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Use of critical point polyacrylamide sols in thermal denaturation experiments with chromatin at physiological ionic strength
ANALYTICAL
BIOCHEMISTRY
( 1988)
172.296-303
Use of Critical Point Polyacrylamide Sols in Thermal Denaturation Experiments with Chromatin at Physiological Ionic Strength MARTINR.RIEHM Department
qf‘Biocherni.str~~. Received
AND
RODNEY E. HARRINGTON
Umversity
yf’Nevada.
November
Rena.
Nevada
89557
12. I987
Low percentage highly crosslinked polyacrylamide gels just above the critical point in the chemically polymerized sol to gel transition are used to generate polyacrylamide sols at critical samples mixed with point concentrations, 7.4 g liter-‘, by mild heating. We find that chromatin these sols induce the sol to gel transition in a process of complex coacervation. In this state, salt insoluble chicken erythrocyte chromatin is stabilized against large scale aggregation and precipitation during thermal denaturation at physiological sodium ion concentrations. The hyperchromic melting behavior of DNA in polyacrylamide sols is reproducible and consistent throughout a wide range of sodium chloride concentrations. Empirical spectroscopic techniques are discussed which isolate temperature-dependent hyperchromic signals at 260 nm due to conformational changes of DNA in chromatin and local environmental changes which promote anomalous light scattering. I’G 198X Academic Prsss, Inc. KEY WORDS: spectrophotometry: thermal denaturation; light scattering: chromatin: histones: nucleic acid structure.
water. Acrylamide and BIS’ were obtained from Bio-Rad. Acrylamide monomer stock solutions were made fresh weekly. Spectrapor dialysis tubing (Grade 1 with a 6000-8000 molecular weight cutoff) was used after a 30min soak in 0.25 tBM EDTA. Polyacrylamide gel slurries were generated by gently homogenizing low percentage, highly crosslinked polyacrylamide gels [ 120 ml, 1.9% (w/v) acrylamide, 0.2% (w/v) BIS, 0.065% (w/v) ammonium persulfate, 0.1% (v/v) TEMED, polymerized under aspirated vacuum for 90 min, plus 200 ml glass-distilled water] using a Thermovac homogenizer (1 min at a setting of 10, 3 min at a setting of 20). Large polyacrylamide particles were removed by centrifugation at 85OOg for 30 min (about 0.4% by weight). Excess acrylamide monomer, TEMED, and ammonium persulfate were removed by exhaustive dialysis of
Thermal denaturation has emerged as one of the more sensitive techniques to probe chromatin structural dynamics (l-3) and is used routinely to assay the structural integrity of the chromatin subunit, the nucleosome. In the past, the technique has been restricted to solutions in the unit millimolar range of sodium ion concentration to maintain the solubility of chromatin fragments and avoid problems with light scattering and aggregation. Here we describe a technique in which high molecular weight polyacrylamide suspensions are used to minimize diffusion-limited chromatin aggregation during heat-induced denaturation at a wide range of sodium ion concentrations. This allows spectroscopic monitoring of the unfolding and denaturation of chromatin which is involved in higher order structures such as the 30-nm fiber (reviewed in (4)). MATERIALS
AND
METHODS ’ Abbreviations
All chemicals used were reagent grade and all solutions were made with glass-distilled 0003-2697188
$3.00
Copyright 0 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.
amide:
amine. 296
TEMED,
used: BIS. N.N’-methylenebisacryiN, N. N’. K’-tetramethylethylenedi-
CHROMATIN
DNA
MELTING
IN
the slurry supernatant against water over a period of several weeks at room temperature. The slurries ‘so generated were quite turbid and maintained a pH of 6.8 to 6.9. After dialysis critical point polyacrylamide sols were generated from the slurries by autoclaving in untreated borosilicate glass bottles. The heat treatment resulted in complete clarification of the slurry and a reduction in pH to 5.0. This critical point sol is stable at room temperature for several months and does not appear to contain ammonia or undergo further hydrolysis which generates ammonium ion. Citrate-treated chicken blood was obtained from Pel-Freleze Biologicals. Chicken erythrocytes and nuclei were prepared according to the method of Libertini and Small (5). A salt insoluble fraction of whole chicken erythrocyte chromatin was extracted from micrococcal nuclease (Worthington)-digested erythrocyte nuclei according to the method of Fulmer and Bloomfield (6) as modified for sucrose density gradient fractionation by Ausio et a!. (7). DNA preparations were made from sucrose density gradient-fractionated salt insoluble chromatin using standard methods of proteinase K (EC 3.4.2 1.14, Boehringer-Mannheim) digestion and phenol/chloroform/isoamyl alcohol extraction with multiple 70% (v/v) ethanol precipitation steps to remove excess phenol. All chromatin and DNA preparations were exhaustively dialyzed to 0.2 mM EDTA before use. For histone analysis sodium dodecyl sulfate-polyacqlamide gel electrophoresis of 0.4 N HCI-extracted histones and high resolution scanning of Coomassie blue R-250stained gels was done as previously described (3). DNA size and distribution were determined using agarose gel electrophoresis (3). Chromatin-polyacrylamide stock sols were prepared at 4°C at concentrations at or above 200 pg ml-’ by diluting chromatin at 4 mg ml-’ 1:;!O with autoclaved polyacrylamide stock. These were gently mixed overnight at 4°C using rotary inversion with a glass bead. Quantitation of the DNA in stock
POLYACRYLAMIDE
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sols was done spectroscopically in 1-mm path length quartz cuvettes using an extinction coefficient of 20 liters g-’ cm-‘. NaCl-polyacrylamide stock sols (5 M) were prepared by dialyzing autoclaved polyacrylamide stock against saturated aqueous NaCl for 1 h at 90°C. The concentrated suspension was then dialyzed against 5 M NaCl for 3 h at the same temperature. The original volume was then reconstituted by adding fresh 5 M NaCl and the suspension clarified by heat treatment at 95°C for 1 h. Final NaCl concentration of the stock sol was determined by volumetric analysis of chloride according to the Fajans method (8). Other, more dilute. stock sols for EDTA and sodium phosphate were prepared by diluting concentrated aqueous solutions 1: 10 to 25 mM Na2EDTA and 1:5 to 100 mM NaH2P0,, pH 7.0, with autoclaved polyacrylamide sol. These salts were minor constituents in samples for thermal denaturation and direct dilution facilitated quantitation of final sodium content. Chromatin and chicken erythrocyte DNA samples for thermal denaturation in polyacrylamide were prepared at 22°C and quantitated gravimetrically in silicon (Sigmacoat)treated 25-ml sidearm flasks. DNA content averaged 0.73 i 0.07 Z4260,l-cm path length. Allowances were made for differences in density of the concentrated NaCl stock ~01s. Before stock NaCl was added, the chromatin samples were mixed on an orbit shaker with a glass bead for 30 s at 300 rpm to break up large micelles of chromatin and polyacrylamide. After salt was added, samples were degassed under mild vacuum for 30 min. Care was taken to avoid evaporation greater than 1%. Ultraviolet spectra were obtained routinely using an IBM Model 9420 dual beam uv-visible spectrophotometer. Thermal denaturation experiments at 260 and 320 nm were done on a Gilford Model 252 uv-visible spectrophotometer with a dynamic range of up to 3 optical density units (+O.OOl) equipped with a Model 2527 thermoprogrammer. Temperature increase was controlled at
29x
RIEHM
AND HARRINGTON
