preparation (26) were both obtained from calf liver.

With regard to nitrogen content, the nucleoprotein preparations listed fall into two distinct groups, one in which the nitrogen content is 16.70/c (4, 7, 11) and one in which the nitrogen content is 15.7 To (6, 25). The nucleoprotein extract falls in this latter group. The nitrogen content of both groups of nucleoproteins is significantly lower than would be expected from the nitrogen content of the components of the nucleoprotein.

Very few deoxyribonucleoprotein preparations have been analyzed for the presence of RNA in spite of the simplicity of the measurement. Luck (7) states that his preparation contains less than 2% R.NA. Dounce and O’Connell (27) found that nucleoprotein prepared by the method of Zubay and Doty

contains 2% RNA by weight. No analyses for RNA are reported for the other preparations listed. The amount of RNA found in the nucleoprotein extract varied considerably, depending on how well the nuclei had been washed before the nucleoprotein was extracted. When the nucleoprotein was extracted from unwashed nuclei, approximately 17% by weight of the extract was RNA. When the nucleoprotein was extracted from nuclei which had been thoroughly washed, as in the procedure previously described, then only approximately 5% by weight of the extract was RNA. In general, the RNA content was measured by means of the orcinol reaction. This reaction actually measures the ribose from purine nucleotides, not RNA directly, and many substances interfere with the test. Therefore, as a check on this method, the RNA content was measured by dialyzing the nucleoprotein extract against an equal volume of 2 N KOH for 30 hours at 37” (28). By analyzing the dialysate for phosphorus, the amount of dialyzable phosphorus could be determined. The amount of RNA will be equal to the amount of dialyzable phosphorus since at alkaline pH values, RNA (but not DNA) is hydrolyzed to dialyzable fragments. As a control, a portion of the nucleoprotein extract was dialyzed against water instead of KOH. No dialyzable phosphorus was found in this control. The value for RNA content obtained by this method was in good agreement with that obtained by means of the orcinol reaction.

No satisfactory methods were found for eliminating this RNA from the nucleoprotein extract. Further washing of nuclei before extraction did not reduce the RNA content. Removal of the RNA by fractional precipitation was complicated by re- strictions on extraction conditions imposed by the instability of the nucleoprotein. For example, the solubility properties of the nucleoprotein extract were permanently altered by exposure to pH values of 6.0 or lower, while exposure to ionic strengths of 1.0 has been shown to cause dissociation of histones from the nucleoprotein molecules (29-31). Within the permissible range of pH values and ionic strengths, the solubility properties of this


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June 1963 S. L. Commerford, M. J. Hunter, and J. L. On&y 2127

RNA (or ribonucleoprotein) and deoxyribonucleoprotein are very similar. Solutes that cause the precipitation of deoxyribonucleoprotein such as calcium chloride, zinc chloride, or ethanol do not discriminate well enough between the RNA and the nucleoprotein to provide a basis for satisfactory fractionation. The best fractionation conditions found were pH 6.5 and r/2 0.15 at +l”. Under these conditions, 57% of the RNA and 15% of the nucleoprotein remained soluble. Since there are good grounds for believing that the nucleoprotein is very heterogeneous, the nucleoprotein which remained soluble at pH 6.5, r/2 0.15, may be quite different from the nucleoprotein that precipitated. As the purpose of this investigation was the extraction and characterization of the total deoxyribonucleoprotein of the cell, no application of these conditions to remove RNA from the nucleoprotein extract was made. Freshly prepared nucleoprotein solutions contained no dialyzable phosphorus.

Physical Properties of Nucleoprotein Preparation

Absorption Spectra-The change in the ultraviolet absorption observed when the nucleoprotein extract was exposed to extreme pH values was similar to that reported for DNA (26) and is shown in Fig. 1. At low pH values, the whole absorption spectrum shifted toward longer wave lengths and the maximal absorption increased by approximately 31%. The same increase in maximal absorption occurred at high pH values, but under these conditions, the absorption curve shifted slightly toward shorter wave lengths.

Electrophoretic Analysis-The schlieren pattern obtained after electrophoresis of a 0.12 g per dl solution of nucleoprotein in Tris-HCl buffer, pH 8.0, I’/2 0.01, showed a symmetrical main peak with an average mobility of -13 x 10m5 cm2 per volt per second. This peak was not electrophoretically homogeneous since it was observed to sharpen in both ascending and descend- ing boundaries when the current was reversed. It accounted for approximately 850/, of the total area, the other 15y0 being present as a trailing shoulder. No material was seen with a mo. bility faster than - 14 x 10m5 or slower than -8 x 10ms cm2 per volt per second.

It was difficult to obtain good schlieren patterns of the electrophoresis of nucleoprotein in Tris-NaCl buffer, pH 8.0, r/2 1.01 due to the high conductivity of the buffer. Nevertheless, the main features of the patterns were always the same. Two peaks of approximately equal size, one with a mobility of approximately -13 x 10h5 cm2 per volt per second and one with a mobility of approximately -7 x 10e5 cm2 per volt per second. No components with a positive mobility were ever seen. It was assumed in calculating these mobilities that the movement of the boundaries was entirely due to electrophoretic migration. Since no buffer anomalies appear when electrophoresis is performed in this buffer, the movement of the boundaries due to other effects (e.g. gassing at the cathode, electro-osmosis, thcr- ma1 expansion) could not be detected, and the calculated mobilities might be seriously in error. To check this possibility, a buffer anomaly was introduced by adding 4 ml of distilled water to 2 liters of the electrophoresis buffer which had previously been brought to dialysis equilibrium with the nucleoprotein solution. No movement of the buffer anomaly thus introduced was observed during electrophoresis. The mobilities of the two nucleoprotein boundaries calculated from this electrophoresis pattern

0 pH 0.3

0 pH 8.0

0 pH 13.0

\Q I I I ,-d ,

240 260 280 300 320

WAVELENGTH (mp 1 FIG. 1. The effect of pH on the ultraviolet absorption spectrum

of deoxyribonucleoprotein.

were the same as those calculated from the patterns which had no buffer anomalies.

