Drug-Nucleic Acid Interactions

672 Pages·2001·11.897 MB·English

by  Jonathan B. Chaires, Michael J. Waring

Preface
Progress in molecular biology and studies of small molecule binding to nu-
cleic acids have been inextricably linked. A testament to that fact is the inclusion of
eight papers directly concerned with drug-DNA interactions among the recently
published list of the 100 most cited articles in the Journal of Molecular Biology.
Few other scientific areas are as well represented on that list. Small molecules have
perhaps taught us more about DNA than DNA has taught us about small molecules.
Watson, for example, notes in the Molecular Biology of the Gene that the "fact that
intercalation occurs so readily indicates that it is energetically favored... and is
additional evidence for the metastability of the double-helical structure--its ability
to assume many inherently unstable configurations that normally revert quickly
back to the standard B conformation." From that point of view, intercalation pro-
vided one of the very first indications of the plasticity of DNA, an area that has
blossomed to reveal an incredible diversity of structural forms. Perhaps the most
widespread interest in small molecules that bind to nucleic acids stems from their
potential as useful pharmaceutical agents. Indeed, some of the very best anticancer
drugs are well-documented DNA binders. While interest in drug-DNA interactions
has at times waned, recent advances in chemical synthesis, analytical instrumenta-
tion to measure binding, and structural biology have greatly enhanced the potential
for rational design of new therapeutic compounds. Accordingly, studies on the in-
teraction of small molecules with nucleic acids have taken on new life and have
helped spawn several emergent biotechnology companies dedicated to exploiting
the promise of making new types of pharmaceuticals targeted at nucleic acids.
The aim of this volume is to consolidate key methods for studying ligand-
nucleic acid interactions, both old and new, into a convenient source. Accordingly,
we have solicited from experts in a variety of disciplines articles that concisely
but completely describe useful methods and strategies for studying small molecule
binding to nucleic acids. Techniques that are useful now range from biophysical
and chemical approaches to methods rooted in molecular and cell biology. We hope
that this volume will serve as a useful compendium of methods both to newcomers
entering the field as well as to scientists already actively engaged in research in
this area.
NAHTANOJ B. SERIAHC
LEAHCIM J. GNIRAW
iiix
Contributors to Volume 340
Article numbers are in parentheses following the names of contributors.
Affiliations listed are current.
NAITSIRHC BAILLY (24, 31), INSERM NEELRAC M. ENAN1LLUC (23), Pharma-
U-524, and Laboratoire de Pharmaco- cology and Developmental Therapeutics
logie Antitumorale du Centre Oscar ,tinU Peter MacCallum Cancer Institute,
Lambret IRCL, 59045 Lille, France airotciV 3002, Australia
TREBLA S. BENIGHT (8), Department ENNAZUS M. CUTrS, (23), Department of
of Chemistry, University of Illinois, Biochemistry, La Trobe University, Bun-
,ogacihC Illinois 60607 and DNA Codes doora, Victoria 3083, Australia
LLC, Chicago, Illinois 10606
SEMAJ C. KAIWORBAD (21), Department of
ECNERWAL A. YELMOTTOB (11), School of Chemistry, Center for Science and -hceT
Chemistry and Biochemistry, Georgia In- nology, Syracuse University, Syracuse,
stitute of ,ygolonhceT Atlanta, Georgia New York 44231
30332
TINA M. DAVIS (2), Department of Chem-
AIHPOS .Y B MEGESUER (10), Laboratoryfi~r istry, Georgia State University, Atlanta,
Fluorescence Dynamics, Department of Georgia 30303
Physics, University of Illinois, Urbana,
Illinois 61801 PETER B. DERVAN (22), Department of
Chemistry, California Institute of -hceT
NAHTANOJ B. CHAIRES (1, 5, 27), De- ,ygolon Pasadena, California 91125
