  Glutaraldehyde (GA) reacts with amino groups in proteins, forming
intermolecular cross-links that, at sufficiently high protein concentration,
can transform a protein solution into a gel. Although GA has been used as a
cross-linking reagent for decades, neither the cross-linking chemistry nor the
microstructure of the resulting protein gel have been clearly established. Here
we use small-angle X-ray scattering (SAXS) to characterise the microstructure
and structural kinetics of gels formed by cross-linking of pancreatic trypsin
inhibitor, myoglobin or intestinal fatty acid-binding protein. By comparing the
scattering from gels and dilute solutions, we extract the structure factor and
the pair correlation function of the gels. The protein gels are spatially
heterogeneous, with dense clusters linked by sparse networks. Within the
clusters, adjacent protein molecules are almost in contact, but the protein
concentration in the cluster is much lower than in a crystal. At the $\sim$1 nm
SAXS resolution, the native protein structure is unaffected by cross-linking.
The cluster radius is in the range 10 - 50 nm, with the cluster size determined
mainly by the availability of lysine amino groups on the protein surface. The
development of structure in the gel, on time scales from minutes to hours,
appears to obey first-order kinetics. Cross-linking is slower at acidic pH,
where the population of amino groups in the reactive deprotonated form is low.
These results support the use of cross-linked protein gels in NMR studies of
protein dynamics and for modeling NMR relaxation in biological tissue.
