Electron spin resonance studies on the conformation and interactions of histones containing cysteine

ARCHIVES

OF

BIOCHEMISTRY

Electron

BIOPHYSICS

408-415 (1977)

183,

Spin Resonance Studies on the Conformation Interactions of Histones Containing Cysteine

Histones ESTEVE

AND

H3 from Calf Thymus

PADR&,*

JAUME

PALAU,*

and H4 from Sea Urchin AND

JEAN-JACQUES

and

Sperm’ LAWRENCEtL

*Institute de Biologia Fundamental, Universidad Autdnoma de Barcelona and Conxjo Superior de Investigaciones Cientificas, Avenida San Antonio M” Claret 171, Barcelona-13, Spain, and tLaboratoire de Biologie Cellulaire, Dkpartment de Recherche Fondamentale, Centre d%tudes NuclCaires de Grenoble, BP 85 Centre de tri, 38041 Grenoble Cedrx, France Received

February

14, 1977

In this study the spin-label method has been used to obtain information about conformational properties of regions containing cysteine of histone H3 from,calf thymus, histone H4 from sperm of the sea urchin Arbacia lixula, and the histone complex H3-H4. It has been found that the microenvironments of histone H3 causing immobilization of the spin labels are sensitive to variations in ionic strength of dilute solutions of phosphate buffer, are partially destroyed by urea, and fully destroyed by proteolytic enzymes. The interaction of spin-labeled histone H3 with histone H4 induces an increase of immobilization of the spin label, indicating an increase in rigidity at the cysteine region of histone H3. The use of a series of spin labels of variable length for histone H3 gives an estimate of 0.8-1.0 nm for the apparent depth of the spin label binding site, a value which does not change upon interaction of histone H3 with H4. Histone H4 from A. lixula sperm causes a similar immobilization of the spin label. As for histone H3, immobilization increases with the ionic strength, and the structures are destroyed by urea and proteolytic enzymes. Upon mixing with histone H3, however, the extent of immobilization appears only slightly changed, and together with sedimentation velocity results, these studies suggest that the spin label attached to histone H4 prevents the complex formation.

The subunit structure found in native chromatin (l-4) appears to be mainly maintained by histone-histone interactions. The subunit itself seems to be built on the basis of an “octamer” structure composed of pairs of the histones H2A, H2B, H3, and H4 (5). Several interacting pairs of heterologous histones, such as H2B-H4 (6, 7), H2B-H2A (7), and H3-H4 (%-lo), have been found to occur either in solution or in native chromatin. The strongest interaction has been reported to occur between H3 and H4 giving rise to a tetramerit structure (H3-H4)2 (10). It seems desirable, at the present stage of research, ’ Part of this work was presented at the Symposium on Chromatin, Glasgow, Scotland, SeptemberOctober, 1975 (Abstract Number 55). p Research worker from the Institute National de la Sante et de la Recherche Medicale.

to obtain as much information as possible on the conformation of histones and on histone-histone interactions. This can throw further light on the knowledge of the structure of chromatin. The presence in calf thymus histone H3 of only two cysteinyl residues makes this protein suitable for spin-label studies when using maleimide labeled with a free radical nitroxide. The use of this label in previous work (11) provided useful information about the microenvironment of the cysteinyl residues in histone H3. Crevices were found in the conformation of this histone which immobilize the spin label. These invaginations are sensitive to conformational changes of the histone molecule induced either by ionic strength or by pH variation. In the present report we examine in 408

Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0003-9861

SPIN-LABEL

STUDIES

OF HISTONES

more detail the histone H3 from calf thymus and extend such studies to histone H4 from sea urchin sperm. This histone possesses characteristics rather similar to those of H4 from calf thymus, but it contains one cysteinyl residue (12, 13). This enables detection of the existence of crevices, if any, within the tertiary structure of histone H4. In addition, the conformational changes due to the interactions within the H3-H4 complexes are considered by studying the pairs H4-SLH3 and H3-SLH4.” MATERIALS

AND

H3 AND

409

H4 MAX,M”M

(1)

