the structure of metaphase chromosomes · chromosomes were removed from ph 3.7 buffer and placed in...

J. Mol. Biol. (1970) 49, 213-229

The Structure of Metaphase Chromosomes I. Electrometric Titration, Magnesium Ion Binding

and Circular Dichroism

KENNETH P. CANTOR AND JOHN E. HEARST

Department of Chemistry and Grwp in Biophysics ad Medical Physics

Univeriiity of Calijorni4x Berkeley, Calij. 94720, U.S.A.

(Received 22 July 1969, and in revised form 9 December 1969)

Isolated mammalian metaphase chromosomes suspended in ion-free or 10d3 Y- N&l solution undergo an irreversible, pH-dependent structural change, as reflected by electrometric titrations, pH-stat experiments and measurements of circular dichroism spectra. Unmasking of titratable groups with pK’s lower than the isoionic pH of chromosomes in ion-free water, pH 66, accompanies the change in conformation. The structural change is inhibited in aqueous solutions of O-001 M- magnesium or calcium ion. Adding magnesium or calcium ions to a final concentration of 10m3 Y results in a lowering of the pH of the water-washed chromosome solution from 56 to 4.9. The binding constant for magnesium ion to chromosomal sites was measured to be 1-l x 106 (moles/liter)-r and the number of binding sites is 16 x lows millimole per milligram of chromosomes.

1. Introduction Acidic solutions and divalent cations stabilize the morphology of metaphase chromosomes as revealed by studies in the light and electronmicroscope (Ris, 1967; Huberman & Attardi, 1966; Chorazy, Bendich, Borenfreund & Hut&son, 1963).

Availability of isolated metaphase chromosomes in milligram quantities (Huberman & Attardi, 1966; Maio & Schildkraut, 1967; Mendelsohn, Moore & Salzman, 1968; Cantor & Hearst, 1966) has opened the door to application of physical and chemical techniques in the study of chromosome structure. We have examined chromosome structure, with special emphasis on the stabilizing role of magnesium ion, by electrometric titrations, pH-stat experiments, circular dichroism spectra and measurement of the binding constant for magnesium.

2. Materials and Methods (a) hokdon of metapham chromosomes

Metaphase chromosomes of L2 mouse ascites tumor cells (Shelton, 1962; Dunham & Stewart, 1963) were isolated by a modification of our earlier method (Cantor & Hearst, 1960). Each female Heston mouse (age 60 to 80 days) was injected intraperitoneally with lo* ascites tumor cells suspended in O-1 ml. TC-199 medium (D&o). After 4 days, 30 pg colchicine (Calbiochem), freshly dissolved in O-3 ml. physiological (0.9%) saline solution were injected intraperitoneally. 17 hr later, the animal was sacrificed by cervical dislocation and its peritoneal cavity opened. The ascitic fluid (approx. 2 ml.) was removed by suction into a flssk immersed in an ice bath. Two l-ml. washings of the peritoneal cavity with

213

214 K. P. CANTOR AND J. E. HEARST

Hanks’ b8lsnced salt solution (Hanks & Wellace, 1949) were pooled with the ascitio fluid. In 8ll steps which followed, temperature ~8s kept between 0 and 4°C. The following volumes, etc., 8pply to the ascitic fluid and washings obtained from 6 mice.

The material ~8s centrifuged at 800 rev&n (80 g) for 6 min in a clinical centrifuge. The pellet, containing cells in metaphase rtrrest, interphase cells 8nd erythrocytes, ~8s washed with 10 ml. H8nks’ solution, centrifuging 8s above. The resulting pellet w8e washed twice with 10 ml. H8nks’ solution which had been diluted with water to 8 quarter strength. Erythrooytes were lysed in this medium, and lysis products renmined in the supernatant fmction during centrifugetion. A&tic lymphocytes were killed and swollen, but few were lysed.

The pellet from the second hypotonic washing ~8s suspended in 176 ml. pH 3-7 buffer, p = O-01 (Perrin, 1963) conteining 1 maa-MgClz (hereafter, pH 3.7 buffer). Cells were homogenized in 8 VirTis model 23 mixer at 9000 rev&in (two-thirds maximum voltage) for 12 min, after 8ddition of 4 g VirTis no. H3640 fine homogenizing beads. Homogermte consisted of free chromosomes, undisrupted cells, interphase nuclei and cellular debris. Homogentlte volume wa8 increased to 200 ml. with pH 3.7 buffer. This ~8s divided equally and sedimented et 1000 rev&in (280 g) in an Interncltionrtl centrifuge, sedimenting most unbroken cells and interph8se nuclei but few chromosomes. Supernatsnt fractions were set aside and each pellet w8e resuspended in 100 ml. pH 3.7 buffer 8nd sedimented under the seme conditions. Supernetrtnt fmctions were pooled with the previous ones, and the pellets resuspended 8s before. This ~8s repeated until the supernatant fraction ~8s cleer (0.D .$&, less than O-6), indiceting that most of the chromosomes had been removed. Five or six spins accomplished this. Chromosomes and remaining interph8se nuclei and unbroken cells were sedimented by centrifuging the pooled supernatant fm&ions at 1900 rev./min (1000 g) for 46 min. After discarding supernatsnt solution, sedimented materi was suspended in 176 ml. pH 3.7 buffer and dispersed by briefly homogenizing in the VirTis 23 mixer at 9000 rev./mm To remove remaining nuclei end undisrupted cells, the sequence of slow-speed (280 g) sedimentation steps ~8s repeated, pooling supern8ttant fractions and resuspending pellets in pH 3*7 buffer between suocessive spins. Suspended chromosomes of the pooled supernatant fmctions were sedimented at 1900 rev&in (1000 g) for 46 min to remove residual soluble material and to reduce suspension volume. After discarding the supern8mnt liquid, the sediment ~8s suspended in pH 3.7 buffer and chromosomes were dispersed in the VirTis mixer. Plate I shows chromosomes from this fln8l step.