partment of Biochemistry, University of
Mississippi Medical Center, Jackson, ANELADGAM NOSSKIRE (4), Department of
Mississippi 39216 Physical Chemistry, Chalmers University
of Technology, Gothenburg SE-41296,
NEY CHOO (30), Gendaq Limited, London Sweden, and Department of BiD-
NW7 lAD, United Kingdom
chemistry, University of Gothenburg,
RUBAB Z. YRHDWOHC (6), School of Chem- Gothenburg SE-40530, Sweden
ical and Life Sciences, University of
EHPOTSIRHC ~IDUCSE (16), Laboratoire
Greenwich, London SE18 6PF, United
de Biophysique, INSERM U201, CNRS
Kingdom
UMR 8646, Museum National d'Histoire
TREBOR M. CLEGG (10), Laboratory for Naturelle, 75231 Paris Cedex ,50
Fluorescence Dynamics, Department of France
Physics, University of Illinois, Urbana,
ALEBAZI FOKT (27), .M .D Anderson Can-
Illinois 10816
cer ,retneC University of ,saxeT Houston,
DLANOD M. SREHTORC (3, 23), Depart- saxeT 77030
ment of Chemistry, Yale University, New
HTIEK R. Fox (20), Division of Biochemistry
Haven, Connecticut 06520-8107
and Molecular Biology, School of Bio-
MARK S. YELREBBUC (28), Department of logical Sciences, University of Southamp-
Chemistry and Biochemistry, University ton, Southampton S016 7PX, United
of ,saxeT Austin, Texas 78712 Kingdom
x CONTRIBUTORS TO VOLUME 340
ES~IR~IHT GARESTIER (16), Laboratoire KISEB I. KANKIA (7), Department of
de Biophysique, 1NSERM U201, CNRS Pharmaceutical Sciences, University ~f
UMR 8646, Museum National d'Histoire Nebraska Medical Center, Omaha,
Naturelle, 75231 Paris Cedex ,50 France Nebraska 68198
YRREJ NAMS1DOOG (21), Department of ATIMSA KUMAR (33), Department of Bio-
Chemistry, Center for Science and -hceT chemistry, University of Mississippi,
nology, Syracuse University, Syracuse, Jackson, Mississippi 39216
New York 13244
DLANOD W. KUPKE (7), Department of
DAVID E. GRAVES (18), Department of
,yrrtsimehC University of ,ainigriV Char-
Chemistry, University of Mississippi,
lottesville, Virginia 10922
,ytisrevinU Mississippi 38677
HTIEK A. IDLAMIRG (17), CRC Drug-DNA WERDNA N. LANE (12), Division of Molecu-
Interactions Research Group, Royal Free lar Structure, National Institute for Med-
and University College Medical School, ical Research, London NW7 IAA, United
University College London, London WI P Kingdom
,TB8 United Kingdom
YROGERG H. LENO (33), lnfgen Incorpo-
R1M1DALV M. VELEUG (28), Department of rated, DeForest, Wisconsin 53532
Chemistry and Biochemistry, "~isrevinU
of ,saxeT Austin, Texas 21787 RETEP T. IEHELLIL (l l), School of Chem-
istry and Biochemistry, Georgia Institute
LUMAHSTHI HAG (6), Krebs Institute of ,ygolonhceT Atlanta, Georgia 30332
for Biomolecular Science, Department
of Chemisto, University of Sheffield, R. SCOTT LOKEY (28), Department of
Sheffield $3 ,FH7 United Kingdom Chemistry and Biochemistr); University
of ,saxeT Austin, Texas 21787
NHOJ A. YELTRAH (17), CRC Drug-DNA
Interactions Research Group, Royal Free
FRANK G. SNEITNOOL (10), Laboratory
and University College Medical School,
for Biochemistry, WEVIO, University of
University College London, London W1P
Gent, Gent 9000, Belgium
,TB8 United Kingdom
PAUL B. HOPKINS (19), Department of NAYR A. LUCE (19), Department of Chem-
istr); University of Washington, Seattle,
Chemistry, University of ,notgnihsaW
notgnihsaW 98195
Seattle, Washington 98195
ECNERUAL H. HURLEY (29), College of EHPOTSIRHC DNAHCRAM (32), Laboratory
Pharmacy, University of Arizona, ,noscuT of Molecular Pharmacology, Division
Arizona 85721 and Arizona Cancer Cen- of Basic Sciences, National Cancer -nI
,ret Tucson, Arizona 85724 stitute, National Institutes of Health,
Bethesda, Maryland 20892
MARK ISALAN (30), Gendaq Limited,
London NW7 laD, United Kingdom SIUL A. MARKY (7), Department of
BRENT L. NOSREVI (28), Department of Pharmaceutical Sciences, University of
Chemistry and Biochemistry, University Nebraska Medical Center, Omaha,