0.25”C mini’. Absolute absorbance and temperature data at 0.25”C intervals were collected as previously described (3). Data analysis included estimation of total hyperchromicity for DNA samples. This involved correcting for low and high temperature linear changes in absorbance according to the method outlined in (9) and deriving the temperature-dependent absorbance data as the temperature-dependent fraction of nucleotide bases no longer hydrogen bonded in the DNA helix (10). We found this approach suitable only for samples to which an unambiguous high temperature baseline could be assigned (generally below 70 mM Na+ or having transitions below 78°C). The transition melting point for these samples was estimated at 50% fraction of nucleotide bases hydrogen bonded and the hyperchromicity was extrapolated, using low and high temperature baselines ofthe original data, back to 22°C. Derivatives with a temperature bandwidth of 2.75”C were obtained by a running linear least-squares computer algorithm as previously described (3). RESULTS AND DISCUSSION
The goal of this work was to develop a method to stabilize chromatin against large scale aggregation at physiological ionic strength. Polyacrylamide was chosen due to its thermal stability, relative transparency in the near uv (the sols are transparent above 225 nm, O.l-cm path length; 235 nm, 1 cm path length), and biological compatibility (10). The polyacrylamide sols are chemically stable up to 100°C and samples can be prepared throughout the temperature range of 0 to 1OO’C and heated or cooled as needed. Chromatin and DNA polyacrylamide stocks were prepared at 4°C to limit sample degradation. Concentrated sodium chloride-polyacrylamide stocks were prepared at temperatures above 90°C and allowed to cool. Melting experiments with polyacrylamide sols were routinely done from 30 to 100°C. The ability of critical point polyacrylamide
sols to greatly minimize diffusion-limited chromatin aggregation is demonstrated in Fig. 1 and discussed further by Ottewill and Williams (11). Using photon correlation spectroscopy in concentrated “hard sphere” dispersions, these workers show that at a volume fraction of 0.5 the system passes a critical point and enters a “gel” phase where long range diffusion of tracer colloids literally freezes. Highly branched polymers, such as polyacrylamide, are most likely to exhibit an even greater ability to limit diffusion since the effective volume fraction is larger than that for polymers behaving as hard spheres. The greatest diffusion limiting effect would be with high molecular weight biopolymers such as DNA and chromatin while small solutes such as salt can still diffuse freely (12). Preliminary estimates based on the electrophoretie behavior of polyacrylamide sols suggests that the polyacrylamide is no less than 2 X IO6 Da in size, contains no more than 2.5 meq carboxylic acid residues per gram dry lyophilized weight, and is highly branched. Figure 1 shows a series of polyacrylamide concentration-dependent thermal denatur-
0 L.--t-+++30
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+-+-c-La 50
60
TEMPERATURE
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100
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FIG. I. Polyacrylamide concentration effects on the thermal denaturation of salt insoluble chicken erythrocyte chromatin at 100 mM sodium chloride. Polyacrylamide concentrations are (a) 7.4 mg ml-‘, (b) 3.1 mg ml-‘, (c) 2.1 mg ml-‘, (d) I .4 mg ml-‘, and (e) no polyacrylamide. For clarity of presentation. the melting curves were corrected to shift initial absorbances at 3o’C to 0.75.
CHROMATIN
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milliliter amounts of water were added to 50ation experiments with chromatin at 100 mM NaCl. With no polyacrylamide [Fig. I, trace ml gel volumes. There were no such problems (e)] the sample precipitates as seen by the loss in clarification when the dialyzed gel slurry was autoclaved. In this case, parts of the susin absorbance at 260 nm. As polyacrylamide concentration increases [Fig. I, traces (d)- pension around the edges of the bottle would (a)], absorbance loss due to precipitation is reach critical point after cooling. These increasingly limited, melting transitions be- would gel and drape off the edges of the glass come more pronounced, and noise in the and finally settle to the bottom of the bulk trace due to large scale aggregation and occlu- sol. This was taken as an indication that the sion of the light beam is subdued. From Fig. polyacrylamide sol was right at the critical 1. trace (a), the sol appears to have a critical point. point which varies roughly between 6.8 and The suspensions can be concentrated to 7.4 g liter-’ in the presence of chromatin with homogeneity beyond the critical point, but a mass average length of 9.5 kb (based upon only at temperatures above 90°C. Attempts estimates of DNA sizes from agarose gels), to concentrate critical point suspensions at depending upon whether gentle (less than room temperature led to extreme turbidity 100°C 3 h) or more severe (autoclaving) heat and generated steep temperature-dependent treatments are used. Gentle heat treatment exponential baselines in thermal denaturalso results in a decrease in pH to approxiation experiments due to optical changes in mately 5.5. Suspensions clarified using gentle the gel phase. Hence, every effort was made to heat treatment evolve ammonia after sitting keep samples at or slightly below the critical for several weeks. This, coupled with the in- point in polyacrylamide. There is some flexcrease in mass necessary to obtain a critical ibility in this because concentrated chromapoint for autoclaved preparations, suggests tin solutions induce the sol to gel phase tranthat the unwinding process and clarification sition, apparently in a process of complex cois a product of self-catalyzed acid hydrolysis acervation ( 12). This may be due in part to of the acrylamide polymer. differential hydration effects of chromatin Chromatin suspensions can be stabilized versus polyacrylamide and specific binding of for spectroscopy at high temperatures when basic histone protein to carboxylic acid resisamples are formulated at or near the critical dues of the polyacrylamide sol. The phase point. Certain details on preparation must be transition is evidenced by macroscopic apemphasized. We have not tried to vary the pearance of chromatin micelles which persist composition of the acrylamide or BIS cross- throughout the low salt range, are stable at linker from the recipe given above. The final temperatures above 100°C. diminish at 0.35 composition was determined by minimizing M NaCl, and completely disappear at 0.6 M, systematically total acrylamide while keeping the point where chromatin itself becomes solBIS constant to a point above which the re- uble. agents could no longer support gel formation The reproducibility of sample preparation after complete polymerization (judged spec- is illustrated in Fig. 2, which shows the derivtroscopically to be 90 min). The strategy ative melting profiles for chicken erythrocyte therefore wa:sto produce gels just above the DNA from 0.2 InM EDTA to 2 M NaCI in critical point for chemically induced gelation polyacrylamide sols normalized to input with maximum pore size and minimum fiber DNA absorbance at 260 nm. Hyperchromicbundle size (13). The amount of water added ity values obtained from 0.3 through 66 mM before homogenization is also important for sodium do not differ significantly between heat treatment and generating critical point DNA melted in polyacrylamide suspension SOIS. Sometimes clarification by gentle heat (36.3 3: 2.8%) and DNA in aqueous solution treatment occurred only when microliter to (36.6 k: 3.6%).