Nucleoprotein-Purine Ribonwleoside Interactions--An attempt was made to obtain more direct evidence for the theory that RNA is synthesized on a DNA-containing template. Since a template mechanism implies specific binding of RNA precursors, the binding of inosine and guanosine to the nucleoprotein was studied. A 0.12 g per dl solution of the nucleoprotcin w~ls dialyzed against an equal volume of 0.02 g per dl of inosine 01 guanosine. The nucleoprotein and the purine ribonucleosides were both made up in 0.02 M Tris-HCl buffer, pH 8.0. After 18 hours, the ultraviolet absorption spectra of the dialysates were determined. No binding of either ribonucleoside to the nucleoprotein was found.

Under the conditions of these experiments, the solution within the dialysis sac contained 1.8 x 10m3 moles of nucleoprotein phosphorus per liter and 3.5 X low4 M guanosine or 3.7 x 10m4 M inosine when dialysis equilibrium was reached. Since the binding of as little as 5 oi: of the total purine ribonucleoside could have been detected, less than 1 molecule of ribonucleoside was bound per 50 nucleoprotcin nuclcotide residues.

An attempt was also made to measure the binding of another purine ribonucleosidc, adenosine, to the nucleoprotein. These experiments failed due to t.he presence of trace amounts of adenosine deaminase in the nucleoprotcin preparations. This en- zyme is normally present in liver in relatively high concentrations (32).

Viscosi&--The relative viscosities of solutions of three different calf liver nucleoprotein preparations (designated DNP-A, DNP-B, and DNP-C) are shown in Table III. The relative viscosities of DNP-A solutions were measured in an Ostwald- Fenske viscometer (flow gradient of approximately 200 SW-~)


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2128 Calf Liver Deozyribwwcleoprotein Vol. 238, No. 6

TABLE III Viscosity of deoxyribonucleoprotein solutions at 25”

Sample DNP-A, Ostwald- Fenske viscometer

(hl = 4.8 dl/s)

gldl

0.0061* 0.0123* 0.0149 0.0246* 0.0296 0.0370* 0.0370

Wative Gscosit: Y

1.026 4.3 1.060 4.9 1.060 4.0 1.093 3.8 1.100 3.4 1.120 3.2 1.114 3.1

s.

-

Mean -

$

6

5

ample DNP-B, Ubbelohdc viscometer, 0.01 ionic

strength Tris-HCI buffer, pH 8.0 (171 = 2.7 dl/g)

-

! s;

:a -1

100 60 25

Relative viscosity

0.0048 g/d1

1.009 1.011 1.017

1.012

0.0143 s/d1

1.029 1.028 1.029

1.029

s g

ample DNP-C Ubbelobde viscometer, 0.0007 b(

potassium phosphate. pH 6.8 ([VI = 5.9 dl/g)

,lS%ar radi- .- ent

wc-

100 60 25

Relative viscosity

0.0104 0.0261 g/d1 g/d1

1.061 1.059 1.057

1.141 1.141 1.140

1.059 1.141

* Done in 0.0007 M potassium phosphate, pH 6.8. All others done in 0.01 ionic strength Tris-HCI buffer, pH 8.0.

I dc E-z 0

20 40 60 60 too

620

l?~a. 2. Curve A, the distribution of sedimentation coefhcients in a 0.9065 g per dl solution of deoxyribonucleoprotein; O--O, 9.3 minutes; O---O, 17.3 minutes; A-A, 25.3 minutes. Curve B, the distribution of sedimentation coefficients of deoxyribonucleoprotein after correction for concentration dependence.

at several different nucleoprotein concentrations and in two different buffers. One of these buffers, 0.7 mM potassium phosphate, pH 6.8, was used by Zubay (11,33) for viscosity measurements on his calf thymus nucleoprotein preparation. The other buffer was 0.01 ionic strength Tris-HCl, pH 8.0. The data in Table III indicate that the viscosity of the nucleoprotein is approximately the same in both buffers. The intrinsic viscosity of this preparation (DNP-A), obtained by extrapolating the reduced specific viscosity to zero concentration, was 4.8 dl per g.

Also shown in Table III are the relative viscosities of DNP-B and DNP-C, each measured at two concentrations and three different shear gradients by means of an Ubbelohde-type viscometer. The relative viscosities showed little dependence on shear gradient over the range from 100 to 25 see-l, except in the case of the solution of sample DNP-B at the lower concentration (0.0048 g per dl). Since this relative viscosity was so low, the significance, if any, of the variation is difficult to assess. Accord- ingly, the mean values of the relative viscosities at each concentration were converted to specific viscosities and these values

extrapolated to zero concentration to obtain intrinsic viscosities. The intrinsic viscosities of DNP-B and DNP-C were found to be 2.7 dl per g and 5.9 dl per g, respectively.