(b) Elect~metric titratim of c~mosomee

Continuous recording electrometric titrations were performed in the microch8mber 8tt8chment of 8 Radiometer SBR2c titrrttor coupled to a Radiometer TPTl pH meter. Titrations were run at ambient tempereture (20 to 23°C) and the oh8mber ~8s continuslly flushed with nitrogen. Chert and pen-drive motors of the titrator were selected so th8t a titration sp8nning 6 pH units took approxinmtely 46 min. A magnetic stirrer, part of the Rrtdiometer equipment, was used in 8ll experiments. Delivery syringes were o8libr8ted by weighing the Bmount of meraury disch8rged per unit trsvel of the pen along the chart (in percentage of total available titmnt added), end converting to volume.

Potassium 8cid phthalate W&B used in standardization of the KOH solutions used 8s beeic titrent (Skoog t West, 1963). Acidic titrant, HCl, ~8s standardized by titrating r&u& standlerdized solutions of KOH. End points were measured eleotrometric8lly.

Approlcimately 4 mg of chromosomes were removed from pH 3.7 buffer 8nd added to ion-free water or aqueous solution with the desired ealt by centrifuging 8 chromosome suspension and taking up the ohromosom81 pellet in the desired solution. Buffer ~8s completely removed by repe8ting this 6 times. The final pellet (volume approx. 0.6 ml.) wae suspended in 7 ml. of the desired solution which had ilrst been boiled to remove carbon dioxide. 6 ml. of suspension were used in the titr8tions. The remeiuing portion ~8s divided into 0.6.ml. portions which were added to 2 ml. of O-1 N-N&OH prior to measurement of o.n.zse. Chromosome concentration ~8s oelcul8ted by using the extinction coefficient of chromosomes (&,) in basic solution (04.9, Centor & Hearst, 1903). The tot81 amount of ohromosomes in the 6-ml. sample was between 16 and 30 O.D.peO units in most experiments.

PLATE I. A micrograph of isolated metaphase chromosomes after all purification steps. ( x 690.)

STRUCTURE OF METAPHASE CHROMOSOMES. I 216

A titration curve of the solvent without chromosomes was taken for each experiment. Titration curves of chromosomes were corrected by subtracting the solvent titration curve after the latter had been adjusted to the volume of the former. Data were read from the titrator output charts for chromosomal and blank samples at intervals of O-2 pH unit. A digital computer was used for the calculations (Cantor, 1969). The calculation included corrections for titrant concentration, the syringe calibration and chromosome concentration so that the data could be expressed as milliequivalents titrant per milligram chromosomes. Included in the computer program was an optional section to calculate the slopes of titration curves. All titration curves and their respective “first derivatives” of this paper are plots from such calculations.

(c) pH-stut experinaenta

A radiometer TTTI pH meter and SBR2c titrator, in pH-stat mode, were used. 6 ml. of chromosome suspension, washed and measured for chromosome concentration as in titrations, were used in a microchamber with water-jacket for temperature control. The chamber was continually flushed with argon which had been passed through a concentrated KOH solution at the same temperature as the reaction chamber. This completely removed carbon dioxide, and brought the water in the gas to the same vapour pressure as that above the suspension, minimizing evaporation or condensation of solution during an experiment. Standardized KOH titrant was added from a syringe calibrated as in the titration experiments. A magnetic stirrer was used. pH was automatically maintained at a value between 5-O and 7.0, depending on the experiment. Chart speed for most experiments was 30 mm/hr. Data were read from the output chart of the titrator at intervals small enough to ensure adequate representation when subsequently plotted. Data were corrected for titrant concentration, syringe calibration and chromosome concentration for expression as milliequivalents titrant per milligram chromosomes as a function of time.

Experiments with blank solutions containing no chromosomes showed a small steady- state addition of titrant, presumably from carbon dioxide contamination. This wae also reflected in a small steady-state addition of titrant to chromosome samples after the initial rapid rate of titrant addition had subsided (Fig. 1). pH-stat data for eaoh experiment were corrected by subtracting the amount of titrant added in steady-state from the total amount added. The resulting points appeared to follow first-order kinetics. Constants A and T in the expression Y = A (1 - exp( - t/r)}, d escribing the reaction, were determined by a curve-fitting computer program. Theoretical curves using these constants, along with experimental data from which they were calculated, are plotted in Fig. 4.

(d) Effect of addition of magneahn chloride to chromosomea mapended in water

Chromosomes were washed in ion-free water and their concentration determined as in titration experiments. 6 ml. of suspension were placed in the pH-stat microchamber, and the pen-syringe drive was adjusted to the moat rapid setting. All other experimental conditions, including syringe calibration, titrant standardization, and use of magnetic stirrer, were as described above. When the pH-stat had commenced regulating at pH 6.25, the argon delivery tube into the microchamber was removed and argon pressure was used to blow a MgCl, solution (0.100 M or O-200 Y) from a lambda pipette into the chromosome suspension. The 6nal MgClc concentration in most experiments was 1 mu. pH immediately decreased, and the amount of released hydrogen ion was reflected by the amount of basic titrant required to bring pH back to its starting value.

(e) Meaawement of the binding con&ant a& number of chrmomrnal binding eitea for magn&um ion

Divalent cation was removed from all glassware by rinsing in 0.1 M-EDTA followed by several rinses in doubly distilled water. Chromosomes were removed from pH 3.7 buffer and placed in double-distilled water as in titration experiments, except the sample was washed a total of 7 instead of 6 times. The final pellet of washed chromosomes was suspended in 27 ml. water. The suspension was divided into five 6*00-ml. portions, and the remaining material was used in determining chromosome concentration by measurement


of o.D.,~,, after dilution with 0.1 N-NaOH. Final chromosome concentration for most experiments wss 8pproximately 0.6 mg/ml.

A fresh solution of Eriochrome Black T wss prepared for esch run of the experiment by dissolving 0*016 g Eriochrome Black T indicator, (Matheson, Coleman & Bell) in 100 ml. double-distilled water. 16 ml. of this solution was mixed with 4 ml. buffer made by diluting 2136 ml. aqueous ammonia (sp. gmvity 030) and 35 g NH&l to 600 ml. with water (Skoog & West, 1903). When diluted fivefold for measurement of Mgl+ concentration (see below), 6nal pH of the Eriochrome Black T buffer solution was 10.12 to 10.18. All MgCls solutions were dilutions of a 1.00 1y1 stock solution.

