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Fluorescence Polarization Kinetic Measurements of Antigen-Antibody Reactions

  • S. A. Levison
  • F. Kierszenbaum
  • W. B. Dandliker
Conference paper

Abstract

The polarization of fluorescent light from solutions has provided important information concerning the size, shape and conformation of macromolecules (1, 2), molecular anisostropy (3), electronic energy transfer (4) and interactions which include dye binding (5) to proteins. During the past few years the measurement of both fluorescence polarization and intensity has been successfully utilized to determine both equilibrium (6, 7, 8, 9) and kinetic parameters (10, 11, 12, 13, 14, 15) for antigen-antibody systems. The basis of this approach involves the tagging of one of the reactants, e. g. the antigen with a small fluorescent molecule which is then used as the detecting and measuring agent for its partner. Changes in either the polarization of fluorescence intensity or the fluorescence intensity itself can then be monitored directly and hence afford a means by which the extent of reaction can be followed. It is important to note that changes in the fluorescence polarization parameter occur even in the absence of fluorescence quenching or enhancement as long as there is a change in rotary brownian motion, which results from the combination of the smaller fluorescent-labeled molecule with its larger unlabeled partner. Hence, fluorescence polarization measurements afford a powerful general approach by which the kinetics and thermodynamics of important macromolecular reactions can be studied. This particular report while including some thermodynamic data, centers mainly on the rates of reaction between antigen and antibody molecules in the primary stages of combination and on the effects of the ionic medium on these rates.

Keywords

Fluorescence Polarization Antibody Molecule Ionic Medium Rotary Brownian Motion Encounter Pair 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Symbols

e,

equilibrium value of parameter

f, b,

free and bound forms, respectively of fluorescent-labeled material

0,

at time approaching zero

(AB),

molar concentration of antibody

(AG),

molar concentration of antigen

F,

molar concentration of fluorescent-labeled material

Fb max,

concentration of cm1 ning sites in unlabeled component b max, as determined by equation (3)

k,

defined by — \( \frac{d\left(AG\right)}{dt} \) = k\( {{\left( AB \right)}^{{{N}_{1}}}}{{\left( AG \right)}^{{{N}_{2}}}} \) (AG) equation (7)

k1,

bimolecular rate constant defined by equation (1)

k−1,

unimolecular rate constant defined by equation (1)

k,

\( \frac{{{k}_{1}}}{{{k}_{-1}}} \) equals equilibrium association constant defined by equation (1)

k′,

empirical rate constant defined by equation (10)

k′′,

empirical rate constant defined by equation (11)

kp,

unimolecular rate constant defined by equation (12)

N1,

order of reaction with respect to (AB)

N2,

order of reaction with respect to (AG)

p,

polarization of fluorescence

Q,

ratio of fluorescence intensity to molar concentration of fluorescent-labeled material

\( \frac{dp}{dt} \)

rate of change of polarization

Ko,

average association constant defined by equation (3)