The viscosities of the nucleoprotein solutions at 25” were quite constant with respect to time. No significant changes in viscosity were found in nucleoprotein solutions which had been left at room temperature for 12 hours.

Sedimentation Studies-The distribution of sedimentation coefficients in a 0.0065 g per dl solution of the nucleoprotein (0.02 M Tris-HCl, pH 8.0) is shown in Fig. 2. Independent calculations of the distribution were made from the sedimentation patterns obtained after centrifugation at 29,500 r.p.m. for 9.3, 17.3, and 25.3 minutes. The fact that there was no systematic trend in the distribution of sedimentation coefficients with time justified one of the assumptions on which the calculations were based, viz. that diiusion of the nucleoprotein can be neglected. The preparation (DNP-B) had a median sedimentation coefficient of 47 S at 0.0065 g per dl.3

It has been found that many linear polymers obey the rela- tionship (34)

s = so/(1 + kc)

in which s is the sedimentation coefficient, s0 is the sedimentation constant, e is the concentration of the polymer, and k is a constant characteristic of the polymer. If this equation is valid for nucleoprotein, a straight line should be obtained when the re- ciprocals of the median sedimentation coefficients are plotted against concentration. In fact, this equation was found to fit the sedimentation behavior of these macromolecules rather well, even when a certain amount of depolymerization had occurred. A well prepared nucleoprotein preparation gave a value of 52 S for so and 33 dl per g for k. Preparations in which the time of isolation was longer or the temperature higher gave lower values for both parameters; for example, one such preparation gave values of 33 S for SO and 21.4 dl per g for k.

With the values of 52 S for so and 33 dl per g for k and on the assumption that k is the same for all species of nucleoprotein present, a distribution curve of sedimentation coefficients corrected for concentration dependence can be calculated after numerical integration of the uncorrected sedimentation distribution curve. Such a distribution curve is also shown in Fig. 2.

The sedimentation behavior of the nucleoprotein was found to be independent of ionic strength in the range of 0.001 to 0.01. At higher ionic strengths, however, the nucleoprotein sedimented at a much slower rate. For example, the median sedimentation coefficient of a 0.012 g per dl of nucleoprotein solution was 26 S in 0.7 mM phosphate buffer, pH 6.8, 18 S in 1 M NaCl, and 6 S in 4 M NaCl. In 1 M NaCl, two components appeared in the sedimentation pattern. One sedimentation pattern was recorded by means of schlieren optics and analyzed to determine the relative amounts of each component. This pattern was obtained

3All the distribution functions shown here have been plotted as (l/c) * (dc/ds). Actually, what is calculated is (l/e) . (de/ds) in which E is the optical density. This will be equal to (l/c). (dc/ds) only if there is no heterogeneity in the nucleoprotein with respect to E. It has been found by Crampton et al. (30) that the e(P) of DNA fractions vary by 10% or more. It is therefore possible that there is also heterogeneity with respect to E in the nucleoprotein. If a correlation exists between the ultraviolet absorption and the sedimentation coefficient, identification of (l/c). (dc/ds) with (l/e)-(d~/ds) will lead to systematic errors.


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from a 0.75 g per dl of solution of the nucleoprotein in 1 M NaCl- 0.01 M Tris-HCl buffer, pH 8.0, which had been centrifuged at 59,780 r.p.m. at 25’. One of the components sedimented at 9 S. On the basis of its shape (a sharp spike), this component was identified as nucleoprotein (or DNA). The other component sedimented very slowly and was never completely resolved i- from the meniscus. This component appeared in the schlieren ! I pattern as a small rounded peak which spread fairly rapidly but i was well separated from the nucleoprotein boundary. When a refractive increment of 0.185 was assumed, it was calculated i that this component represented from 10 to 12% of the total F

colloid on a weight basis. 8

The distribution of DNA in a cesium chloride gradient was determined following the procedure used by Meselson (23). In I I I I I II I I I I I

this experiment, 0.70 ml of a 7.7 molal cesium chloride solution 6.0 6.2 6.4 6.6 6.8 70 22

that contained 5.2 pg of DNA (added as nucleoprotein) was DISTANCE FROM CENTER OF ROTATION (cm)

placed in a standard centrifuge cell and centrifuged at 25” for FIG. 3. The distribution of DNA in a cesium chloride density 40 hours at 44,770 r.p.m. The distribution of DNA in the cen- gradient.

trifuge cell was followed by means of ultraviolet optics. The distribution of cesium chloride was followed by means of schlieren optics. The distribution of DNA in the centrifuge cell after centrifugation had proceeded for 40 hours is shown in Fig. 3. No ultraviolet-absorbing material was seen between the meniscus and the main DNA band. The density of the cesium chloride solution as a function of distance was determined both esperi- mentally from the refractive indes gradient and theoretically by means of the equation relating the activity of a solute to its position in a gravitational field at equilibrium (23, 35). The results of these independent methods were in good agreement, constituting adequate proof that the salt gradient was at equilibrium. The density of the cesium chloride solution at that position in the cell at which the DNA concentration was masi- ma1 (p?.,) was 1.693 g per mlr. The salt gradient at TO, (dpldr),,, was 0.114 g per cm4. The variance of the DNA band (a*) was 0.0026 cm2, a value comparable to that obtained for calf thymus DNA in a similar salt gradient (36). Assuming no heterogeneity with respect to partial specific volume (G), we can determine an apparent molecular weight from this value (23), provided a reasonable estimate of fl can be made. If fi is assigned a value of 0.48 (from the data of Hearst and Vinograd for ‘I’4 bacteriophage DNA (37, 38)) an apparent molecular weight of 1.2 x lo6 is obtained for the anhydrous Cs-DNA. This would correspond to a molecular weight of approximately 2.0 x lo6 for the nucleoprotein. However, since the skewed profile of the distribution of the Cs-DNA in the salt gradient indicates considerable heterogeneity with respect to ii (23), the true molecular weight m.ay be even an order of magnitude greater than the above value which must be regarded as a minimum (36).