Blanks contained 6.00 ml. double-distilled water. A multiple of 10 d. of 6 x 10e3 M- MgCls was added to all but one chromosome sample and its corresponding blank. Additions in a typical experiment were 10,20,30 and 60 4. Tubes containing chromosome suspension were centrifuged at the highest speed in e clinical centrifuge for 16 mm., leaving unbound magnesium ions in the supernatent fraction. 4 ml. of eupernatit solution from each tube containing chromosomes, and its corresponding blank, were removed for assay of Mgs+.

4 ml. each of a series of &&Cl, standards ranging in concentration from 10S6 to 10m4 Y was used for calibration. The standard representing zero concentration Mgl + consisted of 0.01 IJ-EDTA.

1 ml. of Eriochrome Black T-buffer solution was added to each of the chromosomal supernatant solutions, blanks and MgCl, standard solutions. After mixing, the samples were kept at room temperature for 30 mm prior to meeeurement Of 0.D.67g.p and O.D.gpo in a Gary 14 spectrophotometer. 0.D.e20 for the stand&r& was plotted as a function of Mg”+ concentration after correcting the measurement using the light absorb- at the isosbestic wavelength (679.2 mp). Magnesium ion concentration for the chromosome ssmples and corresponding blanks were obtained by reference to this standard curve.

Ultraviolet circular dichroism spectra of chromosomes suspended in ion-free water and in 10m3 M-M~C~, were measured in a model 6001 circular dichroism attachment of a Gary 60 spectropohwimeter. Temperature wse regulated at 10°C. Chromosome samples were removed from pH 3.7 buffer by exhaustive w&ring as in titration experiments. Prior to introduction of a Bemple into the instrument, it was diluted to an O.D.&&,,, of approximately one. After taking a spectrum the sample was removed and divided into several portions for measurement of 0.D.160 in 0.1 N-NaOH for exact deter&n&ion of concentration. A baee line wss taken for each spectrum, which was corrected accordingly.

The Gary 60 is coupled to an on-line PDP 8/S computer (Digital Computer Corp.) which was used to smooth spectra, correct data for chromosome concentration, end correct for base-line shifts. Besults for seveml runs of the same spectrum with different chromosome samples were averaged with a desk calculator.

3. Results (a) Chvmo8omes in the absence of div&nt cation

(i) Titration behavior Chromosomes were stored in pH 3.7 buffer until shortly before the titrations. When

washed chromosomea were suspended in water, either ion-free or containing lOa M- N&l, and placed in the microchamber of the titrator, pH decreased from approximately 7. Decrease in pH was rapid at 6rst, and slowed until, after a few minutes, the rate of decrease was imperceptible between pH 6.6 and 6.7. pH decrease was due to chromosomes, and not to absorption of carbon dioxide, as pH of a control solution without chromosomes remained at 7.

The forward, back and reversal of back titration (reverse back titration) of chromosomes in the pH range 4.7 to 9.0 is shown in F’igure 2. The most striking feature of these curves is their irreversibility. When the suspension was returned, in the back


FIG 1. Addition of titrent to chromosomes in the pH-stat et pH 6.22. The straight line through the last points haa been extrepoleted to t = 0 for oorreotion of the d&8.

titration, to the starting pH of the forward titration (approx 5*6), an average net 2.24 milli-equivalent hydroxyl ions per mg chromosomes had been added to the suspension (average of five experiments), reflecting the release of this number of hydrogen ions in the course of the forward and back titrations (at pH 5.6). When the forward titration was repeated after completion of the back titration (reverse back titration, Fig. 2), the shape of the back titration curve was essentially reproduced. Thus titratable groups unmasked during the forward titrations did not return to their original configurations.

The pH was lowered to 4.98 (average of four experiments, range 4.83 to 5.12) when the same amount (in m-equiv./mg chromosomes) of acidic titrant as basic titrant was added to a chromosome suspension the pH of which had been raised from 5.6 to between 8 and 9 in the forward titration (Fig. 2). From these data, and plots of the slopes to titration curves, it appears that groups normally titrating below pH 6 are unmasked

PH

FIQ. 2. Electrometric titretion curvea of chromosomes in ion-free water. When the a8me number of m-equiv. 8oidic IN basic titrant bee been added, pH is 4.98.


during the forward titration, in which they have an apparent pK of 6-7. Their irreversible exposure to the bulk solution results in the decrease of their apparent pK’s to 5.7.

Titrating chromosomes suspended in 10 - 3 na-NaCl gave the same results as described above.

The irreversible ohange in uhromosomal structure during titration is not solely a function of pH. This was first observed in the titrations plotted in Figure 3. In this

h

PH

FIU. 3. Eleotrometrio titration ourve8 of ohromosomed ~uapended in ion-free water. The reverse beck titration curve of this a& is similar to that of F’ig. 3, ehowing that the irreversibility ia not dely a function of PH.

experiment, chromosomes suspended in water at pH 555 were titrated with base to pH 6.9; a back titration to the starting pH was run; and finally a reverse back titration ourve was taken. The back titration and reverse back titration curves of Figure 3 are not ooinaident, as are the comparable curves of Figure 2. The reverse back titration curves of the two titration experiments above (Figs 2 and 3) are the same within experimental error, showing that groups exposed when the pH was increased from 5.6 to 8.4 and then returned, were also exposed when pH was increased from 555 to 6-9 and returned. This suggests that the structural change which gives rise to different titration behavior might be a slow, time-dependent phenomenon occurring at pH values below pH 6.9.

(ii) pH-&at ezperimmts

As previously discussed, when fully washed chromosomes in suspension were plaoed in the pH-stat microchamber, pH drifted down from 7 at an ever decreasing rate until further pH ohange became imperceptible at pH 5.6. In these experiments, spontaneous decrease of pH was allowed to continue to a value between 5.6 and 7, at which point addition of base was commenced to regulate pH at a constant value. Volume of added titrant as a function of time was recorded.

