Conformational origin of the aggregation of recombinant human factor VIII

Citation
Ao. Grillo et al., Conformational origin of the aggregation of recombinant human factor VIII, BIOCHEM, 40(2), 2001, pp. 586-595
Citations number
52
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
0006-2960 → ACNP
Volume
40
Issue
2
Year of publication
2001
Pages
586 - 595
Database
ISI
SICI code
0006-2960(20010116)40:2<586:COOTAO>2.0.ZU;2-V
Abstract
Aggregation of proteins is a major problem in their use as drugs and is als o involved in a variety of pathological diseases. In this study, biophysica l techniques were employed to investigate aggregate formation in the pharma ceutically important protein, recombinant human factor VIII (rhFVIII). Reco mbinant human factor Vm incubated in solution at 37 degreesC formed soluble aggregates as detected by molecular sieve chromatography and dynamic light scattering. This resulted in a corresponding loss of biological activity. Fluorescence and CD spectra of the thermally stressed rhFVIII samples did n ot, however, suggest significant differences in protein conformation. To id entify conformational changes in rhFVIII that may be involved in rhFVIII ag gregation, temperature and solutes were used to perturb the native structur e of rhFVIII. Far-UV CD and FTIR studies of rhFVIII as a function of temper ature revealed conformational changes corresponding to an increase in inter molecular beta -sheet content beginning at approximately 45 degreesC with s ignificant aggregation observed above 60 degreesC. Fluorescence and DSC stu dies of rhFVIII also indicated conformational changes initiating between 45 and 50 degreesC. An increase in the exposure of hydrophobic surfaces was o bserved beginning at approximately 40 degreesC, as monitored by increased b inding of the fluorescent probe, bis-anilinonaphthalene sulfonic acid (bis- ANS). perturbation by various solutes produced several transitions prior to extensive unfolding of rhFVIII. In all cases, a common transition, charact erized by an increase in the wavelength of the fluorescence emission maximu m of rhFVIII from approximately 330 to 335 nm, was observed during thermal and solute perturbation of factor VIII, Moreover, this transition was corre lated with an increased association of factor VIII upon incubation at 37 de greesC in the presence of various solutes. These results suggest that assoc iation of rhFVIII in solution was initiated by a small transition in the te rtiary structure of the protein which produced a nucleating species that le d to the formation of inactive soluble aggregates.