ISSN:
1573-904X
Keywords:
protein pharmaceuticals
;
liposomes
;
formulation excipient
;
molecular chaperones
;
structured intermediate states
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
Abstract Purpose. The advent of recombinant DNA technology has madepossible the pharmaceutical use of a wide range of proteins and peptides.However, the complex structure of proteins renders them susceptibleto physical instabilities such as denaturation, aggregation andprecipitation. We tested the hypothesis that partial unfolding and exposure ofhydrophobic domains leads to physical instability, and investigatedapproaches to stabilize protein formulations. Methods. KP6 β, an 81 amino acid killer toxin from Ustilago maydis,was used as a model protean. Circular dichroism and fluorescencespectroscopy were used to study the temperature dependent folding/unfolding characteristics of KP6 β. ANS (1,8 anilinonaphthalenesulfonate), a fluorescent probe that partitions into hydrophobic domains,was used to detect exposure of hydrophobic domains. Results. As the temperature was elevated, near-UV CD indicatedprogressive loss of KP6 β tertiary structure, while far-UV CD indicatedretention of secondary structure. Increasing exposure of hydrophobicdomains was observed, as indicated by the penetration of ANS. Atelevated temperatures (60°C), KP6 β conserved most secondarystructural features. However, tertiary structure was disordered, suggestingthe existence of a partially folded, structured intermediate state.Liposomes bound to partially unfolded structures and prevented theformation of aggregates. Conclusions. Partial unfolding resulted in increased exposure ofhydrophobic domains and aggregation of KP6 β, but with preservationof secondary structure. Liposomes interacted with the structuredintermediate state, stabilizing the protein against aggregation. These resultssuggest a general formulation strategy for proteins, in which partiallyunfolded structures are stabilized by formulation excipients that act asmolecular chaperones to avoid physical instability.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1007561308498
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