Purification of granulocyte chemotactic peptide/interleukin-8 reveals N-terminal sequence heterogeneity similar to that of beta-thromboglobulin.

Stimulated human peripheral blood leukocytes produce a chemotactic factor for granulocytes (granulocyte chemotactic peptide/interleukin-8; GCP/IL-8), which is structurally related to platelet-derived beta-thromboglobulin. Analytically pure CGP/IL-8 and beta-thromboglobulin could be obtained after three purification steps, comprising adsorption to silicic acid, heparin-Sepharose chromatography and ion-exchange chromatography. Although GCP/IL-8 and beta-thromboglobulin had a similar affinity for heparin, they could be separated on a cation-exchange column. Both molecules were heterogeneous in that 6-7-kDa protein doublets were detected upon SDS/PAGE. N-terminal amino acid sequence analysis revealed the presence of six immunologically related but differently truncated polypeptides of beta-thromboglobulin, of which only two corresponded to previously described forms. Similarly, apart from a major polypeptide, five minor species of GCP/IL-8 were detected that also differed by N-terminal truncation. The most processed forms of beta-thromboglobulin and GCP/IL-8 were found to have their N-terminus in that region of the primary structure where a significant similarity between the two molecules starts. GCP/IL-8 was found to be chemotactic for granulocytes with a specific activity of 10(5) units/mg, whereas none of the beta-thromboglobulin species possessed detectable chemotactic activity.