300
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The first effect (Fig. 3A) is a decrease in peak intensity at 260 nm due to spectral degradation of the incident light as it impinges upon the turbid solution. Such degradation can result in a 20 to 25% decrease in spectral
200
FIG. 2. Thermal denaturation of chicken erythrocyte DNA in critical point polyacrylamide sols from 0.3 mM to 2 M NaCI. Average polyacrylamide concentration was 7.2 2 0.2 mg ml-‘. DNA stock sols were prepared as discussed under Materials and Methods by diluting DNA 1: IO to a concentration of 100 mg ml-’ with autoclaved polyacrylamide sol. Thermal denaturation samples averaged 0.75 .&,a, l-cm path length. in DNA. [A] The melting curve series plotted as a function of log sodium chloride concentration (x-axis), with absorbance normalized to the input absorbance of DNA. In this case the view is from lower to higher temperatures since the normalized absorbance (Jj-axis) for each curve is projected orthogonally from 30 to 100°C along the temperature axis (zaxis) into the plane of the page. Isotherms are plotted at 1°C intervals parallel to the plane of the page. [B] The derivative melting profiles of the series in [A] plotted as a function of log sodium chloride concentration projected along the z-axis into the plane of the page. The view in this case is from lower to higher NaCI concentration with isotherms plotted at 1°C intervals. except at points where they cross the derivative maxima.
As shown in the normalized spectra of Fig. 3A and in the thermal denaturation experiments of Fig. 4, chromatin scatters a significant amount of light when it forms salt-induced higher order fibers. We have corrected for two effects of light scattering on the optical density of DNA in chromatin at physiological ionic strength based on turbidity at 320 nm.
400
300 WAVELENGTH
km)
0 NDRMALlZED
1.2
.6 hbs.
320nm
FIG. 3. Light-scattering corrections for chicken erythrocyte chromatin based on turbidity at 320 nm. [A] The sodium chloride-dependent spectra of salt insoluble chromatin in solution normalized to the absorbance of the DNA at 260 nm measured in 0.2 mM EDTA buffer. Sodium concentrations are (from bottom to top) 0.4 (0.2 mM Na2EDTA), 1 IO, 120, 140. 150, 160, 170, and 200 mM. [B] A graph ofthe fractional decrease in peak intensity at 260 nm due to spectral degradation as a function of turbidity at 320 nm. The correction for the decrease in peak intensity is one-half the ratio of minimum absorbance (at 236 nm for samples with no turbidity at 0.2 mM EDTA shifting to 244 nm at 200 mM NaCI) to maximum absorbance (at 258 nm at 0.2 mM EDTA shifting to 262 nm at 200 mM NaCI). The solid line represents a linear least-squares polynomial fit to the data of the form j’ = -5.36 X 10m3 + 0.504 (I) -0.527 (x)” + 0.199 (.u)~ with a standard interpolation error of kO.0057 and a correlation coefficient of 0.9972. The negative values at the intercept are a result ofsubtracting the ideal A,,,/J,,, ratio from raw data to obtain the turbidity-dependent correction factors used in this graph. [C] A graph of the turbidity-dependent shifts in spectral baselines above 320 nm due to Raleigh scatter. Data for the intercepts (solid circles) and slopes (solid triangles) of the spectral base lines were obtained by linear regression analysis of normalized spectra, some of which are plotted in [A]. Linear leastsquares fits to these data (solid line, intercepts: dashed line, slopes) all have correlation coefficients above 0.996. The temperature-dependent absorbance at 360 nm from chromatin melting experiments is first normalized to input DNA absorbance at 260 nm and then corrected first for decreases in peak intensity [B] and then baseline shift [C] using the normalized data from parallel thermal denaturation experiments at 320 nm.
CHROMATIN
DNA
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POLYACRYLAMIDE
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A second light-scattering effect is the increase in turbidity for nontransition wavelengths at and above 320 nm which results in a steep baseline. This is due to Raleigh light scattering. To correct for Raleigh baseline shift, we approximated the turbidity increase for the nontransition wavelengths using a linear least-squares fit. In this way we obtained intercepts and slopes of the baseline as linear functions of turbidity at 320 nm (Fig. 3C). With these corrections, it is possible to isolate the signal due to changes in DNA structure which are the result of either DNA melting (normal hyperchromicity) or anomalous light scattering at the nucleotide transition wavelength. First, correction factors due to spectral degradation based on parallel thermal denaturation experiments at 320 nm were obtained and added to the absorbance 109 [Nd] of samples normalized to input absorbance of the DNA at 260 nm at each temperature inFIG. 4. Thermal denaturation of salt insoluble chicken crement. Then the Raleigh baseline shifts erythrocyte chromatin in critical point polyacrylamide sols. Samples contained an average of 7.3 mg ml-’ polywere subtracted, again as a function of noracrylamide and were 0.75 &Or l-cm path length. in maiized turbidity in parallel thermal denaturDNA. [A] The sodium chloride series of melting experiation experiments at 320 nm, from the relaments from 0.2 mM EDTA to 200 mM NaCI. 5 mM tionships determined from Fig. 3C. These NaH,PO.,. 0.2 rflM EDTA monitored at 260 nm. As in were then extrapolated to correct the data at Fig. ?A, the view into the plane of the page is from low to high temperature with isotherms plotted at 1 “C inter260 nm. vals. [B] The parallel series monitored at 320 nm at the The results of these corrections are presame relative sca.le as in [A]. The temperature-dependent sented in Fig. 5 for chromatin at 200 mM soabsorbance at 260 and 320 nm is normalized to input dium chloride. From Fig. 4 it is clear that a DNA absorbance at 260 nm based on gravimetric prepacomparatively small hyperchromicity signal ration ofthe samples with stock sols quantitated spectroscopically as discussed under Materials and Methods. is superimposed upon larger increases in absorbance baseline due to changes in chromatin structure as salt concentration is inintensity at 260 nm (Fig. 3B). This is seen as creased. Also, comparing the derivatives of a general smearing and shifting of the DNA the uncorrected and corrected scans in Fig. 5, K to ?r* transition maximum in relation to the there are sharp turbidometric transitions at trough at 236 nm which separates the 200- 53 and 70°C along with the transition at 84°C nm rz to ir* transition peaks which include which we associate with the melting of the contributions from both DNA and protein. DNA helix (Fig. 5B). The turbidometric tranTo quantitatle this effect we calculated a cor- sition at 70°C is significant and can be associrection factor equal to one-half the ratio of ated with the denaturation and aggregation of minimum to maximum absorbance for chro- histone protein ( 14). The change in hypermatin samples along the salt-induced series chromicity and turbidity has a signal to noise of increasing turbidity. Figure 3B shows this ratio of about 26 for samples at 200 mM Na+ correction factor plotted as a function of ab- (Fig. 5A). The noise level increases somewhat sorption at 320 nm and fitted to a least- when scattering corrections are applied since the 320-nm scans are relatively weak and squares third-order polynomial.
