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Photochemistry of Macromolecules pp 47–65Cite as

Fluorescence Polarization Kinetic Measurements of Antigen-Antibody Reactions

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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.

This work is supported by The John A. Hartford Foundation, The National Science Foundation, The National Institutes of Health, (AM 7508, AM 5458 and Special Fellowhip AM 42568 from The National Institute of Arthritis and Metabolic Diseases.)

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Abbreviations

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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Author information

Authors and Affiliations

  1. Division of Biochemistry, Scripps Clinic & Research Foundation, La Jolla, California, 92037, USA

    W. B. Dandliker

Authors
  1. S. A. Levison
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  2. F. Kierszenbaum
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  3. W. B. Dandliker
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Editor information

Editors and Affiliations

  1. NASA-Ames Research Center, Moffett Field, California, USA

    Ronald F. Reinisch

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© 1970 Plenum Press, New York

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Levison, S.A., Kierszenbaum, F., Dandliker, W.B. (1970). Fluorescence Polarization Kinetic Measurements of Antigen-Antibody Reactions. In: Reinisch, R.F. (eds) Photochemistry of Macromolecules. Springer, Boston, MA. https://doi.org/10.1007/978-1-4684-8035-1_5

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  • DOI: https://doi.org/10.1007/978-1-4684-8035-1_5

  • Publisher Name: Springer, Boston, MA

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  • Online ISBN: 978-1-4684-8035-1

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