Abstract
The complexation of 2-(p-toluidinyl)naphthalene-6-sulfonate (TNS) to the hydrophobic sites of bovine serum albumin (BSA) and the effects of quencher and denaturant were studied by steady-state and dynamic fluorescence spectroscopy. Experimental results show that there are multiple sites in BSA which bind TNS. Fluorescence decay kinetics of BSA-bound TNS were recovered by multifrequency phase-modulation measurements and analyzed by a global analysis scheme. For all the cases, a distributed lifetime model best fits the experimental data—a result of the dynamical nature of BSA/TNS association and of the multiple binding sites in BSA. In addition to the distributed species, a short, discrete component exists, arising from unbound or free TNS. The recovered Stern-Volmer quenching constant for acrylamide from the lifetime data is consistent with the steady-state results. The denaturant, urea, decreases significantly the pre-exponential factor for protein-bound TNS, but affects the center lifetime and the distribution width only at high urea concentrations. The perturbation of probe molecules on the three-dimensional conformation of proteins and the effects of complex (TNS-BSA) age were also studied.
Get full access to this article
View all access options for this article.
References
1.
Carter
D. C.
He
X.
Munson
S. H.
Twigg
P. D.
Gernert
K. M.
Broom
M. B.
Miller
T. Y.
, Science 244 , 1195 (1989 ).
2.
Foster
J. F.
, “Some Aspects of the Structural and Conformational Properties of Serum Albumin,” in Albumin Structure, Function, and Uses ,
Rosenoer
V. M.
Oratz
M.
Rothschild
M. A.
, Eds. (Pergamon , Oxford , 1977 ), pp. 53 –84 .
3.
Kragh-Hansen
U.
, Dan. Med. Bull. 37 , 57 (1990 ).
4.
Dugiaczyk
A.
Law
S. W.
Dennson
O. E.
, Proc. Natl. Acad. Sci. U.S.A. 79 , 71 (1982 ).
5.
Meloun
M.
Moravek
L.
Kostka
V.
, FEBS Lett. 58 , 134 (1975 ).
6.
Brown
J. R.
Shockley
P.
, “Serum Albumin: Structure and Characterization of Its Ligand Binding Sites,” in Lipid Protein Interactons ,
Jost
P. C.
Griffith
O. H.
, Eds. (John Wiley & Sons , New York , 1982 ), Vol. 1 , pp. 25 –68 .
7.
Sargent
T. D.
Yang
M.
Bonner
J.
, Proc. Natl. Acad. Sci. U.S.A. 78 , 243 (1981 ).
8.
Peters
T.
Jr.
, “Serum Albumin,” in Plasma Proteins ,
Putnam
F. W.
Ed. (Academic Press , New York , 1975 ), Vol. 1 , pp. 131 –181 .
9.
Knudsen
A.
Pedersen
A. O.
Broderson
R.
, Arch. Biochem. Biophys. 244 , 273 (1986 ).
10.
Štrop
P.
Mikeš
F.
Havranová
M.
, Collect. Czech. Chem. Commun. 52 , 1362 (1987 ).
11.
Kato
A.
Nakai
S.
, Biochim. Biophys. Acta 624 , 13 (1980 ).
12.
Sklar
L. A.
Hudson
B. S.
Simoni
R. D.
, Biochemistry 16 , 5100 (1977 ).
13.
Keshavarz
E.
Nakai
S.
, Biochim. Biophys. Acta 576 , 269 (1979 ).
14.
McClure
R. J.
Craven
B. M.
, J. Mol. Biol. 83 , 551 (1974 ).
15.
Rao
S. N.
, J. Bio. Chem. 251 , 3191 (1976 ).
16.
Lakowicz
J. R.
, Principles of Fluorescence Spectroscopy (Plenum Press , New York , 1983 ).
17.
Warner
I. M.
Patony
G.
Thomas
M. P.
, Anal. Chem. 57 , 463A (1985 ).