/

LENGTH (n m)

0

N

0.80

N-O

cc 0

1.12

METHODS

Materials The spin labels used in this study (Fig. 11 were purchased from Synvar Associates (Palo Alto, California). All other chemicals were of analytical grade, and were obtained from Merck. Calf thymus histone H3 was obtained according to method 1 of Johns (141, slightly modified in order to remove contaminants (151. The thiol content in this histone is 1.4 molimol of protein (151. Calf thymus histone H4 was prepared following method 1 of Johns (14). Histone H4 (or v2al) from Arhacia lixula sperm was obtained as described by Palau et al., Scheme 1 (161. Amino acid analyses (automatic analyzer Beckman M 119) and polyacrilamide gel electrophoresis (17) were used in order to check the purity of samples. Only a minor proportion of aggregated forms were detected.

Methods Spin labeling of histones. The spin labeling of histones H3 and H4 was performed according to a modification of a published method (111. The final concentration in sodium phosphate buffer before labeling was 24 mM (pH 6.5) in all cases. Under these conditions H3 (18) and H4 (19) have been reported to be extensively structured. The histones were disolved in distilled water at a concentration of about 130 pM, and the solutions were left to stabilize for 3 h at 4°C. All the subsequent operations were performed at 4°C. An equal volume of 48 mM phosphate buffer (pH 6.5) was then added, and the spin label in powder form was immediately added in order to obtain a spin-label concentration of 130 pM for histone H3, and of 65 ELMfor histone H4. The mixture was slowly stirred for 3 h and finally dialyzed against distilled water in order to remove the un‘I Abbreviations used: esr, electron spin resonance; MalNEt, N-ethylmaleimide; SLH3 or SLH4, spin-labeled histones (H3 or H4); uv, ultraviolet.

0

1.27 0 0 i: H C

1.60

0

FIG. 1. Structural formulas of the spin labels used in this work. Distances correspond to the maximum length of the extended molecule and have been taken from Burley et al. (24). reacted spin label. The spin-labeled histone was stored as an aqueous solution at 4°C. Not more than a week elapsed between the histone spin labeling and the esr measurements. Preparation ofhistone solutions. Spin-labeled histone solutions at final concentrations of about 33 PM were obtained by diluting the stock histone solution with distilled water, and by dialyzing overnight at 4°C against the desired buffer. Histones H3 and H4, labeled or unlabeled according to the experiment, were made to interact by mixing the corresponding histone solutions in water (pH 4.01 and leaving them to stabilize 3-4 h at 4°C. The mixture was then dialyzed overnight at 4°C against the appropriate buffer. The final concentration of spin-labeled protein was about 33 FM. Electron spin resonance measurements. The esr spectra were recorded on a Varian E 12 esr spectrometer, operating at 9.4 MHz. The microwave power was 20 mW, and the amplitude of modulation was 2 G. The receiver gain was variable depending on the sample. The histone solutions were maintained at 4°C until introduction into a flat cell, and the esr spectra were recorded within an interval of 15 min. The spectrometer operating temperature was 20 t 1°C. In order to handle the esr spectral data, and following criteria given by different authors (20, 211,

410

PADROS,

PALAU,

several parameters can be defined (see Fig. 2): h,/h, and h,/h,, which are indicative of the motional constraints imposed on the spin labels, h, being associated with a strongly immobilized state, and h, and h, with a partially immobilized state; and M,,,, the separation of the outermost lines, which gives an indication of the variations in the strongly immobiUltrucentrifugation. The determination of the sedimentation velocity coefficients was carried out in a Beckman model E analytical ultracentrifuge, using an A,,,-H rotor, at 60,000 rpm and 20°C. The runs were done under the following conditions: protein concentration, 0.7-1.0 mgiml; solvent, 10 mM phosphate buffer (pH 6.5). The sedimentation boundaries were detected either by the Schlieren or by the uv scanning method. The standard deviation of the computed values in no case exceeded 0.2 S. No corrections were made for the ionic strength of the solvent. RESULTS