RIEHM
AND
ra:
30
53 TEMPERhTURE
70
90 deq.
c
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HARRINGTON
t
50 TEYTRATLRE
78
90 deg.
c
FIG. 5. Uncorrected and corrected thermal denaturation profiles of chromatin at 200 mM sodium chloride. [A] A plot of the melting curve (dotted) and derivative profile (solid line) for salt insoluble chicken erythrocyte chromatin prepared and stabilized in critical point polyacrylamide sols as discussed under Materials and Methods containing 7.42 mg ml-’ polyacrylamide. 209.4 mM sodium (as NaCI, 0.22 mM EDTA. and 5.2 mM NaHZPOJ, pH 7.0). and DNA in chromatin at an .426L1 of 0.716. [B] A plot of the melting curve (dotted) and derivative profile (solid line) ofthe light scatter-corrected data as outlined in Fig. 3.
noisy compared to the 260~nm thermal denaturation data. We were able to alleviate this by using running averages over a temperature bandwidth of 5.25”C along the 320-nm scan for the light-scattering corrections. With this methodology, the signal to noise ratios are reduced to about 7.3 (Fig. 5B) for the corrected absorbance scans. The distinction between normal hyperchromic shifts due to DNA melting and anomalous light scattering at the transition wavelength is more apparent in the salt-dependent thermal denaturation of chromatin shown in Fig. 4. At sodium ion concentrations in the unit millimolar range, the corrected hyperchromic shift is 37.5%. In this case, the change in absorbance is due entirely to DNA melting. At intermediate Naf concentrations, the corrected hyperchromic shift reaches 46.9%, roughly 28% greater than expected. This can be compared to physiological sodium ion concentrations, at which the hyperchromic shift is only about 23.1% (Fig. 5B). We believe that these differences in hyperchromic shifts can provide important and unique information about the structural dynamics of DNA in chromatin during the pro-
cess of denaturation. At low sodium ion concentrations, the conditions under which prior thermal denaturation studies on chromatin have been conducted, the chromatin fiber is extended and its DNA denatures in a spectroscopically clear environment free of large denatured complexes. Above 100 mM sodium, chromatin fibers aggregate into dense bundles ( 15,16) large enough to scatter and spectrally degrade incident light. The hyperchromic effect due to melting of the nucleic acid helix appears to be partially obscured in this environment and is combined with discrete turbidometric transitions which we associate with histone denaturation. At intermediate ionic strengths, the hyperchromic signal appears to be enhanced. From 40 to 75 mM Na+ the extended 11-nm fiber of chromatin folds into the 30-nm fiber ( 17). Although this level of condensation results in a slight increase in light scattering (Fig. 4A), the DNA remains spectroscopically accessible. However, because of increased histone-histone interaction in the 30-nm fiber, large histone aggregates are able to form during thermal denaturation as suggested by the large increase in light scattering monitored at 320 nm at temperatures above 80°C in Fig. 4B. Histone aggregation appears to occur at the same point at which DNA itself is melting (Fig. 4A). The combination of a simultaneous increase in absorbance due to DNA melting and an increase in light scattering due to histone aggregation, all monitored at the nucleotide transition wavelength, seems to result in an amplified hyperchromic shift at intermediate sodium ion concentrations. ACKNOWLEDGMENTS We thank Dr. Jacob Waterborg and Dr. Gerald Fasman for encouragement and many valuable suggestions throughout the course of this work. Research was supported by U. S. Public Service Grant GM 33435. NIH Training Grant T32 CA 09563. and Hatch Project 13 I from the College of Agriculture, University of Nevada, Reno.
REFERENCES 1. Weischet, W. 0.. Tatchell, K.. Van Holde, K. E., and Klump. H. ( 1978) Nwleic .Icids Rex. 5, 139- 160.
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2. Cowman, M. K., and Fasman. G. D. (1980) Biochemisrry 19, 532-541. 3. Riehm. M. R., and Harrington. R. E. (1987) Biochrtnistr~~26, 2878-2886. 4. Felsenfeld. C;., and McGhee. J. D. (1986) Ccl1 44, 375-377. 5. Libertini, L. J.. and Small, E. W. (1980) IVzlclerc Acids Ra. 8, 35 17-3534. 6. Fulmer, A. W.. and Bloomfield. V. A. (198 1) Proc. Null. .-lead Sci. VS.1 78, 5968-5972. 7. Ausio. J., Sari. R.. and Fasman, G. D. (1986) Butchetnistry25. 1981-1988. 8. Skoog, D. A.. and West. D. M. ( 1976) Fundamentals of Analytical Chemistry. 3rd ed.. p. 728, Holt. Rinehart&Winston, New York. 9. Bloomfield. V. A., Crothers, D. M.. and Tinoco, I.. Jr. (1974) Physical Chemistry of Nucleic Acids. p. 302, Harper & Row, New York. 0. Revzin. A.. Ceglarek. J. A., and Garner. M. M. ( 1986) And Bioclwtn 153, 172- 177.
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1 I. Ottewill. R. H., and Williams, N. St. J. (1987)Na1~re (London) 325.232-234. 12. Morawetz. H. (1965) Macromolecules in Solution, High Polymers. Vol. 21, p. 88. 283-284. WileyInterscience, New York. 13. Gordon, A. H. ( 1969) Electrophoresis of Proteins in Polyacrylamide and Starch Gels, p. 12. Amer. Elsevier. New York. 14. Riehm. M. R. (1987) Protein-Dependent and SaltDependent Structural Stability of Eukaryotic Chromatin, Ph.D. dissertation. p. 83-84. University of Nevada. Reno. 15. Ausio. J.. Borochov. N.. Kam. Z.. Reich. M., Seger, D.. and Eisenberg. H. (1983) in Structure. Dynamics, Interactions and Evolution of Biological Macromolecules (Helene. C.. Ed.), pp. 89-100. Reidel, Dordrecht. 16. Widom. J. (1986)J. iwol. Biol. 190,411-424. 17. Harrington, R. E. ( 1985) Biochetnis~rv 24, 201 I2021.
