From spectroscopic data, it has been shown that Cr³⁺ binds tightly to chromodulin (K_f = 10²¹ M⁻⁴), and that the binding is highly cooperative (Hill Coefficient = 3.47).^[7] It has been shown that holochromodulin binds 4 equivalents of Cr³⁺.^[6][7][8] Evidence for this comes from in vitro studies which showed that apochromodulin exerts its maximal activity on insulin receptors when titrated with 4 equivalents of Cr³⁺.^[7][8][10] Chromodulin is highly specific for Cr³⁺ as no other metals are able to stimulate tyrosine kinase activity. It is believed to stimulate the phosphorylation of the 3 tyrosine residues of the β subunits of the insulin receptor.^{[7][8][10][11]} From electronic studies, the crystal field stabilization energy was determined to be 1.74 x 10³ while the Racah parameter B was 847 cm⁻¹. This indicates that chromium binds to chromodulin in the trivalent form.^[11] In addition, magnetic susceptibility studies have shown that chromium does not coordinate to any N-terminal amine groups but rather to carboxylates (although the exact the amino acids involved are still unknown).^[3] These magnetic susceptibility studies are consistent with the presence of a mononuclear Cr³⁺ center and an unsymmetric trinuclear Cr³⁺ assembly with bridging oxo ligands.^[11] In chromodulin isolated from bovine liver, x-ray absorption spectroscopy studies have shown that the chromium (III) atoms are surrounded by 6 oxygen atoms with an average Cr—O distance of 1.98 Å, while the distance between 2 chromium (III) atoms is 2.79 Å. These results are indicative of a multinuclear assembly.^[11] No sulfur ligands coordinate to chromium and instead, it has been proposed that a disulfide linkage between 2 cysteine residues occurs owing to a characteristic peak at 260 nm.^[11]