Journal of Fluorescence

, Volume 3, Issue 1, pp 1–16 | Cite as

Dynamics of nonspecific adsorption of insulin to erythrocyte membranes

  • Robert M. Fulbright
  • Daniel Axelrod


Molecules may arrive at targets (receptors, enzymes, etc.) localized on a membrane surface by first adsorbing onto the surface and then surface diffusing to the targets. The flux rate of molecules arriving at targets via this mechanism depends on the surface diffusion coefficient of the molecules and, in some circumstances, on the adsorption/desorption kinetics. The technique of total internal reflection with fluorescence recovery after photobleaching (TIR-FRAP) was used here to study these rate parameters of fluorescein-labeled insulin (f-insulin) interacting with erythrocyte ghosts. Ghosts were adhered to polylysine coated slides for TIR illumination. Some ghosts became flattened and unsealed on the polylysine so that both extracellular and cytoplasmic sides of the membrane were openly exposed to the solution. An aluminum thin film between the polylysine and the fused silica of a slide quenched ‘background’ fluorescence from f-insulin adsorbed directly onto the polylysine. An interference fringe pattern from two intersecting and totally internally reflecting laser beams provided surface-selective excitation with a spatial variation of illumination intensity across a ghost for surface diffusion measurements. Measured characteristic values of desorption rate constants ranged from 0.043 to 270 s−1. According to a preexisting theoretical model, the largest desorption rate constant in this range would result in some increase in the total flux rate to a perfect sink target due to capture from the surface, provided that the surface diffusion coefficient was ≥ about 10−8 cm2/s. However, based on TIR-FRAP measurements on our system, we estimate that the surface diffusion coefficient is less than about 5×10−10 cm2/s. The combination of novel techniques presented here may prove valuable to other workers seeking to make diffusive and chemical kinetic rate parameter measurements of biomolecules at biological cell membranes.

Key Words

Total internal reflection fluorescence surface diffusion desorption kinetics photobleaching recovery hormone reaction rates 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    G. Adam and M. Delbruck (1968) in A. Rich and N. Davidson (Eds.),Structural Chemistry and Molecular Biology, W. H. Freeman, San Francisco, pp. 198–215.Google Scholar
  2. 2.
    H. C. Berg, and E. M. Purcell (1977)Biophys. J.,20, 193–219.PubMedGoogle Scholar
  3. 3.
    D. Wang, S.-Y. Gou, and D. Axelrod (1992)Biophys. Chem. 43, 117–137.PubMedGoogle Scholar
  4. 4.
    N. L. Thompson, T. P. Burghardt, and D. Axelrod (1981)Biophys. J. 33, 435–454.PubMedGoogle Scholar
  5. 5.
    T. P. Burghardt and D. Axelrod (1981)Biophys. J. 33, 455–468.PubMedGoogle Scholar
  6. 6.
    E. H. Hellen and D. Axelrod (1991)J. Fluoresc. 1, 113–128.Google Scholar
  7. 7.
    R. D. Tilton, A. P. Gast, and C. R. Robertson (1990)Biophys. J. 58, 1321–1326.PubMedGoogle Scholar
  8. 8.
    M. L. Pisarchick, D. Gesty, and N. L. Thompson (1992)Biophys. J., 63:215–223.PubMedGoogle Scholar
  9. 9.
    K. H. Pearce, R. G. Hisket, and N. L. Thompson (1992)Biochemistry 31, 5983–5995.PubMedGoogle Scholar
  10. 10.
    K. K. Gambhir, J. A. Archer, and C. J. Bradley (1978)Diabetes 27, 701–708.PubMedGoogle Scholar
  11. 11.
    S. T. Sui, T. Urumow, and E. Sackmann (1988)Biochemistry 27, 7463–7469.PubMedGoogle Scholar
  12. 12.
    B. S. Jacobson, J. Cronin, and D. Branton (1978)Biochim. Biophys. Acta 506, 81–96.PubMedGoogle Scholar
  13. 13.
    E. H. Hellen and D. Axelrod (1987)J. Opt. Soc. Am. B 4, 337–350.Google Scholar
  14. 14.
    R. R. Chance, A. Prock, and R. Silbey (1975)J. Chem. Phys. 62, 2245–2253.Google Scholar
  15. 15.
    R. R. Chance, A. Prock, and R. Silbey (1978)Adv. Chem. Phys. 37, 1–65.Google Scholar
  16. 16.
    M. R. Philpott (1975)J. Chem. Phys. 62, 1812–1817.Google Scholar
  17. 17.
    H. Morawitz and M. R. Philpott (1974)Phys. Rev. B 10, 4863–4868.Google Scholar
  18. 18.
    M. P. Sheetz and D. E. Koppel (1979)Proc. Natl. Acad. Sci. USA 76, 3314–3317.PubMedGoogle Scholar
  19. 19.
    C. S. Foote (1968)Science 162, 963–970.PubMedGoogle Scholar
  20. 20.
    R. M. Fulbright (1991) Ph.D. thesis, University of Michigan, Ann Arbor.Google Scholar
  21. 21.
    F. Tietze, G. E. Mortimore, and N. R. Lomax (1962)Biochim. Biophys. Acta 59, 336–346.PubMedGoogle Scholar
  22. 22.
    W. W. Bromer, S. K. Sheehan, A. W. Berns, and E. R. Arquilla (1967)Biochemistry 6, 2378–2388.PubMedGoogle Scholar
  23. 23.
    E. R. Arquilla, W. W. Bromer, and D. Mercola (1969)Diabetes 18, 193–205.PubMedGoogle Scholar
  24. 24.
    D. A. Mercola, J. W. S. Morris, and E. R. Arquilla (1972)Biochemistry 11, 3860–3874.PubMedGoogle Scholar
  25. 25.
    P. R. Bevington (1969)Data Reduction and Error Analysis for the Physical Sciences, McGraw-Hill, New York.Google Scholar
  26. 26.
    T. G. Blundell, D. Dodson, D. Hodgkin, and D. Mercola (1972)Adv. Prot. Chem.,26, 279–402.Google Scholar
  27. 27.
    J. R. Abney, B. A. Scalettar, and N. L. Thompson (1992)Biophys. J. 61, 542–552.PubMedGoogle Scholar
  28. 28.
    D. Axelrod, E. H. Hellen, and R. M. Fulbright (1992) in J. Lakowicz (Ed.),Topics in Fluorescence Spectroscopy, Vol. 3: Biochemical Applications, Plenum Press, New York, pp. 289–343.Google Scholar
  29. 29.
    R. M. Weis, K. Balakrishnan, B. A. Smith, and H. M. McConnell (1982)J. Biol. Chem. 257, 6440–6445.PubMedGoogle Scholar
  30. 30.
    J. Davoust, P. F. Devaux, and L. Leger (1982)EMBC J 1, 1230–1238.Google Scholar
  31. 31.
    A. L. Stout and D. Axelrod (1989)Appl. Opt. 28, 5237–5242.Google Scholar

Copyright information

© Plenum Publishing Corporation 1993

Authors and Affiliations

  • Robert M. Fulbright
    • 1
  • Daniel Axelrod
    • 1
  1. 1.Biophysics Research Division & Department of PhysicsUniversity of MichiganAnn Arbor

Personalised recommendations