BIOCHEMISTRY
( 1988)
172.296-303
Use of Critical Point Polyacrylamide Sols in Thermal Denaturation Experiments with Chromatin at Physiological Ionic Strength MARTINR.RIEHM Department
qf‘Biocherni.str~~. Received
AND
RODNEY E. HARRINGTON
Umversity
yf’Nevada.
November
Rena.
Nevada
89557
12. I987
Low percentage highly crosslinked polyacrylamide gels just above the critical point in the chemically polymerized sol to gel transition are used to generate polyacrylamide sols at critical samples mixed with point concentrations, 7.4 g liter-‘, by mild heating. We find that chromatin these sols induce the sol to gel transition in a process of complex coacervation. In this state, salt insoluble chicken erythrocyte chromatin is stabilized against large scale aggregation and precipitation during thermal denaturation at physiological sodium ion concentrations. The hyperchromic melting behavior of DNA in polyacrylamide sols is reproducible and consistent throughout a wide range of sodium chloride concentrations. Empirical spectroscopic techniques are discussed which isolate temperature-dependent hyperchromic signals at 260 nm due to conformational changes of DNA in chromatin and local environmental changes which promote anomalous light scattering. I’G 198X Academic Prsss, Inc. KEY WORDS: spectrophotometry: thermal denaturation; light scattering: chromatin: histones: nucleic acid structure.
water. Acrylamide and BIS’ were obtained from Bio-Rad. Acrylamide monomer stock solutions were made fresh weekly. Spectrapor dialysis tubing (Grade 1 with a 6000-8000 molecular weight cutoff) was used after a 30min soak in 0.25 tBM EDTA. Polyacrylamide gel slurries were generated by gently homogenizing low percentage, highly crosslinked polyacrylamide gels [ 120 ml, 1.9% (w/v) acrylamide, 0.2% (w/v) BIS, 0.065% (w/v) ammonium persulfate, 0.1% (v/v) TEMED, polymerized under aspirated vacuum for 90 min, plus 200 ml glass-distilled water] using a Thermovac homogenizer (1 min at a setting of 10, 3 min at a setting of 20). Large polyacrylamide particles were removed by centrifugation at 85OOg for 30 min (about 0.4% by weight). Excess acrylamide monomer, TEMED, and ammonium persulfate were removed by exhaustive dialysis of
Thermal denaturation has emerged as one of the more sensitive techniques to probe chromatin structural dynamics (l-3) and is used routinely to assay the structural integrity of the chromatin subunit, the nucleosome. In the past, the technique has been restricted to solutions in the unit millimolar range of sodium ion concentration to maintain the solubility of chromatin fragments and avoid problems with light scattering and aggregation. Here we describe a technique in which high molecular weight polyacrylamide suspensions are used to minimize diffusion-limited chromatin aggregation during heat-induced denaturation at a wide range of sodium ion concentrations. This allows spectroscopic monitoring of the unfolding and denaturation of chromatin which is involved in higher order structures such as the 30-nm fiber (reviewed in (4)). MATERIALS
AND
METHODS ’ Abbreviations
All chemicals used were reagent grade and all solutions were made with glass-distilled 0003-2697188
$3.00
Copyright 0 1988 by Academic Press. Inc. All rights of reproduction in any form reserved.
amide:
amine. 296
TEMED,
used: BIS. N.N’-methylenebisacryiN, N. N’. K’-tetramethylethylenedi-
CHROMATIN
DNA
MELTING
IN
the slurry supernatant against water over a period of several weeks at room temperature. The slurries ‘so generated were quite turbid and maintained a pH of 6.8 to 6.9. After dialysis critical point polyacrylamide sols were generated from the slurries by autoclaving in untreated borosilicate glass bottles. The heat treatment resulted in complete clarification of the slurry and a reduction in pH to 5.0. This critical point sol is stable at room temperature for several months and does not appear to contain ammonia or undergo further hydrolysis which generates ammonium ion. Citrate-treated chicken blood was obtained from Pel-Freleze Biologicals. Chicken erythrocytes and nuclei were prepared according to the method of Libertini and Small (5). A salt insoluble fraction of whole chicken erythrocyte chromatin was extracted from micrococcal nuclease (Worthington)-digested erythrocyte nuclei according to the method of Fulmer and Bloomfield (6) as modified for sucrose density gradient fractionation by Ausio et a!. (7). DNA preparations were made from sucrose density gradient-fractionated salt insoluble chromatin using standard methods of proteinase K (EC 3.4.2 1.14, Boehringer-Mannheim) digestion and phenol/chloroform/isoamyl alcohol extraction with multiple 70% (v/v) ethanol precipitation steps to remove excess phenol. All chromatin and DNA preparations were exhaustively dialyzed to 0.2 mM EDTA before use. For histone analysis sodium dodecyl sulfate-polyacqlamide gel electrophoresis of 0.4 N HCI-extracted histones and high resolution scanning of Coomassie blue R-250stained gels was done as previously described (3). DNA size and distribution were determined using agarose gel electrophoresis (3). Chromatin-polyacrylamide stock sols were prepared at 4°C at concentrations at or above 200 pg ml-’ by diluting chromatin at 4 mg ml-’ 1:;!O with autoclaved polyacrylamide stock. These were gently mixed overnight at 4°C using rotary inversion with a glass bead. Quantitation of the DNA in stock
POLYACRYLAMIDE
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sols was done spectroscopically in 1-mm path length quartz cuvettes using an extinction coefficient of 20 liters g-’ cm-‘. NaCl-polyacrylamide stock sols (5 M) were prepared by dialyzing autoclaved polyacrylamide stock against saturated aqueous NaCl for 1 h at 90°C. The concentrated suspension was then dialyzed against 5 M NaCl for 3 h at the same temperature. The original volume was then reconstituted by adding fresh 5 M NaCl and the suspension clarified by heat treatment at 95°C for 1 h. Final NaCl concentration of the stock sol was determined by volumetric analysis of chloride according to the Fajans method (8). Other, more dilute. stock sols for EDTA and sodium phosphate were prepared by diluting concentrated aqueous solutions 1: 10 to 25 mM Na2EDTA and 1:5 to 100 mM NaH2P0,, pH 7.0, with autoclaved polyacrylamide sol. These salts were minor constituents in samples for thermal denaturation and direct dilution facilitated quantitation of final sodium content. Chromatin and chicken erythrocyte DNA samples for thermal denaturation in polyacrylamide were prepared at 22°C and quantitated gravimetrically in silicon (Sigmacoat)treated 25-ml sidearm flasks. DNA content averaged 0.73 i 0.07 Z4260,l-cm path length. Allowances were made for differences in density of the concentrated NaCl stock ~01s. Before stock NaCl was added, the chromatin samples were mixed on an orbit shaker with a glass bead for 30 s at 300 rpm to break up large micelles of chromatin and polyacrylamide. After salt was added, samples were degassed under mild vacuum for 30 min. Care was taken to avoid evaporation greater than 1%. Ultraviolet spectra were obtained routinely using an IBM Model 9420 dual beam uv-visible spectrophotometer. Thermal denaturation experiments at 260 and 320 nm were done on a Gilford Model 252 uv-visible spectrophotometer with a dynamic range of up to 3 optical density units (+O.OOl) equipped with a Model 2527 thermoprogrammer. Temperature increase was controlled at