18.
Bright
F. V.
, Anal. Chem. 60 , 1031A (1988 ).
19.
Bright
F. V.
Betts
T. A.
Litwiler
K. S.
, C.R.C. Crit. Rev. Anal. Chem. 21 , 389 (1990 ).
20.
Demas
J. N.
, Excited-State Lifetime Measurements (Academic Press , New York , 1983 ).
21.
Lakowicz
J. R.
, J. Biochem. Biophys. Meth. 2 , 91 (1980 ).
22.
Chen
R. F.
Scott
C. H.
, Anal. Lett. 18 , 393 (1985 ).
23.
Davidson
R. S.
Hilchenbach
M. M.
, Photochem. Photobiol. 52 , 431 (1990 ).
24.
Chen
R. F.
, Arch. Biochem. Biophys. 133 , 262 (1969 ).
25.
Miller
J. S.
Walsh
M. P.
Nemcek
K.
Johnson
J. D.
, Biochemistry 27 , 991 (1988 ).
26.
Haaijman
J. J.
Coolen
J.
Kröse
C. J. M.
Pronk
G. J.
Ming
Z. F.
, Histochem. 84 , 360 (1986 ).
27.
Sanger
J. W.
Mittal
B.
Sanger
J.
, J. Cell Biol. 99 , 918 (1984 ).
28.
Damodaran
S.
, Biochim. Biophys. Acta 914 , 114 (1987 ).
29.
Auer
H. E.
Glicks
D. M.
, Biochemistry 23 , 2735 (1984 ).
30.
Badea
M. G.
DeToma
R. P.
Brand
L.
, Biophys. J. 24 , 197 (1978 ).
31.
Stryer
L. S.
, J. Mol. Biol. 13 , 445 (1965 ).
32.
DeToma
R. P.
Easter
J. H.
Brand
L.
, J. Am. Chem. Soc. 98 , 5001 (1976 ).
33.
Hlady
V.
Andrade
J. D.
, Coll. Surf. 42 , 85 (1989 ).
34.
Hlady
V.
Andrade
J. D.
, Coll. Surf. 32 , 359 (1988 ).
35.
Kosower
E. M.
Kanery
H.
Dodluk
H.
Striker
G.
Jovin
T.
Bonl
H.
Huppert
D.
, J. Phys. Chem. 87 , 2479 (1983 ).
36.
Grellmann
K. H.
Schmitt
U.
, J. Am. Chem. Soc. 104 , 6267 (1982 ).
37.
Kosower
E. M.
, Acc. Chem. Res. 15 , 259 (1982 ).
38.
Dodluk
H.
Kosower
E. M.
, J. Phys. Chem. 81 , 50 (1977 ).
39.
Fleming
G. R.
Porter
G.
Robbins
R. J.
Synowiec
J. A.
, Chem. Phys. Lett. 52 , 228 (1977 ).
40.
Seliskar
C. J.
Brand
L.
, J. Am. Chem. Soc. 93 , 5414 (1971 ).
41.
Greene
F. C.
, Biochemistry 14 , 747 (1975 ).
42.
Jobe
D. J.
Verrall
R. E.
, J. Phys. Chem. 92 , 3582 (1988 ).
43.
Catena
G. C.
Bright
F. V.
, Anal. Chem. 61 , 905 (1989 ).
44.
Brand
L.
Gohlke
J. R.
, J. Biol. Chem. 246 , 2317 (1971 ).
45.
Andley
U. P.
Liang
J. N.
Chakrabarti
B.
, Biochemistry 21 , 1853 (1982 ).
46.
McClure
W. O.
Edelman
G. M.
, Biochemistry 5 , 1908 (1966 ).
47.
Jones
M.
Keenan
R. W.
Horowitz
P.
, J. Chromatogr. 237 , 522 (1982 ).
48.
Alcala
J. R.
Gratton
E.