Electron Photomicrography-Several electron photomicrographs of a calf liver nucleoprotein preparation were made by Professor C. E. Hall, Massachusetts Institute of Technology. One of

FIG. 4. Electron photomicrograph of calf liver deoxyribonucleoprotein.

these is reproduced in Fig. 4. In this figure, the nucleoprotein molecules are shown magnified 90,500 diameters. The nucleo- spherical particle appearing in Fig. 4 is a polystyrene marker. protein molecules appear as long, smooth, slightly curled threads. This particle is 100 rnp in diameter. From the shadow cast by these molecules, their diameter was estimated by I’rofessor Hall to be 30 f 10 ,4. This value and DISCUSSION

the general shape of the molecule fit well with the generally Preparation of Deoxyribonucleoprotein-Owing to the peculiar accepted structure of the nucleoprotein, namely that it consists structure of deoxyribonucleoprotein, it would be expected that of a single, double stranded DNA molecule with the histone the manner in which the nucleoprotein was isolated from the evenly distributed along its length (11, 39). The largest nucleo- tissue would profoundly affect its physical properties. The protein molecule in Fig. 4 is approximately 5,000 A long. The enzymatic hydrolysis of a few phosphodiester linkages of DNA

June 1963 X. L. Commerford, M. J. Hunter, and J. L. Oncley 2129


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2130 CalJ’ Liver Deoxyribonucleoprotein Vol. 238, Ko. 6

resulting from a brief esposure to the powerful tissue deoxyribo- nucleases might be sufficient to reduce the molecular weight by a factor of two or more. On the other hand, aggregation of nucleoprotein molecules could lead to a significant increase in molecular weight. In the absence of any criterion for recognizing “native” nucleoprotein, therefore, there is no certain way of de- ciding whether any given nucleoprotein preparation is “native,” enzymatically degraded or aggregated. The approach adopted in these experiments was to avoid as far as possible conditions favorable for aggregation and enzymatic degradation in the espectation that the nucleoprotein so obtained would closely approximate the native molecule.

Enzymatic degradation should be materially reduced by iso- lating intact nuclei before nucleoprotein extraction since many of the degradative enzymes are present in the non-nuclear parts of the cell. Because none of the methods available in the literature will permit rapid isolation of clean nuclear preparations in large quantity, the method described in “Experimental Pro- cedure” was developed.

One of its essential features was the mechanical extrusion of the tissue through narrow orifices. This treatment homogenized the tissue and ruptured cells without gross injury to cell components and without chopping the connective tissue into fine flakes which were then difficult to separate from the nuclei. Another essential feature was the removal of cellular debris. I f the liver mince was not diluted with at least a 20-fold buffer volume, the cellular remnants did not sediment well and the resultant nuclei were obviously contaminated with remnants and cytoplasmic components. It was also observed that if the pH was allowed to drop below 7, contamination of the nuclei increased, presumably due to aggregation of the cytoplasmic particles. Nucleoprotein could not be obtained quantitatively or reproducibly from such nuclei.

The isolation of nucleoprotein from the nuclei was greatly facilitated by the EDTA washes. These washes, presumably by removal of calcium, rendered the nuclear membrane more fragile and permitted the extraction and removal of the more soluble nuclear components. The 0.15 ionic strength wash solutions did not extract any deoxyribonucleoprotein from the nuclei, since the nucleoprotein is insoluble at this ionic strength, although relatively soluble in water. The nucleoprotein which was finally obtained on the addition of water, with or without homogenization in a Waring Blendor, was thus uncontaminated by the soluble proteins of the nucleus.

The extraction of nucleoprotein from washed nuclei was com- plete in less than 30 minutes when the nuclei were prepared and extracted by the methods previously described. If, however, the time of processing the tissue were prolonged or if the temperature were allowed to rise, especially at the stages prior to removal of cytoplasmic components, the nucleoprotein became very refractory to extraction. (A somewhat similar observation has been made by Allgen (40) .) It was then necessary to extract for long periods of time to obtain even a small yield of nucleoprotein. The nucleoprotein obtained under these conditions appeared to be extensively degraded since the sedimentation coefficient was less than half that of the nucleoprotein prepared as described previously. It is probable that, in many of the methods reported in the literature for the preparation of deoxyribonucleoprotein, the preliminary treatment of the tissue was such as to make the nucleoprotein similarly refractory to extraction. Although most papers concerning the preparation of the

nucleoprotein mention neither yield nor rate of extraction, the common practice of extracting for from 12 to 36 hours in distilled water (4, 6, 30, 41-43)4 suggests a very slow rate of extraction. Furthermore, in at least two instances (41, 43)) it was explicitly stated that very small amounts of nucleoprotein were extracted within the first few hours,