Electron Spin Resonance Behavior SLH3 Histone from Calf Thymus

of

Figure 2 shows a representative spectrum obtained for histone H3 reacted with the spin label 1. This corresponds to a combination of two types of spectra, one due to strongly immobilized spin labels (peaks 1, 3 in part, and 5 in Fig. 2) with a correlation time of about 100 ns (estimated from 2A,), and another due to weakly immobilized

;fII

i

FIG. 2. Definition of esr parameters. The spectrum corresponds to histone H3 from calf thymus, which was labeled with spin label I, and then dialyzed against 24 mM phosphate buffer (pH 6.5). The parameters are: h,lh, and h,/h,, indicative of the motional constraints imposed on the spin labels, h, being associated with a strongly immobilized state, and h, and h, with a partially immobilized state; and 2A,,,, indicative of the variation in the strongly immobilized state.

AND

LAWRENCE

spin labels (peaks 2, 3 in part, and 4) with a correlation time of 2 to 5 ns (estimated from h,/h,). It was demonstrated earlier that the peaks of strong immobilization come from spin labels attached to cysteinyl residues (11). We have now estimated the contribution of spin labels attached to lysyl residues to the hyperfine signals as being no more than 3%. This was accomplished by a comparison of the labeling efficiency for histone H3 in aqueous solution and for this histone blocked with MalNEt. The low yield in spin labeling of lysyl residues is due in part to the fact that the maleimide nitroxide spin label hydrolyzes in 1 hour (20, and to the fact that at pH 7 the reaction rate of MalNEt with simple thiols is approximately lOOO-fold greater than that with corresponding simple amino compounds (22). Table I shows the existence under various conditions of slight variations in the spin-label mobilities, as monitored by 2A,, and h,lh,. On the other hand, h,lh, increases with ionic strength. No value of 2A,, lower than 62 G was observed, thus indicating that no population of spin labels with an intermediate correlation time is present. The variations of h,lh,, h21h4, and 2A, suggest that the steric hindrances in the neighborhood of the two cysteine residues increase with the rise in the ionic strength of dilute solutions of phosphate buffer. Figure 3a and Table I show the data obtained for SLH3 dissolved in 2.4 mM phosphate buffer-6 M urea (pH 6.5). The parameter h ,lh, becomes zero, whereas h,l h, remains high and almost unchanged when compared with the values obtained in the absence of urea. This demonstrates that urea is not able to eliminate completely the steric factors that cause immobilization of the spin labels. On the other hand, digestion of SLH3 with chymotrypsin or with trypsin (23) gives rise to a mixture of peptide fragments which shows an esr spectrum typical for unrestricted spin-label movement. The use of a series of spin labels of variable distances between the reactive group and the unpaired electron (Fig. 1) enables

SPIN-LABEL TABLE ELECTRON

SPIN

RESONANCE

STUDIES

OF HISTONES

I PARAMETERS

FOR

HISTONE H3 FROM CALF THYMUS (LABELED WITH I) UNDER VARIOUS CONDITIONS OF SOLVENTS AND TREATMENTS (PH 6.5)

.__----

Conditions Hz0 2.4 mM Phosphate-6 M urea 24 mM Phosphate, treated with chymotrypsin” 2.4 mM Phosphate, blocked with MalNEtO 2.4 mM Phosphate 4.8 mM Phosphate 7.2 mM Phosphate 24 mM Phosphate

0.04 0.01

2.78 2.67

-

0.00

1.41

-

0.02

1.25

-

0.09 0.12 0.15 0.33

2.96 3.17 3.42 3.38

62.0 E1.O 63.5 65.5

” SLH3 was mixed with a solution of chymotrypsin (lo%, w/w) in 24 mM phosphate buffer (pH 6.5) and incubated for 2 h at 25°C. h Prior to the spin labeling, histone H3 was incubated with MalNEt (1:2, molimol) in 24 mM phosphate buffer (pH 6.5) for 4 h at 4°C.

