University of Utah Undergraduate Research Abstracts, Volume 3, Spring 2003

Purification of the Vitamin B 12 Binding Protein Haptocorrin and Progress Toward Binding Studies of Bioconjugated Substrates

Todd Gruber Senior Chemistry [e-mail] Faculty Sponsor: Charles B. Grissom Dept. of Chemistry [e-mail] PURIFICATION OF THE VITAMIN B 12 BINDING PROTEIN HAPTOCORRIN AND PROGRESS TOWARD BINDING STUDIES OF BIOCONJUGATED SUBSTRATES A purification scheme for the vitamin B12 (cobalamin) binding protein haptocorrin (HC) has been developed to aid in the study of vitamin B12 linked anti-cancer compounds in cancer therapy. Human breast milk, a source of HC, was subjected to ultracentrifugation. The whey fraction was extracted and loaded onto a size exclusion column. The eluted fractions were then assayed for haptocorrin activity using a charcoal binding assay using radiolabeled 57Co-cobalamin. The active fractions were combined and passed through an an ion exchange (Mono-Q) column on a fast-protein liquid chromatog-raphy (FPLC) system. Eluted fractions were again assayed for activity, and active fractions were checked for purity using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), as well as native-PAGE of haptocorrin bound to radiolabeled cobalamin. Pure HC will allow for studies into how the binding of cobalamin to haptocorrin is affected by chemical conjugation of various molecules to cobalamin itself, which have been produced by other Grissom lab members as a method of targeted drug delivery for cancer therapy. These binding studies will determine whether or not HC can still recognize cobalamin that has been chemically modified, and if it can carry targeted drugs to cancer cells. 56