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Bibliography

  1. 1.
    Weber, G. Biochem. J., 51, 145 (1952).Google Scholar
  2. 2.
    Steiner, R. F. and Edelhoch, H. Chem. Rev., 62, 457 (1962).CrossRefGoogle Scholar
  3. 3.
    Albrecht, A. J. Mol. Spectroscopy, 6, 84 (1967).CrossRefGoogle Scholar
  4. 4.
    Weber, G. and Teale, F. W. J. The Proteins, 3, 445, edited by Neurath, Academic Press, New York ( 1965).Google Scholar
  5. 5.
    Laurence, D. J. R. Biochem. J., 51, 168 (1952).Google Scholar
  6. 6.
    Dandliker, W. and Feigen, G. Biochem. Biophys. Res. Comm., 5, 299 (1961).CrossRefGoogle Scholar
  7. 7.
    Haber, E. and Bennett, J. C. Proc. Nat. Acad. Sci., 48, 1935 (1962).CrossRefGoogle Scholar
  8. 8.
    Dandliker, W. B., Schapiro, H. C., Meduski, J. W., Alonso, R., Feigen, G. A. and Hamrick, J. R. Jr. Immunochem., 1, 165 (1964).CrossRefGoogle Scholar
  9. 9.
    Dandliker, W. B., Halbert, S. P., Florin, M. C., Alonso, R. and Schapiro, H. C. J. Exp. Med., 122, 1029 (1965).CrossRefGoogle Scholar
  10. 10.
    Dandliker, W. B. and Levison, S. A. Immunochem., 5, 171 (1967).Google Scholar
  11. 11.
    Levison, S. A., Jancsi, A. N. and Dandliker, W. B. Biochem. Biophys. Res. Comm. 33, 942 (1968).CrossRefGoogle Scholar
  12. 12.
    Levison, S. A. and Dandliker, W. B. Immunochem., 6, 253 (1969).CrossRefGoogle Scholar
  13. 13.
    Levison, S. A., Kierszembaum, F. and Dandliker, W.B. Fed. Proc., 28, 326 (1969).Google Scholar
  14. 14.
    Tengerdy, R. P. Immunochem., 3, 463 (1966).CrossRefGoogle Scholar
  15. 15.
    Tengerdy, R. P. J. Immunol., 99, 126 (1967).Google Scholar
  16. 16.
    Kierszenbaum, F., Dandliker, J. and Dandliker, W. B. Immunochem., 6, 125 (1969).CrossRefGoogle Scholar
  17. 17.
    Porter, R. R. Biochem. J., 73, 119 (1959).Google Scholar
  18. 18.
    Kierszenbaum, F., Levison, S.A. and Dandliker, W. B Anal. Biochem., 28, 563 (1969).CrossRefGoogle Scholar
  19. 19.
    White, J. U., Williamson, D. E., Levison, S. A. and Dandliker, W. B. (in preparation).Google Scholar
  20. 20.
    Frost, A.A. and Pearson, R.G. Kinetics and Mechanism, p. 186, John Wiley, Inc., New York (1961)Google Scholar
  21. 21.
    von Hippel, P.H. and Wong, K.Y. Science, 145, 577 (1964)CrossRefGoogle Scholar
  22. 22.
    Warren, H.C. and Cheatum, S.G. Biochem., 5, 1702 (1966)Google Scholar
  23. 23.
    Warren, J. C., Stowring, L. and Morales, M. F. J. Biol. Chem., 241, 309 (1966).Google Scholar
  24. 24.
    Pressman, D., N íssonoff, A. and Radzimski, G. J. Immunol., 86, 35 (1961).Google Scholar
  25. 25.
    Dandliker, W. B., Alonso, R., de Saussure, V. A., Kier szenbaum, F., Levison, S.A. and Schapiro, H. C. Biochem., 6, 1460 (1967).CrossRefGoogle Scholar
  26. 26.
    Bunton, C. A. and Robinson, L. J. Am. Chem. Soc., 90, 5965 (1968).CrossRefGoogle Scholar
  27. 27.
    Feinstein, A. and Rowe, A. J. Nature, 205, 147 (1965).CrossRefGoogle Scholar
  28. 28.
    Valentine, K. and Green, N. J. Molec. Biol., 27, 615 (1967).CrossRefGoogle Scholar
  29. 29.
    Laidler, K. J. Chemical Kinetics, p. 198. McGraw-Hill, Inc., New York, N. Y. (1965).Google Scholar
  30. 30.
    Day, L. A., Sturtevant, J. M. and Singer, S. J. Ann. N. Y. Acad. Sciences, 103, 611 (1963).CrossRefGoogle Scholar
  31. 31.
    Dandliker, W.B. and de Saussure, V.A. In The Chemistry of Biosurfaces, Edited by M. Hair, Marcel Dekker, N.Y. (1970).Google Scholar
  32. 32.
    Blps, K. J. Chem. Phys., 16, 490 (1948).Google Scholar
  33. 33.
    Winstein, S., Appel, B., Baker, R. and Diaz, A. The Chemical Society, London, Special Publication No. 19, pp. 109–130, (1965).Google Scholar
  34. 34.
    Eigen, M. Z. Elektrochem., 64, 115 (1960).Google Scholar
  35. 35.
    Eigen, M. and Tamm, K. A. Elektrochem., 66, 107 (1960)Google Scholar
  36. 36.
    Szwarc, M. Accounts of Chemical Research, 2, 87 (1968).CrossRefGoogle Scholar
  37. 37.
    Noelken, M. E., Nelson, C. A., Buckley, C. E. III and Tanford, C. J. Biol. Chem., 240, 218 (1965).Google Scholar
  38. 38.
    Halsey, G. D. J. Chem. Phys., 17, 758 (1949).Google Scholar

Copyright information

© Plenum Press, New York 1970

Authors and Affiliations

  • S. A. Levison
  • F. Kierszenbaum
  • W. B. Dandliker
    • 1
  1. 1.Division of BiochemistryScripps Clinic & Research FoundationLa JollaUSA

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