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. 1998 Aug;75(2):1032–1039. doi: 10.1016/S0006-3495(98)77592-7

Macromolecular diffusion of biological polymers measured by confocal fluorescence recovery after photobleaching.

P Gribbon 1, T E Hardingham 1
PMCID: PMC1299777  PMID: 9675204

Abstract

Fluorescence recovery after photobleaching with an unmodified confocal laser scanning microscope (confocal FRAP) was used to determine the diffusion properties of network forming biological macromolecules such as aggrecan. The technique was validated using fluorescein isothiocyanate (FITC)-labeled dextrans and proteins (molecular mass 4-2000 kDa) at 25 degrees C and with fluorescent microspheres (207 nm diameter) over a temperature range of 5-50 degrees C. Lateral diffusion coefficients (D) were independent of the focus position, and the degree and extent of bleach. The free diffusion coefficient (Do) of FITC-aggrecan determined by confocal FRAP was 4.25 +/- 0.6 x 10(-8) cm2 s-1, which is compatible with dynamic laser light scattering measurements. It appeared to be independent of concentration below 2.0 mg/ml, but at higher concentrations (2-20 mg/ml) the self-diffusion coefficient followed the function D = Do(e)(-Bc). The concentration at which the self-diffusion coefficient began to fall corresponded to the concentration predicted for domain overlap. Multimolecular aggregates of aggrecan ( approximately 30 monomers) had a much lower free diffusion coefficient (Do = 6.6 +/- 1.0 x 10(-9) cm2 s-1) but showed a decrease in mobility with concentration of a form similar to that of the monomer. The method provides a technique for investigating the macromolecular organization in glycan-rich networks at concentrations close to those found physiologically.

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Selected References

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  1. Amu T. C. Activation enthalpy of diffusion for well fractionated dextrans in aqueous solutions. Biophys Chem. 1982 Dec;16(4):269–273. doi: 10.1016/0301-4622(82)87031-2. [DOI] [PubMed] [Google Scholar]
  2. Axelrod D., Koppel D. E., Schlessinger J., Elson E., Webb W. W. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J. 1976 Sep;16(9):1055–1069. doi: 10.1016/S0006-3495(76)85755-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Grodzinsky A. J. Electromechanical and physicochemical properties of connective tissue. Crit Rev Biomed Eng. 1983;9(2):133–199. [PubMed] [Google Scholar]
  4. Hardingham T. E., Ewins R. J., Muir H. Cartilage proteoglycans. Structure and heterogeneity of the protein core and the effects of specific protein modifications on the binding to hyaluronate. Biochem J. 1976 Jul 1;157(1):127–143. doi: 10.1042/bj1570127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hardingham T. E., Muir H., Kwan M. K., Lai W. M., Mow V. C. Viscoelastic properties of proteoglycan solutions with varying proportions present as aggregates. J Orthop Res. 1987;5(1):36–46. doi: 10.1002/jor.1100050107. [DOI] [PubMed] [Google Scholar]
  6. Hardingham T. E., Muir H. The specific interaction of hyaluronic acid with cartillage proteoglycans. Biochim Biophys Acta. 1972 Sep 15;279(2):401–405. doi: 10.1016/0304-4165(72)90160-2. [DOI] [PubMed] [Google Scholar]
  7. Hardingham T. E. The role of link-protein in the structure of cartilage proteoglycan aggregates. Biochem J. 1979 Jan 1;177(1):237–247. doi: 10.1042/bj1770237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hardingham T. Proteoglycans: their structure, interactions and molecular organization in cartilage. Biochem Soc Trans. 1981 Dec;9(6):489–497. doi: 10.1042/bst0090489. [DOI] [PubMed] [Google Scholar]
  9. Harper G. S., Comper W. D., Preston B. N., Daivis P. Concentration dependence of proteoglycan diffusion. Biopolymers. 1985 Nov;24(11):2165–2173. doi: 10.1002/bip.360241111. [DOI] [PubMed] [Google Scholar]
  10. Harper G. S., Preston B. N. Molecular shrinkage of proteoglycans. J Biol Chem. 1987 Jun 15;262(17):8088–8095. [PubMed] [Google Scholar]
  11. Jain R. K., Stock R. J., Chary S. R., Rueter M. Convection and diffusion measurements using fluorescence recovery after photobleaching and video image analysis: in vitro calibration and assessment. Microvasc Res. 1990 Jan;39(1):77–93. doi: 10.1016/0026-2862(90)90060-5. [DOI] [PubMed] [Google Scholar]
  12. Johnson E. M., Berk D. A., Jain R. K., Deen W. M. Diffusion and partitioning of proteins in charged agarose gels. Biophys J. 1995 Apr;68(4):1561–1568. doi: 10.1016/S0006-3495(95)80328-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kitchen R. G., Cleland R. L. Dilute solution properties of proteoglycan fractions from bovine nasal cartilage. Biopolymers. 1978 Mar;17(3):759–783. doi: 10.1002/bip.1978.360170316. [DOI] [PubMed] [Google Scholar]
  14. Mow V. C., Zhu W., Lai W. M., Hardingham T. E., Hughes C., Muir H. The influence of link protein stabilization on the viscometric properties of proteoglycan aggregate solutions. Biochim Biophys Acta. 1989 Aug 18;992(2):201–208. doi: 10.1016/0304-4165(89)90011-1. [DOI] [PubMed] [Google Scholar]
  15. Ohno H., Blackwell J., Jamieson A. M., Carrino D. A., Caplan A. I. Calibration of the relative molecular mass of proteoglycan subunit by column chromatography on Sepharose CL-2B. Biochem J. 1986 Apr 15;235(2):553–557. doi: 10.1042/bj2350553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Poitevin E., Wahl P. Study of the translational diffusion of macromolecules in beads of gel chromatography by the FRAP method. Biophys Chem. 1988 Sep;31(3):247–258. doi: 10.1016/0301-4622(88)80030-9. [DOI] [PubMed] [Google Scholar]
  17. Ratcliffe A., Doherty M., Maini R. N., Hardingham T. E. Increased concentrations of proteoglycan components in the synovial fluids of patients with acute but not chronic joint disease. Ann Rheum Dis. 1988 Oct;47(10):826–832. doi: 10.1136/ard.47.10.826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Reihanian H., Jamieson A. M., Tang L. H., Rosenberg L. Hydrodynamic properties of proteoglycan subunit from bovine nasal cartilage. Self-association behavior and interaction with hyaluronate studied by laser light scattering. Biopolymers. 1979 Jul;18(7):1727–1747. doi: 10.1002/bip.1979.360180711. [DOI] [PubMed] [Google Scholar]
  19. Soumpasis D. M. Theoretical analysis of fluorescence photobleaching recovery experiments. Biophys J. 1983 Jan;41(1):95–97. doi: 10.1016/S0006-3495(83)84410-5. [DOI] [PMC free article] [PubMed] [Google Scholar]

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