Results from two such experiments, at pH 6.22 and 5.73, are shown in Figure 4. The solid curves of this figure are the beet least squares fit of the plotted experimental data to an equation of the form:

Y = A(1 - exp( - t/T)).

This equation describes a first-order &ion, where Y is the total amount of titrant added at time t, A is the asymptotic limit of total added titrant, and T is the time-


0 130 200 300 t:Time hid

FIQ. 4. pH-stat results for addition of basic titrant to chromosomes. The curves are the best least squares fit to the experimental data points shown using Y =

A{ 1 - exp( - t/l)}. The upper two curves are for chromosomes in ion-free water. -O-O--. pH 6.22, + = 33 min, A, = 2.95 x 10 --1 ra-equiv./mg; -.-.-, pH 6.73,~ = 67 min, A = 2.07 x 10 -4 M-equiv./mg. The dotted line (... q . . . 0.e.) represents the result for chromosomes in 10 -3 M-Mgcl,, pH 4.95.

constant of the reaction. The excellent fit of the data to an equation of this form indicates that hydrogen ion release from metaphase chromosomes in the pa-stat is indeed a first-order reaction. The reaction is spontaneous and its rate is pH-dependent. In the pH range over which this experiment was repeated (5.6 to 7*0), the reaction proceeded more rapidly at high than at low pH. This is reflected in the curves of Figure 4.

Following addition of base in the pH-stat, chromosomes are similar, with respect to exposed titratable groups, to post-transition chromosomes of titration experiments. There is little difference between the shapes of the forward and back titration curves of chromosomes after coming to equilibrium in the pH-stat, showing that the transition associated previously with the forward titration occurred during the addition of base in the pH-stat.

A quantitative comparison between the results of pH-stat addition of base to a chromosome suspension in water and electrometric titration curves of chromosomes indicates that the same number of titratable groups is exposed in both processes. At pH 6.22 in the pH-stat, the curve approaches an asymptotic limit of 2.94~ lo-* m-equiv. KOH per milligram chromosomes; at pH 6.73, it approaches 2.07 x lo-* m-equiv. per mg. An average of the data from four titration experiments shows a difference between the forward and reverse-back titration curves at pH 6.22 of 3.25 x lo-* m-equiv. per mg, and at pH 6.73 of 2.3 x IO-* m-equiv per mg. The results of these two methods at each pH are the same within experimental error. We conclude from the above, that release of hydrogen ion in the pH-stat and irreversible titration behavior of chromosomes are two manifestations of the same phenomenon.

(b) Chromosomea in the presence of divalent cu.&ion (i) Isoionic pH and pH-stat re-sults

When chromosomes (which had been thoroughly washed in lob3 &f-MgCl,, 10e3 M- CaCI,, or ion-free water) were suspended in 10m3 M-MgCl, or CaCl, and placed in the


titration chamber, the pH was approximately 4.9 (in five experiments, pH fell in the range 4.83 to 6.02, with an average of 4.93). This contrasts with behavior in suspensions containing no divalent cation, where initial pH was about 7, and drifted down to stabilize at approximately 6.6.

The broken curve of Figure 4 is the pH-stat result from chromosomes suspended in 10S3 a~MgCl,. A suspension of ohromosomes was placed in the temperature-regulated ohamber of the pH-stat and the instrument was sot to maintain overnight a pH of 4.95. There was no unmasking of aaidic groups with pK lower than 4.96, as indicated by an absence of basio titrant addition. In addition, there was no pH increase. Thus there was no observable struotural ohange with time in chromosomes suspended in 10V3 M-

a6g a+4, in contrast to the observations of ohromosomes in salt-free medium or in low3 M-NaCl.

The electrometric titration curves for chromosomes suspended in 10e3 BZ-MgCl, are presented in Figure 5. Hesults for chromosome samples suspended in 10m3 r&aCl,

PH

FIO. 6. Eleotrometrio tit&ion ourvea of metnphaee ohromoeomee suqmded in 10 -B M-Mgcl,. The ourvea are redble below pH 8.

were the same. The large amount of t&ant addition in the pH range 6.5 to 7.0, ocmrring when divalent oation was not present, was not observed in the presence of lo-” X-Mgs+ or Caa + . The curves are reversible, as the bask titration ourve resembles that of the forward titration curve, most olosely in the range below pH 8. First derivatives to the forward and back titrations, indicating the relative number of groups titrating at any pH, are ooincident between pH 5-8 and 8. Thus the same numbers and types of groups titrate over this range during the forward and back titration. Titra- table groups with pK’s in this range inalude the a-amino of N-terminal amino aoids (pK = 7.4 to 7.9) and imidazole of histidines (pK = 6.4 to 7.0). Others sre possible, since the pK is partially dependent on the oharge array surrounding any group. As with ohromosomes in salt-free or monovalent-ion solution, ohromosomes in 10e3 M- Mga+ or Ca” + show irreversible titration behavior above pH 8.

In summary, presence of either MgI + or Ca’ + (10 -a M) results in a forward titration ourve whioh at&x at a pH below that observed in ion-free chromosome suspensions

STRUCTURE OF ‘METAPHASE CHROMOSOMES. I 221

or suspensions containing Ne+ . Presence of divalent cation results in reversible titration ourves over the range where there was marked irreversibility in the absenoe of divalent cation.

(iii) In$unce of divalent cation concentration on chrowwad titration behuvkr

The first derivatives to a series of four forward titration curves of chromosome samples suspended in solution containing from 10m5 to 10S3 M-M~C~, are shown in Figure 6. A plot of the first derivatives refleots the relative number of groups titrating

PH

Fm. 6. First derivativee to the forwerd titration curves of chromoaomea aqended in MgCI, solutiona of the indioated concentrations: (- ) lo-“aa; (----) 2x 10-6&f; (----) 6x10-e& (. . . , . .) lo-SM.

in any pH range and may serve as a guide in determining which groups are titrating. At the lowest concentration, 10Sb Y, the curve is essentially the same aa those for titrations in ion-free media. At the high end of the magnesium concentration range, 10m3 M, the pH 7 peak of the 6rst derivative curve is not present, and there is a broad peak with a maximum at about pH 6-7. As Mga + concentration increases from 10W6 Y, there is a decrease in the height of the pH 7 peak tmd an increase in the pH 6-7 peak. Disappearance of the pH 7 peak in first derivatives to the forward titration, accom- panied by the appearance of a peak at lower pH in the back titration, is typical of titration in solution without divalent cation. The similarity between post-transition titration curves (in media without divalent cation) and titration curves in Oe3 M- MgCl, suggests structural similarity between chromosomes under these conditions.