Chemical Composition-The nucleotide base composition of the nucleoprotein extract is, within experimental error, in keep- ing with that expected on the basis of the Watson-Crick structure. The ratio of thymine to adenine is 1.03, and the ratio of guanine to cytosine is 1.01. Furthermore, the over-all nucleotide base composition is very similar to that found for calf liver DNA by Hurst et al. (26) by means of ion exchange chromatog- raphy. The phosphorus content and the nitrogen-phosphorus ratio also agree quite well with the values reported by Carter (4), Steiner (6), and Zubay and Doty (11). However, the value for the nitrogen content of this preparation of the nucleoprotein agrees neither with theory nor with the values obtained by several other workers (4, 7, 11). All the values for the nitrogen content of deoxyribonucleoprotein which have been reported are significantly lower than the value of 17.601, expected on the basis of the theoretical nitrogen content of DNA and the measured nitrogen content of the histone (7, 44, 45).5 There are several possible explanations for this discrepancy. First, the analytical methods used to determine nitrogen may be at fault. This does not seem likely because, although the nitrogen contained in purines and pyrimidines is not easily converted to ammonia, the sealed tube digestion procedure used has been reported to convert quantitatively all uracil and tryptophan nitrogen into ammonia (13). In addition, two different analytical procedures gave substantially the same results. Another possibility is that the dry weight measurements are in error, due to incomplete removal of water. It has been reported that it is quite difficult to remove all water from DNA (47) and this seems to be true of the nucleoprotein as well since it was found necessary to heat the nucleoprotein in a vacuum at 110” for approximately 30 hours before a constant weight was obtained. However, it is unlikely that the low content of nitrogen found for this preparation of nucleoprotein was due solely to incomplete removal of water since this would require that the nucleoprotein heated to constant weight in a vacuum at 110” contained 11 y0 water by weight.

A third possibility is that the nucleoprotein preparations contained a protein-bound impurity which has a low nitrogen content. The only impurity which has been found in this deoxyribonucleoprotein preparation in anything but trace amounts is

4 One exception to this statement is the preparation of the nucleoprotein from calf thymus by water extraction reported by Zubay and Doty (11, 33).

5 There seems to be some confusion concerning the theoretical composition of DNA. This is probably because the composition of DNA varies considerably, depending on the ratio of sodium to phosphorus assumed and on whether DNA is considered a tetra- nucleotide or a very large polvnucleotide. A very large nolv- nucleotide with an equal-distribution of nucleotide bases and”a molar Na:P ratio of 1 contains 15.9% N. 9.4% P. and 6.95% Na by weight (46). Therefore, in nucleoprotein’m which the molar Na:P ratio is 0.14 (25) the DNA component should contain 16.9% N, 10.0% P, and 0.8yo Na by weight, assuming an equal distribution of nucleotide bases. DNA with a 0.14 M Na:P ratio and the distribution of nucleotide bases experimentally determined for calf liver nucleoprotein should contain 16.6oj/, nitrogen and 10.0% phosphorus.


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June 1903 S. L. Comm,erjord, M. J. Hunter, and ,J. L. On&q 2131

RNA. It is not known whether this RNA is present as the free tissues and species. However, in spite of the abundant evidence acid or as ribonucleoprotein. There are some grounds, however, for t,he heterogeneity of the components of deoxyribonucleopro- for believing that most of this RNA is bound to the nucleoprotein. tein, there is little data in the literature on the heterogeneity of The best evidence for this comes from the sedimentation patterns the nucleoprotein itself. of the nucleoprotein recorded by means of the ultraviolet ab- In the course of this investigation, calf liver nucleoprotein was sorption optical system. These patterns indicated that only 3 % found to be very heterogeneous with respect to sedimentation or less of the total ultraviolet-absorbing material sedimented at a coefficient. At low concentrations of nucleoprotein these were rate slower than that of the nucleoprotein. Since RNA nc- found to range from 25 S to over 200 S. The shape of the sedi- counts for approximately 10% of the total nucleic acid present, mentation distribution curve of calf liver nucleoprotein is very and therefore 10% of the total ultraviolet absorption, 70% or similar to those published by Shumaker and Schachman (22) for more of the total RNA must therefore have sedimented along calf thymus DNA with the exception that the long narrow with the nucleoprotein. Furthermore, the majority of the RNA shoulder leading the nucleoprotein distribution is much more contained in the nucleoprotein preparation has the same solu- pronounced than that observed in the DNA distribution. The bility characteristics as the nucleoprotein. Finally, RNA is position of the nucleoprotein distribution curve is shifted by ap- known to be present in the chromosome (1). According to All- proximately 20 S toward higher sedimentation coefficients with frey, Mirsky, and Stern (48), 8 to 9% of the total nucleic acid in respect to the DNA curve. calf liver chromosomes is RNA. A smaller amount of heterogeneity in calf liver nucleoprotein

The attempt to obtain more direct evidence for the theory was found with respect to electrophoretic mobility. Approxi- that RNA is synthesized on a DN;\-containing template was un- mately 85% of the total calf liver nucleoprotein was found to successful. No significant binding of inosine or guanosine to the have an electrophoretic mobility between -12 x 10e5 and nucleoprotein could be detected.6 -14 X 1OP cm2 volt? set-I. The electrophoretic mobility of