of ,saxeT Austin, Texas 21787 Nebraska 68198
ECNERET C. SNIKNEJ (6), Yorkshire Cancer ERIALC J. MCGURK (17), CRC Drug-DNA
Research Laboratory of Drug Design, Interactions Research Group, Royal Free
Cancer Research Group, University of and University College Medical School,
Bradford, Bradford BD7 1DP, United University College London, London WI P
Kingdom ,TB8 United Kingdom
CONTRIBUTORS TO VOLUME 340 xi
RETEP J. MCHUGH (17), CRC Drug-DNA NOD R. PHILLIPS (23), Department
snoitcaretnI Research ,puorG Royal Free of Biochemistry, LaTrobe ;3tisrevinU
and University College Medical ,loohcS ,aroodnuB airotciV 3083, Australia
ytisrevinU College London, London W1P
SEVY REIMMOP (32), Laboratory of -celoM
,TB8 United Kingdom
ular Pharmacolog); Division of Basic
KRAM .P MCPIKE (21), Department of ,secneicS National Cancer ,etutitsnI Na-
,yrtsimehC Center for Science and -hceT tional Institutes of Health, Bethesda,
nolog); Syracuse University, ,esucaryS Maryland 20892
New York 13244
~lSOJ LAGUTROP (25, 27), Departamento de
HTIDEREM M. MURR (28), Department of aigoloiB Molecular y ,raluleC lnstituto de
Chemistry and ,yrtsimehcoiB Universi~ aigoloiB Molecular de ,anolecraB ,CISC
of ,saxeT Austin, Texas 21787 anolecraB 08034, Spain
IRUON ITAMAEN (32), yrotarobaL of -celoM RAMEDLAW PRIEBE (27), .M .D Ander-
ular Pharmacology, Division of Basic nos Cancer ,retneC University of ,saxeT
,secneicS National Cancer ,etutitsnI -aN ,notsuoH Texas 77030
tional Institutes of Health, Bethesda,
ASERET AKOLWEZRP (27), .M .D Ander-
Maryland 29802
nos Cancer ,retneC University of ,saxeT
VALSORAJ LI~ITESEN (8), Department of ,notsuoH Texas 77030
Applied Mathematics, Faculty of -htaM PETER SSUFNEGER (10), Laboratory for
ematics and Physics, Charles ,~isrevinU Fluorescence Dynamics, Department of
811 0O Praha ,1 Czech Republic Physics, Universi~' of Illinois, ,anabrU
PETER E. NIELSEN (15), Department of sionillI 10816
Medical Biochemistry and Genetics, ehT GNOSNIJ NER (5), Department of -mehcoiB
Panum Institute, University of Copen- ,yrtsi University of Mississippi Medical
,negah Copenhagen DK-2200, Denmark ,retneC ,noskcaJ Mississippi 39216
TGNEB NORD~N (4), Department of Phys- RETEP .V RICCELLI (8), Department
ical Chemistry, Chalmers University of Chemistry, University of ,sionillI
of ,ygolonhceT Gothenburg ,69214-ES ,ogacihC Illinois 60607, and AND Codes
Sweden ,CLL ,ogacihC Illinois 10606
RICHARD OWCZARZY (8), Department DRAHCIR D. YDRAEHS (26), Department of
of Chemistry, University of ,sionillI yrtsimehC and ,yrtsimehcoiB Seton Hall
,ogacihC Illinois 60607, and Integrated ;)tisrevinU South Orange, New Jersey
AND Technologies, Coralville, Iowa 97070
14225
ALEGNA M. SNOW (26), Memorial High
PETR AKSO~(NAP (8), Department of -mehC ,loohcS Elmwood ,kraP New Jersey 07407
,yrtsi University of Illinois, ,ogacihC
SELRAHC H. SPINK (9), Department of
Illinois 60607, and Center for Discrete
,yrtsimehC State ytisrevinU of New ,kroY
,scitamehtaM Applied Computer Science
,dnaltroC New kroY 54031
and Applications DIMAT1A, Charles
,ytisrevinU Prague, Czech Republic, and UYKEAD NUS (29), Institute for Drug De-
AND Codes LLC, Chicago, Illinois ,tnempolev naS Antonio, Texas 54287
10606
GNEHS-NAIJ NUS (16), Laboratoire de
MARY HTEBAZILE PEEK (13), School of Biophysique, INSERM U201, CNRS
yrtsimehC and ,yrtsimehcoiB Georgia -nI RMU 8646, Museum National d'Histoire
stitute of ,ygolonhceT Atlanta, Georgia Naturelle, 75231 Paris Cedex ,50
23303 ecnarF
xii CONTRIBUTORS TO VOLUME 340
LEAHCIM J. TILBY (17), Cancer hcraeseR bridge, Cambridge 2BC IQJ, United
,tinU Medical School, University of New- modgniK
castle Upon Tyne, Newcastle NE2 4HH,
NASUS E. WELLMAN (9), Department
United Kingdom
of Pharmacology and Toxicolog), Uni-
JOHN W. TRAUGER (22), Department versity of Mississippi Medical ,retneC
of Chemistry, California Institute ,noskcaJ Mississippi 39216
of Technology, Pasadena, California