The effect of magnesium ion on chromosomal structure is not due to ionic strength of the suspending medium, but is specifIcelly a function of the cation valency. In 10S3 Y- N&l (ionic strength, CL, = 10m3), the forward titration curve and associated lirst- derivative curve are similar to those for chromosomes suspended in ion-free water. In the first derivative to the forward titration the ratio of groups titrating at pH 5.7 to those titrating at pH 6.5 to 7 was much less than 1.0. In contrast, this ratio in the presence of MgCl, at ionio strength 1.6 x10-* (6~10~~ M) was 1-O and increased with ionic strength. Thus, at ionic strength 10 -3, titration behavior of chromosomes in MgCl, was different from that in N&l, and the difference is specifically attributable to the valency of the respective ions.

222 K. P. CANTOR AND J. E. HEARBT

(iv) Add&ion of ntugne.sium ion.9 to dromoeome.9 in h-free medium

As discussed above, the pH of a chromosome suspension in lob3 M-MgCl, (- 4.9) was below that of a sample in ion-free solution or in 10m3 nf-NaCl. To study this further, MgCl, was added to chromosomes in ion-free medium at pH 6.25 in the pH- stat. In most experiments, magnesium ion concentration of the chromosome suspension was brought to lOa M. When this was done, there was an immediate release of hydrogen ion from ohromosomes. The time constant for this release was less than 10 seoonds, the most rapid rate measurable with our apparatus. In the pH-stat, bringing MgCl, concentration to 10m3 Y resulted in the release, at pH 6.26, of 3.4 x lo-* m-mole hydrogen ion per milligram chromosome (range of five experiments, 3-l x 10 - * to 3-6 x 10 - 4 m-mole/mg). When the final Mga + concentration was greater than 10S3 M, the amount of released H + was in the above range, indicating saturation of magnesium binding sites at 10e3 M-Mg a + . A calculation, from the number of binding sites and binding constant for magnesium, of chromosomal sites (see below) conlirms this.

The number of hydrogen ions released upon titrating chromosomes in ion-free solution to basic pH, and then back to acidic pH is the same, within experimental error, as the number released upon addition of Mga+ to 10e3 M. The diiIerenoe at pH 6-25 between the forward and back titration curves of chromosomes in water in five experiments was 3-O to 4.0 x lo-’ m-mole/mg, with an average of 3.3 x 10e4 m-mole per milligram chromosomes.

(v) Protection of chromosomd &u&we by mqne.sium ions

The structural state of chromosomes is protected by magnesium ions. When chromosomes were washed exhaustively in 10S3 M-MgCl, and then titrated while in this medium, the titration curves were reversible. When, prior to titration, magnesium ion was removed by several washings in ion-free water, the resulting titration curves were the same as those of chromosomes in water; i.e. there was irreversible titration behavior. Thus, the structural change induced by the interaction of met&phase chromosomes with magnesium ion is a reversible one. The chromosomal structure which gives rise to reversible titration behavior in the presence of magnesium ion is lost when Mga+ is removed.

(c) T&&able amino acids of chromoeomd proteins

Figure 7 is a reverse titration curve over the maximum pH range experimentally available. The number of certain titratable amino acids in chromosomal protein may be calculated using the amount of dry weight protain in chromosomes, 68.3% (Cantor t Hearst, 1966), an average amino acid residue weight of 109 (Walker, 1965), and the back titration curve of Figure 7. Taking Walker’s (1965) range for the titration of carboxyl groups of glutamic and aspartic acid (pH 4-O to 6.76) and correcting for the oontribution of DNA and RNA to the titration curve in this range (Gulland, Jordan & Taylor, 1947; Cox, Jones, Marsh & Peacooke, 1956), we calculate a total of 124 exposed carboxylic acid groups per 1000 amino aoids. In nucleohistone, 32 groups titrate in this region (Walker, 1965).

Analysis of the titration curve above pH 6.76 is not as straightforward. The pH ranges for titration of imidazole groups of histidine (pK = 6.4 to 7-O), a-amino groups of N-termina 1 amino aaids (pK = 7-4 to 7.9), and sulfhydryl groups of cysteine (pK = 8-3) overlap (Edsall & Wyman, 1958), and the contribution from each group separately


PH

FIG. 7. Electrometric baok titration ourve of chromosomes in 10 --3 aa-MgCl, over the maximum pH range experimentally available.

cannot be determined from the titration data alone. 5’7 groups per 1000 amino acids in chromosomes titrate between pH 6.75 and pH 9-O. If the average length of chromosomal protein is 150 ammo acids, approximately seven of these are a-amino’s of N- terminal amino acids. Histidine and cysteine together thus comprise 50 groups per 1000 amino acids. On the basis of titration results and amino acid analysis, Walker (1965) found in nucleohistone 18 histidine residues and no sulfhydryls. In chromosomes, groups in excess of those found in nucleohistone must be in the non-hi&one protein.

(d) Binding of magnesium by chmmm.mea

Eriochrome Black T is a dye which, upon binding magnesium, undergoes spectral shifts in the visible wavelength range. Stoichiometry of the Eriochrome Black T- magnesium complex is dependent on pH and the relative concentrations of cation and dye (Harvey, Kormamy & Wyatt, 1953; Young & Sweet, 1955; Sohwarzenbaoh & Biedermann, 1948; Cantor, manuscript in preparation). Because of the various complexes of Eriochrome Black T with magnesium ion, a simple method was used for measuring magnesium concentration. The o.D.~~,, was measured for each of a set of solutions containing a known concentration of Mga + (1 x 10-s to 1 x 1O-‘3 M) and the same amount of Eriochrome Black T and buffer used for unknowns. Absorbance was plotted as a function of magnesium ion concentration, and Mg2+ concentration of unknowns was read from this plot after measuring their absorbance at 620 mp.