FIG. 3. Electron spin resonance spectra of histone SLH3 from calf thymus, under various conditions (pH 6.5): (a) SLH3 in 6 M urea-2.4 mM phosphate; (bl SLH3 treated with chymotrypsin for 2 h at 25”C, in 24 mM phosphate buffer; (c) SLH3 treated with trypsin for 2 h at 25”C, in 24 mM phosphate buffer.

an estimate to be made of one possible dimension of the spin label binding site. Figure 4 shows, for histone SLH3 in 2.4 mM and in 24 mM phosphate buffer (pH 6.51, the plots of h,lh, and h,lh, as a func-

H3 AND

H4

411

tion of the maximal extended length of the spin labels (24). It is apparent that the h,l h, values show an abrupt drop between 0.8 and 1.0 nm of spin-label length. This argues in favour of an assignment of a depth of about 0.8-1.0 nm for the solid structure that strongly immobilizes the spin labels. On the other hand, h,lh, shows a gradual variation throughout all the range of distances, indicating that the structure that weakly immobilizes the spin labels is less defined, or more fluid, than the one corresponding to highly restricted motion of the spin labels. Electron Spin Resonance Behavior of SLH4 Histone from Sea Urchin Sperm Electron spin resonance spectra of histone H4 from A. lixula sperm labeled with the spin label I are presented in Fig. 5, and Table II shows the values of esr parameters under various conditions. The behavior is very similar to that found for histone SLHS, and the results can be interpreted to show the appearance of more immobilizing structures as the ionic strength increases. Electron spin resonance spectra of histone H4 from calf thymus show that, in the absence of thiol groups, there is some labeling of lysyl residues. The signals are three hyperfine multiplets. This demonstrates that the peaks arising from restricted motions in the case of histone H4 from sea urchin sperm, are due to spin labels attached to cysteinyl residues. No evaluation was made on the extent of lysyl labeling in the presence of thiol groups. Denaturation and degradation have similar effects to those found for SLH3 (see Table II): The effect of urea is strong on h,l h, values, but not on h,lh,. The enzymatic treatment causes the disappearance of a great deal of the structures that immobilize the spin labels. The H3-H4

Interactions

Preliminary experiments were carried out by complexing histones H4 and SLH3, both from calf thymus. The results were comparable to those obtained by using histone H4 from A. Zixula sperm. The experiments reported in this section were there-

412

PADROS,

MAXIMUM

LENGTH

PALAU,

(nm)

AND

LAWRENCE

MAXIMUM

LENGTH

(nm)

FIG. 4. Electron spin resonance parameters as a function of the maximum extended length of the spin labels I-V (pH 6.51: O-O, histone SLH3 from calf thymus in 2.4 mM phosphate buffer; A-A, histone SLH3 from calf thymus in 24 InM phosphate buffer; 0-G, histone H4 from A. lixula sperm plus SLH3 from calf thymus, in 4.8 mM phosphate buffer.

fore carried

out with

histone

H4 from A.

lixula sperm.

V

cu 10 G FIG. 5. Electron spin resonance spectra of histone SLH4 from A. lixula sperm, dissolved in H,O, and in phosphate buffer (pH 6.5). TABLE

II

ELECTRON SPIN RESONANCE PARAMETERS FOR HISTONE H4 FROM A. LIXULA SPERM (LABELED WITH I) UNDER DIFFERENT CONDITIONS OF SOLVENTS AND TREATMENTS (PH 6.5) Conditions H,O 2.4 mM Phosphate-4 M urea 24 mM Phosphate, treated with chymotrypsin” 2.4 mM Phosphate 4.8 mM Phosphate 7.2 mM Phosphate 24 mM Phosphate -~ ” See footnote

h,lh,

hh

0.01 0.01

2.55 2.68

-

0.01

1.37

-

0.05 0.08 0.11 0.45

2.82 2.87 2.90 2.80

63 64 66 65

a of Table I.

fir,, (Gauss!