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0.25”C mini’. Absolute absorbance and temperature data at 0.25”C intervals were collected as previously described (3). Data analysis included estimation of total hyperchromicity for DNA samples. This involved correcting for low and high temperature linear changes in absorbance according to the method outlined in (9) and deriving the temperature-dependent absorbance data as the temperature-dependent fraction of nucleotide bases no longer hydrogen bonded in the DNA helix (10). We found this approach suitable only for samples to which an unambiguous high temperature baseline could be assigned (generally below 70 mM Na+ or having transitions below 78°C). The transition melting point for these samples was estimated at 50% fraction of nucleotide bases hydrogen bonded and the hyperchromicity was extrapolated, using low and high temperature baselines ofthe original data, back to 22°C. Derivatives with a temperature bandwidth of 2.75”C were obtained by a running linear least-squares computer algorithm as previously described (3). RESULTS AND DISCUSSION
The goal of this work was to develop a method to stabilize chromatin against large scale aggregation at physiological ionic strength. Polyacrylamide was chosen due to its thermal stability, relative transparency in the near uv (the sols are transparent above 225 nm, O.l-cm path length; 235 nm, 1 cm path length), and biological compatibility (10). The polyacrylamide sols are chemically stable up to 100°C and samples can be prepared throughout the temperature range of 0 to 1OO’C and heated or cooled as needed. Chromatin and DNA polyacrylamide stocks were prepared at 4°C to limit sample degradation. Concentrated sodium chloride-polyacrylamide stocks were prepared at temperatures above 90°C and allowed to cool. Melting experiments with polyacrylamide sols were routinely done from 30 to 100°C. The ability of critical point polyacrylamide
sols to greatly minimize diffusion-limited chromatin aggregation is demonstrated in Fig. 1 and discussed further by Ottewill and Williams (11). Using photon correlation spectroscopy in concentrated “hard sphere” dispersions, these workers show that at a volume fraction of 0.5 the system passes a critical point and enters a “gel” phase where long range diffusion of tracer colloids literally freezes. Highly branched polymers, such as polyacrylamide, are most likely to exhibit an even greater ability to limit diffusion since the effective volume fraction is larger than that for polymers behaving as hard spheres. The greatest diffusion limiting effect would be with high molecular weight biopolymers such as DNA and chromatin while small solutes such as salt can still diffuse freely (12). Preliminary estimates based on the electrophoretie behavior of polyacrylamide sols suggests that the polyacrylamide is no less than 2 X IO6 Da in size, contains no more than 2.5 meq carboxylic acid residues per gram dry lyophilized weight, and is highly branched. Figure 1 shows a series of polyacrylamide concentration-dependent thermal denatur-
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FIG. I. Polyacrylamide concentration effects on the thermal denaturation of salt insoluble chicken erythrocyte chromatin at 100 mM sodium chloride. Polyacrylamide concentrations are (a) 7.4 mg ml-‘, (b) 3.1 mg ml-‘, (c) 2.1 mg ml-‘, (d) I .4 mg ml-‘, and (e) no polyacrylamide. For clarity of presentation. the melting curves were corrected to shift initial absorbances at 3o’C to 0.75.
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milliliter amounts of water were added to 50ation experiments with chromatin at 100 mM NaCl. With no polyacrylamide [Fig. I, trace ml gel volumes. There were no such problems (e)] the sample precipitates as seen by the loss in clarification when the dialyzed gel slurry was autoclaved. In this case, parts of the susin absorbance at 260 nm. As polyacrylamide concentration increases [Fig. I, traces (d)- pension around the edges of the bottle would (a)], absorbance loss due to precipitation is reach critical point after cooling. These increasingly limited, melting transitions be- would gel and drape off the edges of the glass come more pronounced, and noise in the and finally settle to the bottom of the bulk trace due to large scale aggregation and occlu- sol. This was taken as an indication that the sion of the light beam is subdued. From Fig. polyacrylamide sol was right at the critical 1. trace (a), the sol appears to have a critical point. point which varies roughly between 6.8 and The suspensions can be concentrated to 7.4 g liter-’ in the presence of chromatin with homogeneity beyond the critical point, but a mass average length of 9.5 kb (based upon only at temperatures above 90°C. Attempts estimates of DNA sizes from agarose gels), to concentrate critical point suspensions at depending upon whether gentle (less than room temperature led to extreme turbidity 100°C 3 h) or more severe (autoclaving) heat and generated steep temperature-dependent treatments are used. Gentle heat treatment exponential baselines in thermal denaturalso results in a decrease in pH to approxiation experiments due to optical changes in mately 5.5. Suspensions clarified using gentle the gel phase. Hence, every effort was made to heat treatment evolve ammonia after sitting keep samples at or slightly below the critical for several weeks. This, coupled with the in- point in polyacrylamide. There is some flexcrease in mass necessary to obtain a critical ibility in this because concentrated chromapoint for autoclaved preparations, suggests tin solutions induce the sol to gel phase tranthat the unwinding process and clarification sition, apparently in a process of complex cois a product of self-catalyzed acid hydrolysis acervation ( 12). This may be due in part to of the acrylamide polymer. differential hydration effects of chromatin Chromatin suspensions can be stabilized versus polyacrylamide and specific binding of for spectroscopy at high temperatures when basic histone protein to carboxylic acid resisamples are formulated at or near the critical dues of the polyacrylamide sol. The phase point. Certain details on preparation must be transition is evidenced by macroscopic apemphasized. We have not tried to vary the pearance of chromatin micelles which persist composition of the acrylamide or BIS cross- throughout the low salt range, are stable at linker from the recipe given above. The final temperatures above 100°C. diminish at 0.35 composition was determined by minimizing M NaCl, and completely disappear at 0.6 M, systematically total acrylamide while keeping the point where chromatin itself becomes solBIS constant to a point above which the re- uble. agents could no longer support gel formation The reproducibility of sample preparation after complete polymerization (judged spec- is illustrated in Fig. 2, which shows the derivtroscopically to be 90 min). The strategy ative melting profiles for chicken erythrocyte therefore wa:sto produce gels just above the DNA from 0.2 InM EDTA to 2 M NaCI in critical point for chemically induced gelation polyacrylamide sols normalized to input with maximum pore size and minimum fiber DNA absorbance at 260 nm. Hyperchromicbundle size (13). The amount of water added ity values obtained from 0.3 through 66 mM before homogenization is also important for sodium do not differ significantly between heat treatment and generating critical point DNA melted in polyacrylamide suspension SOIS. Sometimes clarification by gentle heat (36.3 3: 2.8%) and DNA in aqueous solution treatment occurred only when microliter to (36.6 k: 3.6%).
