Some viscoelastic properties of human erythrocyte spectrin networks end-linked in vitro

Abstract

We have succeeded in making macroscopic networks of end-linked human erythrocyte spectrin. The network junctions were made using erythrocyte protein 4.1 irreversibly attached to 5 nm (diameter) colloidal gold particles. Rotary shadowing electron microscopy verifies that the protein 4.1-labelled colloidal gold particles bind only to the tail end of the spectrin molecules. Electron micrographs of protein 4.1-labelled colloidal gold particles incubated at 4°C with spectrin dimers reveal that 1–5 spectrin dimers attach to each protein 4.1-labelled colloidal gold particle yielding a spider-like appearance of these complexes. Incubation with a low concentration of spectrin tetramers instead of dimers leads to extensive formation of spectrin microaggregates whereas use of spectrin concentrations higher than 3 mg/ml and a molar ratio between spectrin tetramers and protein 4.1/Au of 4 leads to formation of macroscopic spectrin networks. We have quantitated the viscoelastic properties of such end-linked macroscopic spectrin networks using a gravitational pendulum viscoelastometer. We find that in vitro end-linked spectrin networks can be described by linear viscoelastic theory. The dynamic storage modulus increases almost linearly with the spectrin-protein 4.1 / gold particle concentration when the spectrin concentration exceeds about 3 mg/ml and the molar ratio between spectrin tetramers and protein 4.1 / Au is 4. At a spectrin concentration of 6 mg/ml and the same ratio between spectrin and protein 4.1 / Au, we find a dynamic storage modulus at low frequency of about 80 dyn/cm 2. This is in adequate agreement with what is predicted by simple elastomer theory.