NEROL DEAN WILLIAMS (13), School of
52119
yrtsimehC and ,yrtsimehcoiB Georgia -nI
NHOJ O. TRENT (14, 27), James Gra- stitute of ,ygolonhceT Atlanta, Georgia
ham Brown Cancer ,retneC Department 23303
of Medicine, University of Louisville,
Louisville, Kentucky 40202 W. DAVID WILSON (2), Department of
Chemistry, Georgia State ,ytisrevinU
PETER M. VALLONE (8), Department Atlanta, Georgia 30303
of Chemistry, University of Illinois,
Chicago, Illinois 60607 and National 1HZGNOH XU (33), Department of -mehcoiB
Institute of "sdradnatS and ,ygolonhceT ,yrtsi University of Mississippi, ,noskcaJ
Biotechnology Division, ,grubsrehtiaG Mississippi 39216
Mao, land 20899
NEVETS M. ZEMAN (3), Department of
LEAHCIM J. WARING (20, 24), Department ,yrtsimehC Yale ,ytisrevinU New ,nevaH
of Pharmacology, University of Cam- Connecticut 06520
1 STSYLANA FO LIGAND-DNA GNIDNIB SMREHTOSI 3
1 Analysis and Interpretation of Ligand-DNA
Binding Isotherms
By NAHTANOJ B. SER1AHC
Introduction
To attain a reasonable understanding of any ligand-receptor interaction, it is
necessary to answer the questions posed by Scatchard ~ more than 50 years ago:
"How many? How tightly? Where? Why? What of it?" The first two Questions (and
in part the third) can be answered by equilibrium binding studies, and are the pri-
mary focus of this chapter. The remaining questions concisely express the concerns
of structural and functional studies, and may be addressed by X-ray crystallogra-
phy, nuclear magnetic resonance (NMR) techniques, molecular modeling, and a
variety of chemical and molecular biological methods. Macromolecular binding
is a phenomenon of general interest, and the underlying general principles are the
same for ligand binding to proteins or to nucleic acids. A number of excellent
general treatments of macromolecular binding are available that explain the un-
derlying physical chemistry in detail .2-6 What distinguishes the binding of small
molecules to DNA from their binding to proteins is the need to account for behav-
ior arising from the lattice properties of linear DNA molecules. Various neighbor
exclusion models have evolved to cope with that complexity, and are described.
An excellent discussion of the principles of nucleic acid binding interactions is
provided by Bloomfield et al. 7
Determination of the binding constant K allows the binding free energy change,
AG, to be calculated by the standard Gibbs equation, AG = - RT In K, where R
is the gas constant and T is the temperature in degrees Kelvin. From studies of the
temperature dependence of the binding constant, or (preferably) by calorimetric
studies, the binding enthalpy (AH) may be obtained. The binding free energy may
then be partitioned into its enthalpic and entropic components, AG = AH -- TAS,
where AS is the entropy change. Knowledge of these thermodynamic parameters
I G. Scatchard, Ann. N.Y. Acad. Sci. 51,660 (1949).
2 j. .T Edsall and J. Wyman, "Biophysical Chemistry." Academic Press, New York, 1958.
3 j. Wyman and S. J. Gill, "Binding and Linkage." University Science Books, Mill Valley, California,
1990.
41. M. Klotz, "Ligand Receptor Energetics." John Wiley & Sons, New York, 1997.
5 E. diCera, "Thermodynamic Theory of Site-Specific Binding Processes in Biological Macro-
molecules." Cambridge University Press, Cambridge, 1995.
6 G. Weber, "Protein Interactions." Chapman & Hall, New York, 1992.
7 .V A. Bloomfield, D. M. Crothers, and J. Ignacio Tinoco, "Nucleic Acids: Structures, Properties and
Functions," 1st Ed. University Science Books, Sausalito, California, 2000.
Copyright © 2001 by Academic Press
All rights of reproduction in any form reserved.
METHODS IN ENZYMOLOGY, VOL. 340 0076-6879/00 $35.00
4 LACISYHPOIB SEHCAORPPA 11
provides a firm foundation for understanding the molecular forces that govern the
binding reaction, allowing one to begin to address Scatchard's question "Why?"