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The first effect (Fig. 3A) is a decrease in peak intensity at 260 nm due to spectral degradation of the incident light as it impinges upon the turbid solution. Such degradation can result in a 20 to 25% decrease in spectral
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FIG. 2. Thermal denaturation of chicken erythrocyte DNA in critical point polyacrylamide sols from 0.3 mM to 2 M NaCI. Average polyacrylamide concentration was 7.2 2 0.2 mg ml-‘. DNA stock sols were prepared as discussed under Materials and Methods by diluting DNA 1: IO to a concentration of 100 mg ml-’ with autoclaved polyacrylamide sol. Thermal denaturation samples averaged 0.75 .&,a, l-cm path length. in DNA. [A] The melting curve series plotted as a function of log sodium chloride concentration (x-axis), with absorbance normalized to the input absorbance of DNA. In this case the view is from lower to higher temperatures since the normalized absorbance (Jj-axis) for each curve is projected orthogonally from 30 to 100°C along the temperature axis (zaxis) into the plane of the page. Isotherms are plotted at 1°C intervals parallel to the plane of the page. [B] The derivative melting profiles of the series in [A] plotted as a function of log sodium chloride concentration projected along the z-axis into the plane of the page. The view in this case is from lower to higher NaCI concentration with isotherms plotted at 1°C intervals. except at points where they cross the derivative maxima.
As shown in the normalized spectra of Fig. 3A and in the thermal denaturation experiments of Fig. 4, chromatin scatters a significant amount of light when it forms salt-induced higher order fibers. We have corrected for two effects of light scattering on the optical density of DNA in chromatin at physiological ionic strength based on turbidity at 320 nm.
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FIG. 3. Light-scattering corrections for chicken erythrocyte chromatin based on turbidity at 320 nm. [A] The sodium chloride-dependent spectra of salt insoluble chromatin in solution normalized to the absorbance of the DNA at 260 nm measured in 0.2 mM EDTA buffer. Sodium concentrations are (from bottom to top) 0.4 (0.2 mM Na2EDTA), 1 IO, 120, 140. 150, 160, 170, and 200 mM. [B] A graph ofthe fractional decrease in peak intensity at 260 nm due to spectral degradation as a function of turbidity at 320 nm. The correction for the decrease in peak intensity is one-half the ratio of minimum absorbance (at 236 nm for samples with no turbidity at 0.2 mM EDTA shifting to 244 nm at 200 mM NaCI) to maximum absorbance (at 258 nm at 0.2 mM EDTA shifting to 262 nm at 200 mM NaCI). The solid line represents a linear least-squares polynomial fit to the data of the form j’ = -5.36 X 10m3 + 0.504 (I) -0.527 (x)” + 0.199 (.u)~ with a standard interpolation error of kO.0057 and a correlation coefficient of 0.9972. The negative values at the intercept are a result ofsubtracting the ideal A,,,/J,,, ratio from raw data to obtain the turbidity-dependent correction factors used in this graph. [C] A graph of the turbidity-dependent shifts in spectral baselines above 320 nm due to Raleigh scatter. Data for the intercepts (solid circles) and slopes (solid triangles) of the spectral base lines were obtained by linear regression analysis of normalized spectra, some of which are plotted in [A]. Linear leastsquares fits to these data (solid line, intercepts: dashed line, slopes) all have correlation coefficients above 0.996. The temperature-dependent absorbance at 360 nm from chromatin melting experiments is first normalized to input DNA absorbance at 260 nm and then corrected first for decreases in peak intensity [B] and then baseline shift [C] using the normalized data from parallel thermal denaturation experiments at 320 nm.
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A second light-scattering effect is the increase in turbidity for nontransition wavelengths at and above 320 nm which results in a steep baseline. This is due to Raleigh light scattering. To correct for Raleigh baseline shift, we approximated the turbidity increase for the nontransition wavelengths using a linear least-squares fit. In this way we obtained intercepts and slopes of the baseline as linear functions of turbidity at 320 nm (Fig. 3C). With these corrections, it is possible to isolate the signal due to changes in DNA structure which are the result of either DNA melting (normal hyperchromicity) or anomalous light scattering at the nucleotide transition wavelength. First, correction factors due to spectral degradation based on parallel thermal denaturation experiments at 320 nm were obtained and added to the absorbance 109 [Nd] of samples normalized to input absorbance of the DNA at 260 nm at each temperature inFIG. 4. Thermal denaturation of salt insoluble chicken crement. Then the Raleigh baseline shifts erythrocyte chromatin in critical point polyacrylamide sols. Samples contained an average of 7.3 mg ml-’ polywere subtracted, again as a function of noracrylamide and were 0.75 &Or l-cm path length. in maiized turbidity in parallel thermal denaturDNA. [A] The sodium chloride series of melting experiation experiments at 320 nm, from the relaments from 0.2 mM EDTA to 200 mM NaCI. 5 mM tionships determined from Fig. 3C. These NaH,PO.,. 0.2 rflM EDTA monitored at 260 nm. As in were then extrapolated to correct the data at Fig. ?A, the view into the plane of the page is from low to high temperature with isotherms plotted at 1 “C inter260 nm. vals. [B] The parallel series monitored at 320 nm at the The results of these corrections are presame relative sca.le as in [A]. The temperature-dependent sented in Fig. 5 for chromatin at 200 mM soabsorbance at 260 and 320 nm is normalized to input dium chloride. From Fig. 4 it is clear that a DNA absorbance at 260 nm based on gravimetric prepacomparatively small hyperchromicity signal ration ofthe samples with stock sols quantitated spectroscopically as discussed under Materials and Methods. is superimposed upon larger increases in absorbance baseline due to changes in chromatin structure as salt concentration is inintensity at 260 nm (Fig. 3B). This is seen as creased. Also, comparing the derivatives of a general smearing and shifting of the DNA the uncorrected and corrected scans in Fig. 5, K to ?r* transition maximum in relation to the there are sharp turbidometric transitions at trough at 236 nm which separates the 200- 53 and 70°C along with the transition at 84°C nm rz to ir* transition peaks which include which we associate with the melting of the contributions from both DNA and protein. DNA helix (Fig. 5B). The turbidometric tranTo quantitatle this effect we calculated a cor- sition at 70°C is significant and can be associrection factor equal to one-half the ratio of ated with the denaturation and aggregation of minimum to maximum absorbance for chro- histone protein ( 14). The change in hypermatin samples along the salt-induced series chromicity and turbidity has a signal to noise of increasing turbidity. Figure 3B shows this ratio of about 26 for samples at 200 mM Na+ correction factor plotted as a function of ab- (Fig. 5A). The noise level increases somewhat sorption at 320 nm and fitted to a least- when scattering corrections are applied since the 320-nm scans are relatively weak and squares third-order polynomial.
