We initially investigated the prope

We initially investigated the properties of several different peptides to determine the best candidate for the modification of IGF-1. All of these experiments were conducted with a FLAG (DYKDDDDK)-tagged IGF-1 protein, as shown in Figure 2. Although the colors of the YKYKY and HHHHHH (H6) proteins remained the same for all of the pH values tested (4.5–8.5), the color of the XKXKX protein was found to be pH-dependent. For example, the XKXKX protein turned from transparent to yellow at high pH (pH > 7). After 24 h the color change completed. The spectral change is similar to that of mixture of catechol and gamine (glycine) (Figure S11 in the Supporting Information). The turbidity of a solution of XKXKX in PBS was also found to be dependent on the pH value (Figure 2 b), with the mixture becom- ing increasingly turbid at high pH (pH > 7). It is noteworthy that these properties are similar to those reported for solutions of dopamine and DOPA.[9] This increase in the turbidity of dopamine solutions at high pH has been attributed to the occurrence of Michael addi- tion or Schiff base reactions.[10] Furthermore, the reaction of the catechol moiety of DOPA with the g-amino groups of the lysine residues in the peptide would make a considerable contribution to reactions of this type and, indeed, the turbidity. The oxidation reaction of catechol is considered to resulted in chemical crosslinking.