Labeled histone H3 (spin label I) was mixed with unlabeled histone H4, as described under Materials and Methods. In order to monitor the formation of complexes between the two histones, sedimentation velocity techniques were used. For the two pairs H3-H4 and SLH3-H4 the same uncorrected sedimentation coefficient, 3.5 S, was obtained for its major component, which corresponds to 80 and 60% of the material, respectively. For the slow component of isolated H3 and H4 (83 and 20% of the material, respectively), lower sedimentation coefficients (2.5 S for H3 and 1.7 S for H4) were obtained. The values obtained for the complexes are somewhat higher than those reported in the literature. For example, Kornberg and Thomas found a value of 3 S for the native complex H3-H4 (8). Nevertheless, such a discrepancy may be explained as due to the low histone concentration we use, and1 or to the low ionic strength of the solvent. From these studies we conclude that the spin labels reacted with histone H3 do not appear to prevent the complex formation between the two histones. Figure 6 shows, for the complex SLH3H4 and for SLHS, the esr parameters h,lh, and h,lh, varying with ionic strength. The results demonstrate that the spin labels are more immobilized within the complex than within the isolated histone SLH3. This is an indication that the region where

SPIN-LABEL

STUDIES

OF HISTONES

the cysteinyl residues are located participates in, or is sensitive to, the interaction. In order to get information on the stoichiometry of this interaction, as visualized by the spin-label method, water solutions of histone H4 at variable concentrations were mixed with water solutions of SLH3 at a constant concentration. The mixed solutions were then dialyzed against 4.8 mM phosphate buffer (pH 6.5). The results obtained are shown in Fig. 7. The plot shows that the parameter h,lh, becomes stabilized at a molar ratio H4/SLH3 of 1, indicating the formation of a one-to-one complex between the two histones. Further characterization of the H3/H4 complex was made by studying the interaction between histones H3 and SLH4. In this case, sedimentation velocity studies do not demonstrate any complex formation between the two histones, as the slow component of the pair H3-SLH4 (60% of the material) shows a sedimentation coefficient of 2.5 S, which is low compared with the value of 3.5 S for the unmodified pair H3-H4 under the same conditions. Figure 8 shows, for the complex SLH4-

H3 AND

05

0

MOLAR

413

H4

10

RATIO

15

20

H4/SLH3

7. Variation of the h,/h, parameter for the histone complex H4 + SLH3 as a function of the molar ratio H4iSLH3. The SLH3 concentration was 33 pM, and the ionic strength was 4.8 mM phosphate buffer (pH 6.5). FIG.

[PHOSPHATE]

(mb.4)

0.4 I----

f. i

0.2

,, /.-(,-

1~’ (63 .‘.

// 21 0

0.0

10 [PHOSPHATE]

O

[pHo:PHATE;“(mMj

20b----T-

(mM)

FIG. 8. Electron spin resonance parameters of histone SLH4 from A. lixula sperm, as a function of the ionic strength (phosphate buffer, pH 6.5): O-0, histone SLH4 from A. lixula sperm; O-O, histone H3 from calf thymus + SHL4 from A. lixula sperm, at an equimolar ratio.

10

[PHOSPHATE]

20

(mM)

FIG. 6. Electron spin resonance parameters of calf thymus histone SLHS, plotted as a function of the ionic strength (phosphate buffer pH 6.5): O-0,. histone SLHJ, from calf thymus; O--O, histone H4 from A. lixula sperm plus histone SLH3 from calf thymus, at an equimolar ratio.

H3 and for SLH4, plots of h,lh, and h,lh, versus ionic strength. A coincidence of values for both systems can be observed. The depth of the binding site of histone H3 in the complex H3-H4 was estimated by using a series of spin labels of different lengths (Fig. 1). The results corresponding to 4.8 mM phosphate buffer (pH 6.5) are plotted in Fig. 4 . It is apparent that no gross change in the esr parameters has

414

PADROS,

PALAU,

occurred, thus indicating that the overall dimensions of the spin-label environments undergo almost no change upon the interaction. DISCUSSION

