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| Content Provider | Springer Nature Link |
|---|---|
| Author | Zhang, Aming Qi, Wei Singh, Satish K. Fernandez, Erik J. |
| Copyright Year | 2011 |
| Abstract | The impact of freeze-thaw (F/T) on structure integrity of protein therapeutics is poorly understood, partially due to lack of methods to detect protein structural perturbations during F/T processing in the frozen state.A new approach of hydrogen/deuterium exchange was developed to separate and distinguish the specific impact of single freezing and F/T cycling on protein structure, using lactate dehydrogenase (LDH) as model system.In the freezing process, a fraction of LDH molecules that was inversely dependent on protein concentration was observed to partially denature its structure. Local structural perturbations were localized by peptide level HX analysis to the surface residues in segments 91–132, 170–237 and 288–331. In contrast, F/T cycling led to irreversible LDH aggregation with global structural unfolding. Residual solvent-protected structure was only detected in the aggregates for three segments, 13–31, 109–117 and 133–143, that were coincident with the consensus aggregation hotspots predicted by four different algorithms.Results indicate freezing preferentially disturbs local structure at the surface residues, consistent with ice-solution interface-mediated denaturation mechanism. F/T-induced aggregation begins as partial denaturation during freezing, but is accompanied by more comprehensive structural rearrangement during F/T cycling. |
| Starting Page | 1179 |
| Ending Page | 1193 |
| Page Count | 15 |
| File Format | |
| ISSN | 07248741 |
| Journal | Pharmaceutical Research |
| Volume Number | 28 |
| Issue Number | 5 |
| e-ISSN | 1573904X |
| Language | English |
| Publisher | Springer US |
| Publisher Date | 2011-02-08 |
| Publisher Place | Boston |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Biochemistry Biomedical Engineering Pharmacy Pharmacology/Toxicology Medical Law |
| Content Type | Text |
| Resource Type | Article |
| Subject | Organic Chemistry Pharmacology Molecular Medicine Pharmacology (medical) Biotechnology Pharmaceutical Science |
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