Somewhat similar experiments have been reported by &bay the rest of the nucleoprotein was in the range of -8 x lo-” to (33) and by Jardetzky (49). I3oth authors investigated the -12 X lo-” cm2 volt+ see-I. Heterogeneity was also demon- possibility that DNA is a template for protein synthesis by strated in the partial specific volume of calf liver DNA by the measuring the binding of amino acids to DNA. Measurable cesium chloride density gradient experiment. A similar hetero- binding was found only with the basic amino acids and peptides geneity in the partial specific volume of calf thymus DNA has (49). This binding was quite weak and was probably due to been reported by Meselson (56). nonspecific electrostatic interaction between the positively Molecular Weight of Calf Liver Nucleoprotein-The measure- charged amino acids and the negatively charged phosphate rcsi- ments performed on the nucleoprotein do not permit the assigna- dues of DNA. In addition, Zubay investigated the hypothesis tion of a definite molecular weight to any of the nucleoprotein that DNA is a template for its own synthesis by measuring the preparations. However, some minimal values for the molecular binding of deoxyadenylic acid to DNA. No significant binding weight can be obtained from the cesium chloride density gradient was found either with native or heat-denatured DNA. study and the electron photomicrographs. The distribution of

Absorption Spectra-The E (P) of native DNA is given as 6000 calf liver DNA in a cesium chloride density gradient establishes a by Beaven (50) and as 6600 by Chargaff (51). Since the content lower limit of approximately 2 x lo6 for the molecular weight of of aromatic amino acids in histone is quite small (44,52), the con- the nucleoprotein. Since it has been shown that the technique tribution of histone to the ultraviolet absorption is negligible by which the nucleoprotein specimens were prepared for electron compared with the contribution of DNA. Therefore, the value microscopy causes cleavage of the nucleoprotein into smaller of 6150 obtained for this preparation is quite reasonable. When molecules (66). the molecular weight of the nucleoprotein deter- . ., DNA is heated or exposed to extremes of pH, the maximal value mined by electron microscopy can also be regarded as a lower of the ultraviolet absorption increases by over 30% (50). The limit. The longest nucleoprotein molecule shown in Fig. 4 is same effect has been reported for the nucleoprotein, both in this 5,000 A in length. This corresponds to a molecular weight of paper and by others (53,54). It is generally considered that this approximately 2- to 3,000,OOO. increase in absorption signifies that the specific hydrogen bonding A better estimate of the molecular weight of the nucleoprotein between nucleotide bases has been irreversibly altered. can be calculated from the equation introduced by Mandelkern

Heterogeneity of Calf Liver L>eoxyribonucleoprotein-There is and Florv (67) which relates the molecular weight of a flexible or “ .

considerable evidence that both DNA and histones are very het- rigid macromolecule to its intrinsic viscosity and sedimentation erogeneous. It has been shown that DNA is heterogeneous (55) constant with respect to partial specific volume (56), nucleotide base composition (30), and sedimentation coefficient (22, 43). Histones Nhl” 70 s Ma = ___ have been shown to be heterogeneous (57-59) with respect to PO - 64 molecular weight (45, 60, 61), solubility (45, 60, 62), amino acid composition (44, 45, 63), sedimentation coefficient (45, 63), and in which /3 is a parameter for which the value depends on the

electrophoretic mobility (45, 63, 64). Moore (65) has recently shape of the macromolecule. summarized the present information on the constitution of the On the basis of its light-scattering envelope, Zubay and Doty lysine-rich and arginine-rich fractions of histones from various (11) have concluded that thymus deoxyribonucleoprotein has

the configuration of a random coil. For a macromolecule with 6 However, since the nucleoprotein extract contains approxi-

mately 5y0 RNA, it may well be already saturated with ribonucleo- this configuration, the parameter /3 has a value of 2.5 X 106.

side residues, and the experiment should be repeated with nucleo- The molecular weights calculated from the above equation

protein which has been largely freed of it,s RNA by pretreatment should, however, be viewed with some reservations as there are with ribonuclease. several uncertainties involved in its use. The equation itself is


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2132 Calf finer Deozyribonucleoprotein

TABLE IV

Vol. 238, No. 6

Certain physical properties of various deoxyribonucleoprotein preparations*

Preparation

I. Extracted in 1 M NaCl Luck (7, 9) “nonfibrous” Welsh (70) “nonfibrous”

Welsh (70) “fibrous”

Frick (71)

II. Extracted in wat,er Carter and Hall (4) Carter (5) Steiner (6) Zubay and Doty (11) Bayley et a2. (39) “Nucleoprotein extract”

171

d/g

s.ot

0.36 26.5t

5t

0.55 3.0t 0.33

34.0 30.0

5

s s,n

11.5t

2.15t

Rigid ellipsoid Random coil B = ml) f3 = 2.5 x 10%

s, hi

20 s.2t

20

43

25-30t

0.75t

15.9t

WH (4)t

(l.o)t

(28)t

(6)t

311 21 50

5.8 19 20.5

@It c5.vt (0.94) (1.1)

(23) 40

52 11 16.5

M x 10-6 (light-

scattering) M x 10-s -

M x 10-6

* Unless otherwise stated, measurements were performed in low salt, pH 6 to 8. All molecular weights in parentheses were calculated by us. A value of O.i6 was assigned to in. -

t Measurements performed in 1 M NaCl.

somewhat empirical. Although it has been applied successfully to many linear polymers (67, 68), it is not certain that it can be applied equally well to nucleoproteins. Furthermore, if calf liver nucleoprotein does not have the random coil configuration assigned by Zubay and Doty to their calf thymus nucleoprotein, the value used for /3 could be in error. The magnitude of this error would not be great, however, as fl is not very sensitive to molecular shape (69). Finally, the sedimentation constant used in this equation should be the weight average rather than the median sedimentation constant. Since a small fraction of the nucleoprotein molecules have very high sedimentation constants, the weight average sedimentation constant will be larger than the median sedimentation constant.