From the relative contributions of fast and slow conformational changes (18, 19), it can be inferred that the extent of selfaggregation of histones H3 and H4 in water at pH near 7 is negligible. Under these conditions, the esr spectra from the spinlabeled histones show the existence of some strongly immobilized components. This reinforces the view that the esr outermost signals are due to folded states of the molecule rather than to a process of aggregation, At high ionic strengths, self-aggregation may contribute in some way to the burial of the spin label within the histone molecules. At the present stage of our research such a contribution cannot be completely discarded. The present experiments on histone H3 confirm earlier work (11) which suggested the existence of a globular region in the tertiary structure of histone H3. This region appears to be destroyed by urea and proteolytic enzymes. The cysteine residues are located, within the globular region, in crevice(s) of about 0.8-1.0 nm, as measured by the series of spin labels. Histone H4 is almost invariant through evolution (25), and the sequence data for this protein from sperm of the sea urchin Parechinus angulosus (13) indicates that the unusual cysteine residue is located at position 73. The assignment of a structure necessary for hindering the spin labels to a region close to the position 73 in the sequence of SLH4 is in agreement with recent studies. Lewis et al. (26) described the 25-67 region as being the critical one for the folding and association of histone H4. They also propose a model for this histone in which the helix is located between residues 49 and 73. The present esr studies indicate that the folded region might include residue 73. As in the case of histone H3, histone SLH4 dissolved in water (pH 6.5) shows the presence of some structures that immobilize the spin labels (Fig. 5), a result

AND

LAWRENCE

which is in agreement with the existence of some nonrandom structure in water as demonstrated by Lewis et al. (26). The results on the H3-H4 interaction confirm other studies in the sense that, by using mild procedures, these histones interact at a ratio of one to one (10, 27). Recently, Lewis has reported (28) that if histone H3 is allowed to become intramolecularly oxidized and then left to interact with histone H4, no H3-H4 complex is formed. He suggests a vital role for the sequence 96-110 of histone H3 in the assembly of the complex subunits. In the present work, we have found that the immobilization of the spin labels in SLH3 increases upon interaction with H4, a fact which indicates that the involved structures of histone H3 become more constrained. Furthermore, the depth measured by using the series of spin labels on the SLH3 structures, does not change appreciably upon interaction with histone H4 (Fig. 4). This suggests that, although the rigidity of the structures that immobilize the spin labels increases, the interaction does not take place directly through the whole region containing both cysteines (residues 96-110); otherwise the largest spin labels IV and V would be significantly constrained in their movement. The properties observed for the SLH3-H4 complex would therefore be accounted for by an allosteric-type behavior, i.e., the conformational changes that take place in the interacting region of SLH3 would induce the conformational changes detected by the spin labels at the cysteine region. There is no evidence of relevant changes in the esr parameters of the SLH4 histone upon mixing with histone H3, and this is in agreement with sedimentation velocity experiments which do not prove any complex formation between H3 and SLH4. It seems likely, therefore, that the spin label attached to the cysteine of histone H4 prevents the H3-SLH4 complex from forming. The tetrameric structure of the complex H3-H4 reported by several authors (8, 10, 29) suggests the presence of two modes of interaction between the two histones. One pair of interacting sites would give rise to a dimer which is known to be in dynamic