The mathematical model used for determinin g the binding constant and number of chromosomal binding sites for Mga+ assumes (1) the number of magnesium binding sites on chromosomes is constant and does not change as more sites are occupied, (2) the binding constant for all sites is the same, and (3) magnesium ion binds to chromosomal sites in a 1 : 1 ratio. Since the magnesium ion assay was effective for concentrations greater than 1 x 10m6 M, the assumptions listed above, especially the first dealing with the absence of co-operative effects, could not be tested below this Mg2+ concentration.


The binding constant between magnesium ion and chromosomal sites is defined as

K = ~@I -, where [PlgS] is the concentration of bound Mga+ or of occupied sites, [Mgl [Sl

[Mg] is the concentration of unbound magnesium, and [S] is the conoentration of unoccupied chromosomal sites. We define #? as the fraction of total magnesium bound

@&3Sl to chromosomal sites: p = [Mg] + ms]. Thus, the fraction of total magnesium which

remains free is 1 - /3, and the expression for K may be rewritten R = P (1-P) [Sj The

total concentration of binding sites, both free and occupied, is [S], = [S] + [MgS], where [S] and [MgS] are the concentrations respectively of unbound and bound sites. Substituting for [S] in the expression for K, and rearranging terms, we obtain:

wi3Sl = - j$ (k) + F%

Experimentally, we measured the concentration of total magnesium and of free magnesium, [Mg], for suspensions oontaining different conoentrations of magnesium ion but the same concentration of chromosomes. MgS] and b were aalculated from these data and [MgS] was plotted as a function of /?/(l -/I) after the data had been adjusted to a chromosome conoentration of 1 mg/ml. (Fig. 8). A straight line is the best fit to the data points. This result suggests that the assumptions used in deriving the above equation are oorreot, since ourves of other shapes would be expeoted if they were not.

As oaloulated from the slope of Figure 8, the binding constant, K, is 1.06 x lo5 (moles/liter)-l. The concentration of binding sites is 1~59~10-~ mole per gram chromosomes. Accounting for experimental error leads to the conclusion that K = 1~1~10~f0~1x10~(moles/liter)~~andthenumberofeitesL1~6x10-~f.0~1x10-~ millimole per milligram chromosomes.

Pm. 8. WS] veruue j/( 1 - ,9) for the meaure mat with Eriochrome Black T of the binding con&ant and number of ohromoaomal binding pitea for magnesium ion. A complete explanation of thia Figure is given in the text.

K = l-06 x lo6 (melee/liter) -I. [S], = 1.69 x 10 -6 m-mole sites

mg chromosomes


(e) Circulur dichroimn spectra of metaphme chmmosome-s Circular dichroism spectra of chromosomes were taken under solvent conditions

corresponding to those used in titration experiments. The circular dichroism spectra of chromosomes in water without divalent cation at different pH’s (see below) are presented in Figure 9. The two scales reflect the respective contributions of chromosomal nucleic acid and protein, so that our results may be compared with published circular dichroism spectra of nucleic acids, synthetic polypeptides and proteins.

Mean residue ellipticity is defined as:

[S] = fg$

In this expression, [e], mean residue ellipticity, is in deg.-cma/decimole; M,, mean residue weight, is in g/mole; 8”, measured ellipticity, is in degrees; c, sample concentration, is in g/ml.; end 1, pathlength, is in cm.

Wavelength (mp)

FIG. 9. Ultrwiolet circuler diohroism spectra of chromosomes in ion-free water: apeotrum A, at pH 6-6; epectrmn C, after titration to pH 7.0, and qsectrum B, after back titration to pH 6.6. Explanations of the error bare and scales are given in the text.

In calculating the protein (left side) scale of Figure 9, we used a mean residue weight of 109 (Walker, 1966), the protein content of our metaphase chromosome sample, 68.3% (Cantor & Hearst, 1966), and the chromosome concentration of each sample from a measurement of O.D.ZBo of chromosome concentration of each sample from a measurement of O.D.,,o of chromosomes in basic solution. For the nucleic acid scale (right side, Fig. 9), we used 340 for the mean residue weight of sodium DNA and RNA nucleotides, and the analytical result for total chromosomal nucleic acid, 27.0% (Cantor & Hearst, 1966). DNA and RNA were considered together, since the mean residue weight of ribo-and deoxyribonucleotides differ by only 5%, and both are found in equal &mounts in ascites tumor mettlphase chromosomes (Cantor & Hearst, 1966). Circular dichroism were first plotted in degrees ellipticity after normalizing the data to a chromosome concentration of one milligram per millimeter. This scale was then adjusted, using the infornmtion above, to express results as ellipticity per decimole amino acid and per decimole of nucleic acid.

16


There was some aggregation and precipitation of chromosomes in the quartz circular diohroism cell, mostly occurring as the sample came to temperature equilibrium prior to a scan. When removing chromosomes from the quartz cells for concentration measurement, care was taken to pipette only suspended material. This was not always possible, and sometimes chromosomes which had not contributed to the observed ellipticity were included in the sample used to measure concentration.

Each spectrum of Figure 9 represents an average of three speotra of separate samples taken under identical conditions. Error bars indicate the range of value within each set of averaged spectra. The unusually large magnitude of the error is a reflection of the dif6culty in accurately measuring chromosome concentration in these experiments. Error flags of F’igure 9 reflect uncertainty in the absolute residual ellipticity at the indicated wavelengths. They do not indicate the error in relative ellipticity within any one curve. Overlap of the error flags of spectra B and C might be misleading, since in each separate experiment there was a decrease in the height of the negative circular dichroism peak at 226 to 227 rnp when the pH of the sample was lowered from 7 to 5.6.