Some of the viscosity and sedimentation data obtained by various workers for their deoxyribonucleoprotein preparations are shown in Table IV. Detailed comparison of these results is impossible due to differences in experimental conditions. How- ever, it can be seen that the preparations of Carter and Hall and of Steiner, in which the extraction with water was prolonged, have much lower molecular weights than the three preparations which were rapidly estracted with water (Zubay and Doty (11); Rayley, Preston, and Peacocke (39); “nucleoprotein extract”).

Bayley, Preston, and Peacocke (39) prepared calf thymus nucleoprotein by the method of Zubay and Doty. The light- scattering and intrinsic viscosity data on these two preparations are in excellent agreement. Calf thymus nucleoprotein has also been prepared by a modification of the method of Zubay and Doty by Fredericq (72). He obtained two nucleoprotein fractions, a gel fraction (G-fraction) and a soluble fraction (S-fraction). The relative proportions of these fractions depended on the exact extraction procedure. The S-fraction had sedimentation and viscosity properties similar to those of the preparation of Zubay and Doty.

The extraction procedure employed in the preparation of the “nucleoprotein extract” was similar to that employed by Zubay

and Doty. With the liver protein, however, prior separation of the nuclei was found to be necessary to obtain satisfactory extraction of the nucleoprotein. Gel formation was never apparent during the extraction procedure (unlike the thymus nucleoprotein extraction procedure), and over 90 y0 of the nuclear nucleoprotein could be solubilized to give a reproducible nucleoprotein extract.

The viscosity behavior of this liver nucleoprotein, however, was quite different from that of the thymus protein, although in other respects, the two preparations were very similar. Viscosity determinations on seven different “nucleoprotein extract” preparations were performed, The maximal value for the intrinsic viscosity which could be derived from this viscosity data was approximately 7 dl per g.

An explanation for this lower value of the intrinsic viscosity of the calf liver preparation in terms of organ difference, enzymic hydrolysis, or denaturation is impossible with the information at present available and will not be attempted at this time.

Dissociation of Histone in 1 M NaCl-It is agreed by most investigators that histones are dissociated from deoxyribonucleoprotein in 1 M NaCl. The extent of this dissociation, however, is not clear (11, 29, 31, 73). It has been shown in this paper, by means of the ultracentrifuge schlieren pattern, that in 1 M NaCl, approximately 10% of the total colloid (presumably histone) was dissociated from the nucleoprotein and had a sedimentation CO-

efficient of approximately 1 S. It is known that, if all the histone is removed from the nucleoprotein and placed in 1 M NaCl, approximately 80% (by weight) of the histone will polymerize to form particles with large sedimentation coefficients (61). This material would be present in the same sedimenting boundary as DNA. Thus, the histone with a sedimentation coefficient of approximately 1 S may represent the nonpolymerizing, lysine-rich component described by Ui (45).

No conclusions concerning the extent to which the polymeriz- ing histone component dissociates from the nucleoprotein in 1 M NaCl solution can be derived from centrifugal studies since under


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*Tune 1963 S. L. Commerjord, M. J. Hunter, and J. L. Oncley 2133

these conditions the nucleoprotein (or DNA) is very viscous and sediments slowly. Due to the viscosity of these solutions, only substances which sediment more slowly than the nucleoprotein will appear as an independent boundary; the polymerized histone will, therefore, sediment with the nucleoprotein (or DNA) boundary whether it is dissociated or not.

These difficulties do not obtain in electrophoretic analyses since, at pH 8, any histone which is dissociated from the nucleoprotein should appear in an electrophoresis pattern as a component with positive mobility whether it is polymerized or not, whereas the nucleoprotein (or DNA) should appear as a component with negative mobility. The failure to observe any component with a positive mobility in 1 M NaCl is somewhat puzzling since from the sedimentation studies discussed above, a component with positive mobility and corresponding in size to at least 2On/, of the total histone or 10% of the total nucleoprotein should have been found. Other investigators have reported components with positive mobility in 1 M NaCl. Frick (74) found a small component with a mobility of +6.9 x lo-” cm2 volt-’ see-‘. Apparently, this component represented less than 5% of the total material. Gajduseck (75) obtained three components, one of which had a mobility of +8 x 10m5 cm2 volt+ se@. Luck (9) found a very small component with positive mobility in 0.8 M NaCl.

4.

5. 6. 7.

8.

9.

10.

11. 12.

13.

14.

15.

16. 17. 18.

CARTER, R. O., AND HALL, J. L., J. Am. Chem. Sot., 62, 1194 (1940).

CARTER, R. O., .I. Am. Chem. Sot., 63, 1960 (1941). STEINER, R. F., Trans. Faraday Sot., 48,1185 (1952). LUCK, J. M., KUPKE, I>. W., RHEIN, A., AND HURD, M., J.

