title: Effect of metal cations on the conformation and inactivation of recombinant human factor VIII.
abstract: Heavy metals have been implicated in the aggregation of proteins and the pathophysiology of several neurodegenerative diseases. Herein, we describe the interaction of recombinant human factor VIII (rhFVIII) with Al(+3), Tb(+3), Co(+2), and Fe(+3) using a combination of intrinsic fluorescence, circular dichroism, and high-resolution fourth-derivative absorbance analysis. rhFVIII in solution was titrated with the metal cations and the properties of the resulting complexes were examined. rhFVIII has a tendency to aggregate and inactivate slowly over time under physiological conditions, but this aggregation process is greatly accelerated in the presence of metals with Al(+3) being the most efficient. This leads to a complete loss of activity of the protein. Al(+3)-induced conformational changes in the protein were small but detectable with limited changes seen in secondary and tertiary structure. Because rhFVIII is a multidomain protein with subunits linked through divalent metal cations, the small intramolecular changes seen may be attributed to rearrangements of the subunits to an aggregation-competent conformer that is very similar to that of the native form.
authors.affiliation: Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047, USA.
authors.forename: Tiffany S
authors.initials: TS
authors.lastname: Derrick
authors.forename: Ramesh S
authors.initials: RS
authors.lastname: Kashi
authors.forename: Manzer
authors.initials: M
authors.lastname: Durrani
authors.forename: Ashish
authors.initials: A
authors.lastname: Jhingan
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
mesh_terms: D000535:Aluminum / Q000737:chemistry; D002412:Cations / Q000737:chemistry*; D002942:Circular Dichroism; D003035:Cobalt / Q000737:chemistry; D004355:Drug Stability; D005169:Factor VIII / Q000737:chemistry*; D006801:Humans; D007501:Iron / Q000737:chemistry; D008027:Light; D008670:Metals / Q000737:chemistry*; D019999:Pharmaceutical Solutions / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013696:Temperature; D013725:Terbium / Q000737:chemistry
chemical_list: D002412:Cations; D008670:Metals; D019999:Pharmaceutical Solutions; D011994:Recombinant Proteins; D013725:Terbium; D003035:Cobalt; D005169:Factor VIII; D000535:Aluminum; D007501:Iron