Details of attempts to parse binding free energies for ligand-DNA interactions in
order to understand the contribution of various molecular forces are described in
publications from this and other laboratories, s- 21
The aim of this chapter is to offer a concise guide for the analysis and interpre-
tation of ligand-DNA binding isotherms. Methods for experimentally obtaining
binding data are not discussed because detailed, practical descriptions of experi-
mental protocols are available. 31 51 In this chapter, examples of binding data are
taken from results obtained in the author's laboratory with the anticancer agent
daunomycin (daunorubicin). Daunomycin is perhaps the best-characterized DNA
intercalator, and its binding to a wide variety of DNA sequences and structures has
been thoroughly investigated. ~ 71,6
Model-Independent Approaches
Figure 1 shows the results from two types of binding experiments, each of
which addresses one of Scatcbard's queries as directly as possible. The method of
continuous variations 1a-sI may be used to construct a so-called Job plot (Fig. 1A).
Binding stoichiometries may be determined from such plots without recourse to
any assumed binding model. For the data shown in Fig. 1A for the interaction
of daunomycin with calf thymus DNA, an inflection near 0.2 mol fraction ligand
indicates a binding stoichiometry of one ligand per 3 or 4 base pairs. The exact
stoichiometry from the inflection at 0.21 mol fraction is (1.0 - 0.21)/0.21 =
3.76 base pairs. This value represents the predominant binding mode, although an
s j. B. Chaires, Anticancer Drug Des. 11,569 (1996).
9 j. B. Chaires, Biopolymers 44, 201 (1997).
l01. Haq, J. E. Ladbury, B. Z. Chowdhry, T. C. Jenkins, and J. B. Chaires, J. Mol. Biol. 271,244 (1997).
II j. Ren, T. C. Jenkins, and J. B. Chaires, Biochemistry 39, 8439 (2000).
21 S. Mazur, .F A. Tanious, D. Ding, A. Kumar, D. W. Boykin, I. J. Simpson, S. Neidle, and W. D.
Wilson, J. Mol. Biol. 300, 321 (2000).
31 X. Qu and J. B. Chaires, Methods Enzymol. 321, 353 (2000).
4L T. C. Jenkins, in "Drug-DNA Interaction Protocols" (K. R. Fox, ed.), Vol. 90, pp. 195-218. Humana
Press, Totowa, New Jersey, 1997.
51 .p C. Dedon, in "Current Protocols in Nucleic Acid Chemistry" (S. L. Beaucage, D. E. Bergstrom,
G. D. Glick, and R. A. Jones, eds.), Vol. ,1 pp. 8.2.1-8.2.8. John Wiley & Sons, New York, 2000.
61 j. B. Chaires, in "Advances in DNA Sequence Specific Agents" (L. H. Hurley, ed.), Vol. 2, pp. 141-
167. JAI Press, Greenwich, Connecticut, 1996.
71 j. B. Chaires, Biophys. Chem. 35, 191 (1990).
81 E Job, Ann. Chim. (Paris) 9, 113 (1928).
91 C. .Y Huang, Methods Enzymol. 87, 509 (1982).
o2 A. Waiters, Biomed. Biochim. Acta 44, 132t (1985).
12 E G. Loontiens, E Regenfuss, A. Zechel, L. Dumortier, and R. M. Clegg, Biochemistry 29, 9029
(1990).
1
ANALYSIS OF LIGAND-DNA BINDING ISOTHERMS 5
/
1.2 ,
I I I I I , , , , ,
200
1.0
g
t
0.8
-200
0.6
-400
0.4
-600
0.2
-800 o.o • B
, I , I I I
0.0 0.2 0.4 0.6 0.8 -20 -18 -16 -14 -12 -t0
Mole Fraction Daunomycin nI Cf
FIG. I. Daunomycin binding to calf thymus DNA. (A) Job plot obtained from fluorescence titration
studies. A F is the difference in fluorescence emission intensity between solutions of daunomycin alone
and in the presence of DNA. The minimum indicates a binding stoichiometry of 3 or 4 base pairs. (B)
Binding isotherm for the daunomycin--calf thymus DNA interaction. The fractional saturation was
calculated assuming a 3-bp binding site. The abscissa is the natural logarithm of the free daunomycin
concentration.
inflection near 0.5-0.6 mol fraction indicates an additional binding mode at higher
drug concentrations. The results shown here, based on fluorescence data, agree well
with data based on absorbance changes. °2 The Job plot thus answers the question
"How many?" directly. In studies of ligand-DNA interactions, this method has
been underutilized and its advantages largely unappreciated. In the case of multiple
binding modes, the method of continuous variations is particularly valuable, and
clearly reveals complexities in the binding process. Published examples for the
groove-binder Hoechst 3325821 and for the bisintercalating anthracycline WP63122
illustrate the value of the method in cases of complicated, multimode binding
interactions.