Biol. Chem., 206, 235 (1953). SHOOTER, K. V., DAVISON, P. F., AND BUTLER, J. A. V., Bio-

chim. et. Biophys. Acta, 13, 192 (1954). KXJPKE, D. W., ELDREDQE, N. T., AND LUCK, J. M., J. Biol.

Chetn., 210, 295 (1954). ROTIIERHAM, J., SCHOTTELIUS, D. D., IRVIN, J. L., AND IRVIN,

E. M., J. Riol. Chem., 223. 817 (1956). ZUBAY, c., AND J)OTY, P., J: MoZ&ular Biol., 1, 1 (1959). KIRK. P. L.. Quantitative ultramicroanalusis. John Wilev and

So&, Inc.: I?ew York, 1950. .I I .s

GRUNBAUM, B. W., SCHAFFER, F., AND KIRK, P. L., Anal. Chem., 24, 1487 (1952).

CONWAY, E. J., Microdiffusion analysis and volumetric error, D. Van Nostrand Co., Inc., New York, 1947.

LOWRY, 0. H., ROBERTS, N. R., LEINER, K. Y., WV, M. I,., AND FARR, A. I,., J. Biol. Chem., 207, 1 (1954).

CERIOTTI, G., J. Biol. Chem., 214, 59 (1955). KECK, K., Arch. Biochem. Biophys., 63, 446 (1956). DISCHE, Z., in E. CHARGAFF AND J. N. DAVIDSON (Editors),

The nlzleic acids, Vol. Z, Academic Press, Inc., New York, 1955, p. 287.

WYATT, G. R., Biochem. J., 48, 584 (1951). REICHMANN, M. E., RICE, 9. A., THOMAS, C. A., AND DOTY,

The histone must certainly dissociate from the nucleoprotein at higher ionic strengths, however, since the position at which the nucleic acid component floated in a cesium chloride density gradient indicated that no significant amounts of histone remain associated under these conditions (i.e. 6 ht CsCI).

P., J. Am. Chem. Sot., 76, 304? (1954). MERRINC~TON. A. C.. Viscometru. Edward Arnold and Co..

SUMMARY

London, 1949, p. lb. “I

SCHUMAKER, V. A., AND SCHACHMAN, H. K., Biochim. et Bio- phys. Acta, 23, 628 (1957).

MESELSON, M., STAHL, F. W., AND VINOGRAD, J., Proc. 1\Tatl. Acad. Sci. U. S., 43, 581 (1957).

HALL, C. E., ANI) LITT, M., J. Bioph.ys. Biochem. Cytol., 4, 1 (1958).

A method was developed for the large scale separation of calf liver nuclei which were free from gross contamination by other cellular particles and whole cells. The internal structure of the nuclei, as observed microscopically, was only slightly altered by the isolation procedure.

DAVISON, P. F., AND BUTLER, J. A. V., Biochim. et Biophys. Acta, 21, 568 (1956).

HURST, R. 0.. MARKO, A. M., AND BUTI~ER, G. C.. J. Biol. Chem., 204, 847 (1955).

DOUNCE. A. L.. AND O’CONNELL. M.. J. Am. Chem. Sot.. 80.

The isolated nuclei were washed several times with 0.15 ionic strength, pH 8 ethylenediaminetetraacetic acid buffer to remove soluble nuclear proteins. The washed nuclei were suspended in water. On removal of the nuclear remnants by centrifugation, a solution which contained over 900/;, of the nuclear deosyribonucleoprotein was obtained.

2013 (i958). ’ , , , ,

DAVIDSON, J. X., AND SMEIUE, R. M. S., Biochem. J., 62,594 (1952).

PETERMANN, M. L., AND LAMB, C. M., J. Biol. Chem.., 176, 685 (1948).

CRAMPTON, C. F., LIPSHITZ, R., AND CHARGAFF, E., J. Biol. Chem., 206, 499 (1954).

MIRSKY, A. E., ANI) POUISTER, A. W., J. Gen. Physiol., 30,117 (1946).

The nucleoprotein extract appeared to be free of most of the obvious cellular contaminants but always contained at least 5% ribonucleic acid.

19. 20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32. SCNMIDT, G., in E. CHARGAFF AND J. N. DAVIDSON (Editors), The nucleic acids, lrol. I, Academic Press, Inc., New York, 1955, p. 555.

The sedimentation and viscosity behavior of the nucleoprotein was studied in some detail, and the results were compared with those obtained from other deosyribonucleoprotein preparations.

ZIJBAY, G., Ph.D. thesis, Harvard University, Cambridge, Massachusetts (1957).

WIIXAMS, J. W., VAN HOLDE, K. E., BALDWIN, R. L., AND FUJITA, H., Chem. Rev., 68, 715 (1958).

IFFT, J. B., VOET, 11. H., AND VINOGRAD, J., J. Phys. Chem., 66, 1138 (1961).

Ac~~cnowledgments-We would like to thank Dr. Paul Doty, Harvard University, for the use of a Spinco Analytrol and an TJbbelohde viscometer, and Dr. Cecil Hall, Massachusetts Insti- tute of Technology, for the electron photomicrographs of a nucleoprotein preparation.

SUEOKA, N., Proc. n:atl. Acad. Sci. U. S., 46, 1480 (1959). HEARST, J. E., ANI) VINOGRAD, J., Proc. Matl. Acad. Sci. 7J. S.,

47, 825 (1961).

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S. L. Commerford, M. J. Hunter and J. L. OncleyThe Preparation and Properties of Calf Liver Deoxyribonucleoprotein

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