The circular dichroism spectra of Figure 9 are of chromosomes (A) suspended in water after exhaustive washing by repeated centrifugation and suspension (pH approximately 6.6); (C) the sample of (A) brought to pH 7-O with KOH; and (B) the sample of (C) returned to pH 6-6 with HCl. Chromosomes in water at pH 56 (A) are in the pre-transition state observed in electrometric titrations. Chromosomes of the sample used for spectrum C are in the post-transition conformation. When pH was raised to 7.0 in titration experiments, the irreversible transition had nearly gone to oompletion and experiments in the pH-stat indicate that when chromosomes are maintained at this pH for 16 minutes or more, the transition goes to completion. These were the conditions under which spectrum C was taken. The pH was returned to 6.6 before spectrum B was taken to see whether conformational changes indicated by the ciroular dichroism speotra were irreversible or whether they were a simple reversible phenomenon assooiated with pH.

The circular dichroism spectra reveal a marked change iu chromosome structure when pH of a suspension is raised from 6.6 to 7-O. They also show that chromosomes do not return to their original conformation when pH is returned to 5.6. The most striking difference between the speotra is in the magnitude of the negative band appearing between 226 and 230 mp. Prior to addition of titrant, pre-transition chromosomes at pH 6-6 have an elliptioity at 230 rnp of N - 400 deg.-cm”/dmole (protein scale). Upon increasing the pH to 7.0, there is a blue shift of the peak to 226 rnp, and the magnitude of the band increases to approximately - 2300 deg.-cm’/ dmole. After taking the suspension back to the starting pH, 6.6, with HCl, negative ellipticity decreases to -1700 deg..cma/dmole, and the peak shifts slightly, to 228 rnp. A tentative analysis of the full abromosomal oircular dichroism spectrum is given by Cantor (1969).

It is possible that the height of the 226 to 230 rnp negative peak refle&s order in chromosomal protein. Ciroular dichroism spectra of nuclei0 aoids taken under a wide range of solvent, and hence structural, conditions do not show as great a variability in oiroular dichroism in the 226 to 230 rnp range as we observe for chromosomes (Wolfe, Cikawa & Kay, 1968; Sarker & Yang, 1967; D. Carroll, personal communication). Protein speotra, on the other hand, show a large struoture-dependent peak in this wavelength range (Hobwarth & Doty, 1966; Momma&s, 1966; Sarker & Doty,


1966; Timasheff & Gorbunoff, 1967). Another interpretation of these results is that of Urry & Ji (1968), who attribute similar low-wavelength band-shifts to distortions accompanying changes in the state of aggregation of particulate systems. The titration experiment demonstrates that a large number of titratable groups are exposed when going from conditions of spectrum A to spectrum C of Figure 9. This indicates that at least a portion of the increase in height of the 225 to 230 rnp band results from conformational changes in chromosomal protein.

4. Discussion (a) Post-transition chromosomes in ion-free medium

and chromosomes in 10e3M magnesium or calcium ions

A comparison of chromosomes following the irreversible titration transition in ion- free water (or in 10d3 M-NaCl) with chromosomes in the presence of 10e3 M-Mga+ or Ca2+ reveals similarity with respect to exposed titratable groups. Back and reverse- back titration curves of chromosomes in ion-free medium are similar in shape to titration curves taken in the presence of Mg2 + (Fig. lo), indicating that the types and numbers of exposed titratable groups are the same. The starting pH for titrations in

FIQ. ( . . . . .

E

PH

10. (-) F orward titration curve of chromosomes in 10 -3 M-M~C~, . ) the back titration curve of chromosomes in ion-free solution (from

(from Fig. Fig. 2).

5) and

divalent cation is 4.93. The pH of chromosomes suspended in water after addition of the same amount of basic and acidic titrant (raising pH to between 8 and 9, and then lowering it) was 4.98. This again points to the possibility that the same number and types of groups are exposed in the two cases. When a chromosome suspension in ion- free medium is made 10e3 M in magnesium ion, the chromosomes release 3-l x 10e4 m-mole hydrogen ion per milligram at pH 6-25. The difference, at pH 6.25, between the forward and back titration curves of chromosomes in water is 3-3 x 10m4 m-mole/ mg. Thus addition of magnesium ions to chromosomes results in exposure of the same number of titratable groups exposed when pH of a chromosome suspension in ion-free medium is raised above 7.

By the above criteria, metaphase chromosomes following the irreversible transition in ion-free solution and chromosomes suspended in lob3 M-Mg2 + are indistinguishable. The similarity of chromosomes under the two conditions does not extend to their respective circular dichroic spectra. When pH of a chromosome suspension in ion-free


solution was raised from 5.6 to 7 and then returned, the negative 226 to 230 rnp circular dichroism band increased in magnitude, reflecting a change in the conformation of chromosomal protein. Preliminary circular dichroism experiments with chromosomes suspended in 10m3 nr-Mgcl, show that a similar exposure of chromosomes to pH 7 and return results in no change in the circular dichroism spectrum. The circular dichroism spectrum of chromosomes in 1O-3 M-Mgcl, resembles that of chromosomes in ion-free medium at pH 5.6 before the irreversible transition. These experiments show that there are similarities between post-transition chromosomes in ion-free medium and chromosomes in 10e3 M-divalent cation, but chromosomes under the two conditions are not in identical structural states.

