Date of Award
Honors Thesis (Open Access)
Colby College. Chemistry Dept.
Thomas W. Shattuck
Metal substitution has been proposed as an aid in the characterization of the structure of Low-Molecular-Weight Chromium-binding substance (LMWCr), which appears to be the biologically active chromium species. Preparations of bovine liver were diced and suspended in an isolation solution containing either a cobalt or a chromium salt. Peptide samples from these preparations were isolated by precipitation and centrifugation and purified by liquid chromatography. Several methods were used to analyze these samples, which may contain LMWCr or the cobalt-substituted form (LMWCo), in order to determine their purity, molecular weight (MW), and metal-to-peptide ratio. Methods used to determine MW included sodium dodecyl sulfate polyacrylamide gel electrophoresis (5DS-PAGE), and high performance liquid chromatography (HPLC). Inductively coupled plasma atomic emission spectroscopy (ICP-AES) and fluorescence spectrophotometry were used to determine the metal-to-peptide ratio. The MW of the peptide appears to be between 3000 and 6000, and the metal-to-peptide ratio appears to be between 3.3 and 3.7 (previous reports suggest that LMWCr contains about 4 Cr(IIT) ions). Amino acid analyses have shown both peptides to contain significant amounts asp. glu, gly, and cys. However, the cobalt peptide has higher concentrations of cys than anticipated and the chromium peptide also has large amounts of ser, ala, lys, and pro. Initial attempts at N-terminal sequencing of the cobalt peptide have been unsuccessful.
Peptides -- Analysis, Chromium -- Physiological effect, Cobalt -- Physiological effect
Recommended CitationJose, Michael J., "Isolation and characterization of chromium- and cobalt-binding peptides" (2001). Honors Theses. Paper 130.
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