Figure B1 shows a titration binding isotherm for the daunomycin-calf thymus
DNA interaction. In this form, the fractional occupancy of binding sites is shown as
a function of the natural logarithm of the free daunomycin concentration .)-tC( The
fractional occupancy was calculated from the experimentally determined binding
22 F. Leng, W. Priebe, and J. B. Chaires, yrtsimehcoiB 37, 1743 (1998).
6 LACISYHPOIB SEHCAORPPA 1
ratio r (moles daunomycin bound per mole base pair) and the binding stoichio-
metry was determined from the Job plot shown in Fig. 1A. The form of the plot
shown in Fig. B1 is regarded by some 3 as the most fundamental representation of
binding data because the logarithm of the free ligand activity is proportional to the
chemical potential of the ligand. For simple binding to identical, noninteracting
sites, titration binding curves should be symmetric about a midpoint located at
a ligand concentration that is the reciprocal of the association binding constant,
and should cover a span of 8.1 lOgl0 units (4.14 In units) in going from 0.1 to 0.9
fractional saturation. 3'4'6 The data shown in Fig. B1 cover a span of 5.4 in units (2.4
log~0 units) and represent an essentially complete binding titration curve. The span
is greater than expected for simple binding, which indicates negative cooperativity,
neighbor exclusion, or heterogeneity of binding sites. Perhaps the main advantage
of the data shown in Fig. B1 is that they may be analyzed in a model-independent
way by using the Wyman concept of median ligand activity. 5'3 The free energy of
ligation (AGx) to go from a state where no ligand is bound to a degree of saturation
of ?k is given by Eq. (1):
2
P
AGx = RT oJ In Cfg~" (1)
where RT has its usual meaning. The pronounced advantage of Eq. (1) is that it
provides a free energy estimate to attain any degree of saturation without recourse
to any specific binding model. Numerical integration of the data in Fig. 1 B yields an
estimate of AGx = -7.8 kcal mol I for the full ligation of a daunomycin binding
site. Free energies derived from binding constants obtained by curve fitting to
specific models must agree with this model-independent value if the model is
reasonable.
Neighbor Exclusion Models
Figure 2 shows data for the daunomycin-calf thymus DNA interaction in the
form of a Scatchard plot, J by far the most common representation of binding data
for ligand-DNA interactions. To explain the curvature in such plots, a variety
of neighbor exclusion models were proposed, 42'32 and these have become the
most commonly used models for the interpretation of binding isotherms. Neighbor
exclusion models assume (in their simplest form) that the DNA lattice consists of
an array of identical and noninteracting potential binding sites. The base pair is
commonly defined as the lattice binding site for duplex DNA. Ligand binding
to any one site occludes neighboring sites from binding as defined by the site
size n. As the lattice approaches saturation, the probability of finding a stretch
32 .D .M ,srehtorC sremylopoiB ,6 575 .)8691(
42 j. .D eehGcM dna R .H noy ,leppiH .J .loM .loiB ,68 964 .)4791(
1 ANALYSIS OF LIGAND-DNA BINDING ISOTHERMS 7
8.0x10 5 , , , I , , I
6.0xl 0 s
"',O •
4.0xl 0 s • o~',~
G~
3C
2.0x10 5
e ~ - . .
0.0
I I I I I I I
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
r
FIG. 2. Scatchard plot for the daunomycin-calf thymus DNA interaction. The solid line is the best
fit of the neighbor exclusion model Eq. (2) to the experimental data yielding the parameters shown
in Table I. The dashed line is the best fit with the exclusion parameter constrained to an integral value
of 3.
of unoccupied DNA n base pairs long decreases, producing the curvature seen
in Fig. 2. The curvature does not result from a decrease in the intrinsic binding
affinity, but rather arises from the decreased probability of finding a free site of the
appropriate size. McGhee and von Hippe124 derived a closed form equation that
embodies the neighbor exclusion model Eq. (2):
r I 1--"r °'
-- = K(1 - nr) 1 ; J (2)
where K is the association constant for ligand binding to an isolated lattice site, n is