(b) A model for irreversible titratz& behavior of chromosomes

The first derivatives to the forward titration curves of chromosomes in ion-free medium (Fig. 2) and pH-stat results (Fig. 4) suggest a mechanism for changes in chromosome structure. There is a sharp peak at about pH 6.7 in the first derivative to the forward titration curve of chromosomes in ion-free medium, indicating that many groups release H+ around this pH. In the back titration, a broader peak appears at pH 5.7. We postulate that certain ionizable groups, titrating around pH 6.7, act as “triggers” in a structural transition. When these groups become ionized, due to an increase in pH (as in a forward titration) or because of statistical charge fluctuations when pH is near their pK (as in pH-stat experiments), they lose the ability to maintain their key bonding role, and there are changes in polypeptide conformation in their vicinity. When this occurs, many previously masked groups with pK’s less than 6.7 (e.g. carboxyl functions of aspartic or glutamic acid) are exposed to the bulk medium and immediately lose their hydrogen ions. In the back titration, these newly exposed groups titrate at a lower pH. pH-stat experiments show that liberation of hydrogen ion is a first-order reaction, and the rate of H+ release increases with pH over the range pH 5.6 to 7.0. Both of these observations support a model in which groups titrating in this pH range act as triggers in initiating large-scale structural changes, resulting in exposure of other, more acidic groups. With an increase in pH, the probability increases that any particular trigger residue will become ionized and an increase in the rate of H + release is in fact observed. The imidazole group of histidine has a pK between 6*4 and 7-O in most proteins (Edsall & Wyman, 1958), suggesting that histidine residues act as the postulated triggers. The large number of histidine residues indicated by analysis of the chromosomal titration curve (Fig. 7) supports this hypothesis.

(c) Magnesium bind@ sites of chromosomes

When a chromosome suspension in water at pH 6.26 is made 10e3 M in Mg2+, 3.4 x lo-* m-equiv. hydrogen ion per mg chromosomes is released. From the binding constant and number of magnesium binding sites of chromosomes, we calculate that more than 99% of the sites are occupied by Mga+ under the experimental conditions employed. Since there are 1.6~10-~ f O-1 ~10~~ millimole binding sites (Mg2+) per milligram chromosomes, 21 + 3 hydrogen ions are released for each bound magnesium ion. The release of this number of hydrogens for each magnesium ion means that the mechanism for liberation of H + is not due to competition for the same sites between Mg2 + and H + . Were this true, no more than two hydrogens per magnesium would be released. A more reasonable model has Mg2+ binding to sites of funda-


mental importance to chromosomal structure. The effect of magnesium suggests that Mg2 + binds to those sites which trigger the irreversible transition in ion-free solutions. This is an attractive possibility, because of the similarities between post-transition chromosomes and chromosomes in the presence of magnesium ions. A difference in the two states is indicated by the fact that chromosomal structure returns to its typical pre-transition state when Mg2 + is first added and then removed; but the structural changes caused by increasing pH or adding basic titrant at constant pH are irreversible.

The experiments described in this paper were performed by Kenneth P. Cantor in partial fuhilment of the requirements for the degree of doctor of philosophy at the Univer- sity of California at Berkeley. The work was supported primarily by U.S. Public Health Service grants no. GM 11180 and no. GM 15661. Support was also received from the American Cancer Society grant no. IN-87, NASA grants no. NsG 243 and NsG 479 and the John Simon Guggenheim Memorial Foundation. One of us (J.E.H.) is a John Simon Guggenheim Memorial Fellow (1969). We gratefully acknowledge the technical assistance of Laura Kayfetz with the preparation of the isolated metaphase chromosomes. We thank Professor I. Tinoco, Jr. for providing access to the Cary 60 spectropolarimeter.

REFERENCES

Cantor, K. P. (1969). Ph.D. thesis, University of California, Berkeley. Cantor, K. P. & Hearst, J. E. (1966). Proc. Nat. Ad. Sci., Wmh. 55, 642. Chorazy, M., Bendich, A., Borenfreund, E. & Hutchison, D. (1963). J. Cell Biol. 19, 59. Cox, R. A., Jones, A. S., Marsh, C. E. & Peacocke, A. R. (1956). Biochim. biophys. Acta, 21,

576. Dunham, L. J. & Stewart, H. L. (1953). J. Nut. Cancer In&. 13, 1203. Edsall, J. T. & Wyman, J. (1958). BiophyeicaZ Chemistry, vol. 1, p. 534. New York:

Academic Press. Gulland, J. M., Jordan, D. 0. & Taylor, H. F. W. (1947). J. Chem. Sot. 1131. Hanks, J. H. & Wallace, R. E. (1949). PTOC. Sot. Ezp. BioE. Med. 71, 196. Harvey, A. E., Jr., Kormamy, J. M. & Wyatt, G. M. (1953). Andyt. Chem. 25, 498. Holzwarth, G. & Doty, P. (1965). J. Amer. Chem. Sot. 87, 218. Huberman, J. A. & Attardi, G. (1966). J. CeEZ BioZ. 31, 95. Maio, J. J. & Schildkraut, C. L. (1967). J. Mol. BioZ. 24, 29. Mendelsohn, J., Moore, D. E. & Salzman, N. P. (1968). J. Mol. BioZ. 32, 101. Mommaerts, W. F. H. M. (1966). J. Mol. BioZ. 15, 377. Per&, P. D. (1963). Austrul. J. Chem. 16, 572. Ris, H. (1967). In Regulation of Nucleic Acid Synthe&e, ed. by V. V. Koningsberger and

L. Bosch, p. 11. Amsterdam; Elsevier Pub. Co. Sarker, P. K. & Doty, P. (1966). Proc. Nut. Acad. Sci., Wash. 55, 981. Sarker, P. K. t Yang, J. T. (1967). In Conformation of Biopolymera, ed. by G. N. Rama-

chandran, vol. 1, p.197. New York: Academic Press. Schwarzenbach, G. & Biedermann, W. (1948). Helv. chim. Acta, 31, 678. Shelton, E. (1952). J. Nat. Cancer In&. 12, 1203. Skoog, D. A. & West, D. M. (1963). Fundamental8 of Analytica Chemistry. New York:

Holt, Rinehart and Winston. Timasheff, S. N. t Gorbunoff, M. J. (1967). Ann. Rev. Biochem. 36, 13. Palo Alto: Annual

Reviews, Inc. Urry, D. W. & Ji, T. H. (1968). Arch. Biochem. Biophys. 128, 802. Walker, I. 0. (1965). J. Mol. BioZ. 14, 381. Wolfe, F. H., Oikawa, K. & Kay, C. M. (1968). Biochemistry. 7, 3361. Young, A. & Sweet, T. R. (1955). Anulyt. Chem. 27, 418.

the structure of metaphase chromosomes · chromosomes were removed from ph 3.7 buffer and placed in...

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