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Diffusion of Insulin-Like Growth Factor-I and Ribonuclease through Fibrin Gels

Jess V. Nauman ^* , Phil G. Campbell ^* , Frederick Lanni ^*  and John L. Anderson 

^* Molecular Biosensor and Imaging Center, Institute for Complex Engineered Systems, and Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213; and Department of Chemical Engineering, Case Western Reserve University, Cleveland, Ohio 44106

Correspondence: Address reprint requests to Jess Nauman, Tel.: 412-268-9881; E-mail: jvn{at}andrew.cmu.edu; or Phil G. Campbell, E-mail: pcampbel{at}ices.cmu.edu.

A fluorescence-based method for simultaneously determining the diffusion coefficients of two proteins is described, and the diffusion coefficient of insulin-like growth factor (IGF-I) and ribonuclease (RNase) in a 0.27% fibrin hydrogel is reported. The method is based on two-color imaging of the relaxation of the protein concentration field with time and comparing the results with a transport model. The gel is confined in a thin (200 µm) capillary and the protein is labeled with a fluorescent dye. The experimentally determined diffusion coefficient of RNase (D = 1.21 x 10^–6 cm²/s) agrees with literature values for dilute gels and bulk aqueous solutions, thus indicating the gel and the dye had a negligible effect on diffusion. The experimental diffusion coefficient of IGF-I (D = 1.59 x 10^–6 cm²/s), in the absence of binding to the fibrin matrix, is consistent with the dimensions of the molecule known from x-ray crystallography and a correlation between D and molecular weight based on 14 other proteins. The experimental method developed here holds promise for determining molecular transport properties of biomolecules under a variety of conditions, for example, when the molecule adsorbs to the gel or is convected through the gel by fluid transport.