Prendergast
F. G.
, Biophys. J. 51 , 587 (1987 ).
49.
Bright
F. V.
Catena
G. C.
Huang
J.
, J. Am. Chem. Soc. 112 , 1343 (1990 ).
50.
Catena
G. C.
Bright
F. V.
, J. Fluor. 1 , 31 (1991 ).
51.
Yeager
M. D.
Feigenson
G. W.
, Biochemistry 29 , 4380 (1990 ).
52.
Libertini
L. J.
Small
E. W.
, Biophys. Chem. 34 , 269 (1989 ).
53.
Siemiarczuk
A.
Ware
W. R.
, J. Phys. Chem. 93 , 7609 (1989 ).
54.
Siemiarczuk
A.
Wagner
B. D.
Ware
W. R.
, J. Phys. Chem. 94 , 1661 (1990 ).
55.
Carraway
E. R.
Demas
J. N.
, Anal. Chem. 63 , 332 (1991 ).
56.
Bright
F. V.
Betts
T. A.
Litwiler
K. S.
, Anal. Chem. 62 , 1065 (1990 ).
57.
Betts
T. A.
Catena
G. C.
Huang
J.
Litwiler
K. S.
Zhang
J.
Zagrobelny
J.
Bright
F. V.
, Anal. Chim. Acta 246 , 55 (1991 ).
58.
Bright
F. V.
Litwiler
K. S.
Vargo
T. G.
Gardella
J. A.
Jr.
, Anal. Chim. Acta 262 , 323 (1992 ).
59.
Spencer
R. D.
Weber
G.
, J. Chem. Phys. 52 , 1654 (1970 ).
60.
Lakowicz
J. R.
Cherek
H.
Balter
A.
, J. Biochem. Biophys. Meth. 5 , 131 (1981 ).
61.
Feddersen
B. A.
Piston
D. W.
Gratton
E.
, Rev. Sci. Instrumn. 60 , 2929 (1989 ).
62.
Jameson
D. M.
Gratton
E.
Hall
R. D.
, Appl. Spectrosc. Rev. 20 , 55 (1986 ).
63.
Lakowicz
J. R.
Laczko
G.
Gryczynski
I.
Sqmacinski
H.
Wiczk
W.
, J. Photochem. Photobiol. B: Biol. 2 (29 ), 5 (1988 ).
64.
Gratton
E.
Jameson
D. M.
Hall
R. D.
, Ann. Rev. Biophys. Bioeng. 13 , 105 (1984 ).
65.
Beechem
J. M.
Gratton
E.
Mantulin
W.
, Globals Unlimited, User and Technical Reference Manuals (University of Illinois , Urbana , 1990 ), pp. 7 –47 .
66.
Beechem
J. M.
Gratton
E.
, Proc. SPIE 909 , 70 (1988 ).
67.
Beck
M. T.
Nagypal
I.
, Chemistry of Complex Equilibria , Translated by
Williams
D. R.
(John Wiley & Sons , New York , 1990 ), pp. 112 –121 .
68.
Huang
J.
Bright
F. V.
, J. Phys. Chem. 94 , 8457 (1990 ).
69.
Creighton
T. E.
, Protein Functions: A Practical Approach (Oxford University , New York , 1989 ), Chap. 2.
70.
Blatt
E.
Husain
A.
Sawyer
W. H.
, Biochim. Biophys. Acta 871 , 6 (1986 ).
71.
Eftink
M. R.
Ghiron
C. A.
, Biochemistry 16 , 5546 (1977 ).
72.
Chmelík
J.
, Collect. Czech. Chem. Commun. 54 , 2543 (1989 ).
73.
Chmelík
J.
Anzenbacher
P.
Chemlíková
J.
Matějčkovà
M.
Kalous
V.
, Collect. Czech. Chem. Commun. 53 , 411 (1988 ).
74.
Litwiler
K. S.
, Ph.D. Thesis , State University of New York at Buffalo (1991 ).