Effects of Additives on the Stability of Recombinant Human Factor XIII during Freeze-Drying and Storage in the Dried Solid

Kreilgaard, Lotte; Frøkjær, Sven; Flink, James M.; Randolph, Theodore W.; Carpenter, John F.

doi:10.1006/abbi.1998.0948

Cited by 104 publications

(83 citation statements)

References 35 publications

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“…Fibrils were generated in vitro as described by Solomon et al (18), except the sample volume was 0.15 ml. Samples were assayed immediately after preparation and daily thereafter for fibrils (19) and for soluble protein (20,21). For detection of fibrils, 20 l of 1 mg/ml protein solution were added to 1.98 ml of a 10 M solution of thioflavin T prepared in 50 mM glycine buffer (pH 9.0).…”

Section: Methodsmentioning

confidence: 99%

Thermodynamic Modulation of Light Chain Amyloid Fibril Formation

Kim¹,

Wall²,

Meyer³

et al. 2000

Journal of Biological Chemistry

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133

131

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To obtain further insight into the pathogenesis of amyloidosis and develop therapeutic strategies to inhibit fibril formation we investigated: 1) the relationship between intrinsic physical properties (thermodynamic stability and hydrogen-deuterium (H-D) exchange rates) and the propensity of human immunoglobulin light chains to form amyloid fibrils in vitro; and 2) the effects of extrinsically modulating these properties on fibril formation. An amyloid-associated protein readily formed amyloid fibrils in vitro and had a lower free energy of unfolding than a homologous nonpathological protein, which did not form fibrils in vitro. H-D exchange was much faster for the pathological protein, suggesting it had a greater fraction of partially folded molecules. The thermodynamic stabilizer sucrose completely inhibited fibril formation by the pathological protein and shifted the values for its physical parameters to those measured for the nonpathological protein in buffer alone. Conversely, urea sufficiently destabilized the nonpathological protein such that its measured physical properties were equivalent to those of the pathological protein in buffer, and it formed fibrils. Thus, fibril formation by light chains is predominantly controlled by thermodynamic stability; and a rational strategy to inhibit amyloidosis is to design high affinity ligands that specifically increase the stability of the native protein.

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Section: Methodsmentioning

confidence: 99%

Thermodynamic Modulation of Light Chain Amyloid Fibril Formation

Kim¹,

Wall²,

Meyer³

et al. 2000

Journal of Biological Chemistry

Self Cite

133

131

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“…The glycol band is preferentially decreased as the urea concentration is increased. The bottom panel (panel C) in all three figures shows how subsequent to the addition of urea, the addition of 0.5 M trehalose, a disaccharide known to stabilize native protein conformations (11,17,(53)(54)(55)(56)(57)(58)(59), impacts the urea induced changes. It can be seen that in all three cases, the addition of trehalose reversed the effect of adding urea, resulting in enhancement of the peak associated with the absence of proton dissociation (435/460 nm).…”

Section: Hpt Fluorescence As a Function Of Added Glycerol: The Influementioning

confidence: 99%

Molecular Level Probing of Preferential Hydration and Its Modulation by Osmolytes through the Use of Pyranine Complexed to Hemoglobin

Roche

Guo

Friedman

2006

Journal of Biological Chemistry

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Two spectroscopic probes are used to expose molecular level changes in hydration shell water interactions that directly relate to such issues as preferential hydration and protein stability. The major focus of the present study is on the use of pyranine (HPT) fluorescence to probe as a function of added osmolytes (PEG, urea, trehalose, and magnesium), the extent to which glycerol is preferentially excluded from the hydration shell of free HPT and HPT localized in the diphosphoglycerate (DPG) binding site of hemoglobin in both solution and in Sol-Gel matrices. The pyranine study is complemented by the use of vibronic side band luminescence from the gadolinium cation that directly exposes the changes in hydrogen bonding between first and second shell waters as a function of added osmolytes. Together the results form the basis for a water partitioning model that can account for both preferential hydration and water/osmolyte-mediated conformational changes in protein structure.It is becoming ever more apparent that water plays a major role both in stabilizing protein structures and in modulating protein dynamics (1-15). Many of the impressive advances in understanding the interplay between water and proteins and the resulting impact on protein stability have been derived from thermodynamic analyses and simulations (14,16,17). Molecular level probing of the hydration shell of proteins under conditions that either enhance or destabilize native structures has proven to be a more daunting task due to the relative dearth of suitable probes and probe techniques. In the present study we utilize pyranine (HPT) 2 as a site-specific fluorescent probe that provides direct spectroscopic detail relating to interactions within hydration layers. In addition, the hydration shell information provided by pyranine is supplemented with Gd 3ϩ vibronic side band data that expose how hydrogen bonding between first and second hydration shell waters is influenced by osmolytes. Together the results from these two probes directly relate to issues such as preferential hydration and osmolyteinduced changes in protein stability. The results are consistent with a relatively simple model that partitions waters and the associated water interactions into three domains: surface waters directly interacting with the hydrated molecule (e.g. protein, cation, chelate etc.), waters within the hydration layer that are impacted by these surface waters, and bulk solvent. The results also support the concept of at least two modes through which osmolytes can alter water interactions within the hydration layer: traditional preferential hydration mechanisms and alteration of hydrogen bonding patterns.The fluorescence from pyranine (8-hydroxy-1,3,6-pyrene trisulfonate, referred to as HPT in the subsequent text) is highly responsive to the composition of its solvent shell. This sensitivity arises from the dissociation/recombination behavior of the single ionizable hydroxyl on the HPT fluorophore (18,19). For the electronic ground state of HPT, the pK a of this prot...

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“…124 Another example is provided by a study of the effects of additives on the storage stability of recombinant human factor XIII in the solid state. 125 Here, the dextran formulation had a high T g but underwent extensive degradation during storage, while both sucrose and trehalose stabilized even though the T g of the sucrose formulation was much lower. Interestingly, the protein structure in the dextran formulation was perturbed more than in the formulation without additives.…”

mentioning

confidence: 95%