SPIN-LABEL

STUDIES

OF HISTONES

equilibrium with forms of higher molecular weight (29). As the ionic strength increases, a second pair of interacting sites would play a complementary role by participating in the formation of the tetramer. On this line of thought our results on H3H4 interactions appear to give a partial view of the different interaction modes existing within the complex, and they indicate that the molecular assembly of histones H3 and H4 may be sensitive to the presence of small molecules used as spectroscopic probes. ACKNOWLEDGMENTS We thank Dr. F. Climent for the supply of sea urchin histone H4, Mr. A. Jeunet for help in the use of the esr equipment, and Miss P. Brachet for technical assistance. We also thank Prof. A. Rassat for the use of the esr instrumentation, Dr. A. Albert (Instituto de Quimica-Fisica Rocasolano, Madrid) for the use of the analytical ultracentrifuge, Prof. L. H. Piette for critical reading of the manuscript, and Dr. J. Ellis for revising the English. E.P. greatly acknowledges the invitation and financial support from the Department de Recherche Fondamentalc (C.E.N. Grenoble), France. REFERENCES 1. HEWISH, D. R., AND BURGOYNE, L. A. (1973) Biochem. Biophys. Res. Commun., 32, 504510. 2. RILL, R., AND VAN HOLDE, K. E. (1973) J. Biol. Chem. 248, 1080-1083. 3. OLINS, A. L., AND OLINS, D. E. (1974) Sciencr 183, 330-332. 4. OUDET, P., GROSS-BELLARD, M., AND CHAMBON, P. (1975) Cell 4, 281-300. 5. THOMAS, J. O., AND KORNBERG, R. D. (1975) Proc. Nat. Acad. Sci. USA 72, 2626-2630. 6. MARTINSON, H. G., AND MCCARTHY, B. J. (19751 Biochemistry 14, 107331078. 7. VAN LENTE, F., JACKSON, J. F., AND WEINTRAUB, H. (1975) Cell 5, 45-50. 8. KORNBERG, R. D., AND THOMAS, J. 0. (1974) Science 184, 865-868. 9. BONNER, W. M., AND POLLARD, H. B. (1975) Bio-

H3 AND

H4

415

them. Biophys. Res. Commun. 64, 282-288. 10. D’ANNA, J. A., JR., AND ISENBERG, I. (1974) Biochemistry 13, 4992-4997. 11. PALAU. J., AND PADROS, E. (1972)FEBSLrtt. 2i. 157-160. 12. SUBIRANA, J. A. (1971) FEBS L&t. 16, 133-136. 13. STRICKLAND, M., STRICKLAND, W. N., BRANDT, W. F., AND VON HOLT, C. (1974) FEBS L&t. 40. 346-348. 14. JOHNS. E. W. (1964) Biochem. J. 92, 55-59. 15. PALAU, J., AND DABAN, J. R. (1974) Eur. J. Biochem. -19, 151-156. 16. PALAU. J., RUIZ-CARRILLO, A., AND SUBIRANA, J. A. (1969)Eur. J. B&hem. 7, 209-213. 17. PANYIM, S., AND CHALKLEY, R. (1969)Arch. Biochcm. Bioph~~s. 130, 337-346. 18. D’ANNA, J. A., JR., AND ISENBERG, I. (1974) Biochemistry 13, 4987-4992. 19. LI, H. J.. WICKETT, R., MORRIE CRAIG, A., AND 11, 375-397. ISENBERG, I. (1972) Biopolymers 20. HAMILTON, C. L., AND MCCONNELL, H. M. (19681 in Structural Chemistry and Molecular Biology (Rich. A., and Davidson, N., eds.), pp. 115-149. W. H. Freeman, San Francisco. 21. DWEK, R. .A. (1973) Nuclear Magnetic Resonance in Biochemistry: Applications to Enzyme Systems, pp. 285-327, Clarendon Press, Oxford. 22. MEANS, G. E., AND FEENEY, R. E. (19711 Chemical Modification of Proteins, p. 112. HoldenDay, San Francisco. 23. PHILLIPS, D. M. P. (1971) in Histones and Nucleohistones (Phillips, D. M. P., ed.), p. 48, Plenum Press, London and New York. 24. BURLEY, R. W., SEIDEL, J. C., AND GERGELY, J. (1971) Arch. Biochem. Biophys. 146, 597-602. 25. DELANGE, R. J., FAMBROUGH, D. M., SMITH, E. L., AND BONNER, J. (1969) J. Biol. Chem. 244, 5669-5679. 26. LEWIS, P. N., BRADBURY, E. M.. AND CRANEROBINSON, C. (1975) Biochemistry 14, 33913400. 27. D’ANNA, J. A., JR., AND ISENBERG, I. (1974) Biothem. Biophys. Res. Commun. 61, 343-347. 28. LEWIS, P. N. (1976) Biochem. Biophys. Res. Commun. 68, 329-335. 29. SPERLING, R., AND BUSTIN, M. (1975) Biochemistry 14, 3322-3331.