Effect of metal cations on the conformation and inactivation of recombinant human factor VIII.
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.
Derrick
Tiffany S
TS
Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, KS 66047, USA.
Kashi
Ramesh S
RS
Durrani
Manzer
M
Jhingan
Ashish
A
Middaugh
C Russell
CR
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
D002412:Cations; D008670:Metals; D019999:Pharmaceutical Solutions; D011994:Recombinant Proteins; D013725:Terbium; D003035:Cobalt; D005169:Factor VIII; D000535:Aluminum; D007501:Iron