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FIG. 5. Uncorrected and corrected thermal denaturation profiles of chromatin at 200 mM sodium chloride. [A] A plot of the melting curve (dotted) and derivative profile (solid line) for salt insoluble chicken erythrocyte chromatin prepared and stabilized in critical point polyacrylamide sols as discussed under Materials and Methods containing 7.42 mg ml-’ polyacrylamide. 209.4 mM sodium (as NaCI, 0.22 mM EDTA. and 5.2 mM NaHZPOJ, pH 7.0). and DNA in chromatin at an .426L1 of 0.716. [B] A plot of the melting curve (dotted) and derivative profile (solid line) ofthe light scatter-corrected data as outlined in Fig. 3.
noisy compared to the 260~nm thermal denaturation data. We were able to alleviate this by using running averages over a temperature bandwidth of 5.25”C along the 320-nm scan for the light-scattering corrections. With this methodology, the signal to noise ratios are reduced to about 7.3 (Fig. 5B) for the corrected absorbance scans. The distinction between normal hyperchromic shifts due to DNA melting and anomalous light scattering at the transition wavelength is more apparent in the salt-dependent thermal denaturation of chromatin shown in Fig. 4. At sodium ion concentrations in the unit millimolar range, the corrected hyperchromic shift is 37.5%. In this case, the change in absorbance is due entirely to DNA melting. At intermediate Naf concentrations, the corrected hyperchromic shift reaches 46.9%, roughly 28% greater than expected. This can be compared to physiological sodium ion concentrations, at which the hyperchromic shift is only about 23.1% (Fig. 5B). We believe that these differences in hyperchromic shifts can provide important and unique information about the structural dynamics of DNA in chromatin during the pro-
cess of denaturation. At low sodium ion concentrations, the conditions under which prior thermal denaturation studies on chromatin have been conducted, the chromatin fiber is extended and its DNA denatures in a spectroscopically clear environment free of large denatured complexes. Above 100 mM sodium, chromatin fibers aggregate into dense bundles ( 15,16) large enough to scatter and spectrally degrade incident light. The hyperchromic effect due to melting of the nucleic acid helix appears to be partially obscured in this environment and is combined with discrete turbidometric transitions which we associate with histone denaturation. At intermediate ionic strengths, the hyperchromic signal appears to be enhanced. From 40 to 75 mM Na+ the extended 11-nm fiber of chromatin folds into the 30-nm fiber ( 17). Although this level of condensation results in a slight increase in light scattering (Fig. 4A), the DNA remains spectroscopically accessible. However, because of increased histone-histone interaction in the 30-nm fiber, large histone aggregates are able to form during thermal denaturation as suggested by the large increase in light scattering monitored at 320 nm at temperatures above 80°C in Fig. 4B. Histone aggregation appears to occur at the same point at which DNA itself is melting (Fig. 4A). The combination of a simultaneous increase in absorbance due to DNA melting and an increase in light scattering due to histone aggregation, all monitored at the nucleotide transition wavelength, seems to result in an amplified hyperchromic shift at intermediate sodium ion concentrations. ACKNOWLEDGMENTS We thank Dr. Jacob Waterborg and Dr. Gerald Fasman for encouragement and many valuable suggestions throughout the course of this work. Research was supported by U. S. Public Service Grant GM 33435. NIH Training Grant T32 CA 09563. and Hatch Project 13 I from the College of Agriculture, University of Nevada, Reno.
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2. Cowman, M. K., and Fasman. G. D. (1980) Biochemisrry 19, 532-541. 3. Riehm. M. R., and Harrington. R. E. (1987) Biochrtnistr~~26, 2878-2886. 4. Felsenfeld. C;., and McGhee. J. D. (1986) Ccl1 44, 375-377. 5. Libertini, L. J.. and Small, E. W. (1980) IVzlclerc Acids Ra. 8, 35 17-3534. 6. Fulmer, A. W.. and Bloomfield. V. A. (198 1) Proc. Null. .-lead Sci. VS.1 78, 5968-5972. 7. Ausio. J., Sari. R.. and Fasman, G. D. (1986) Butchetnistry25. 1981-1988. 8. Skoog, D. A.. and West. D. M. ( 1976) Fundamentals of Analytical Chemistry. 3rd ed.. p. 728, Holt. Rinehart&Winston, New York. 9. Bloomfield. V. A., Crothers, D. M.. and Tinoco, I.. Jr. (1974) Physical Chemistry of Nucleic Acids. p. 302, Harper & Row, New York. 0. Revzin. A.. Ceglarek. J. A., and Garner. M. M. ( 1986) And Bioclwtn 153, 172- 177.
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1 I. Ottewill. R. H., and Williams, N. St. J. (1987)Na1~re (London) 325.232-234. 12. Morawetz. H. (1965) Macromolecules in Solution, High Polymers. Vol. 21, p. 88. 283-284. WileyInterscience, New York. 13. Gordon, A. H. ( 1969) Electrophoresis of Proteins in Polyacrylamide and Starch Gels, p. 12. Amer. Elsevier. New York. 14. Riehm. M. R. (1987) Protein-Dependent and SaltDependent Structural Stability of Eukaryotic Chromatin, Ph.D. dissertation. p. 83-84. University of Nevada. Reno. 15. Ausio. J.. Borochov. N.. Kam. Z.. Reich. M., Seger, D.. and Eisenberg. H. (1983) in Structure. Dynamics, Interactions and Evolution of Biological Macromolecules (Helene. C.. Ed.), pp. 89-100. Reidel, Dordrecht. 16. Widom. J. (1986)J. iwol. Biol. 190,411-424. 17. Harrington, R. E. ( 1985) Biochetnis~rv 24, 201 I2021.