Fourier-transform infrared spectroscopic investigation of protein stability in the lyophilized form

Costantino, Henry R.; Griebenow, Kai; Mishra, Prashant; Langer, Robert; Klibanov, Alexander M.

doi:10.1016/0167-4838(95)00156-o

Cited by 115 publications

(101 citation statements)

References 15 publications

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“…3B). This phenomenon is characteristic of aggregating proteins (36,37). The deconvolution into Gaussian components yielded reconstructed spectra indistinguishable from the original ones.…”

Section: Resultsmentioning

confidence: 99%

Secondary Structure and Ca2+-induced Conformational Change of Calexcitin, a Learning-associated Protein

Ascoli¹,

Luu²,

Olds³

et al. 1997

Journal of Biological Chemistry

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Calexcitin/cp20 is a low molecular weight GTP-and Ca 2؉-binding protein, which is phosphorylated by protein kinase C during associative learning, and reproduces many of the cellular effects of learning, such as the reduction of potassium currents in neurons. Here, the secondary structure of cloned squid calexcitin was determined by circular dichroism in aqueous solution and by Fourier transform infrared spectroscopy both in solution and on dried films. The results obtained with the two techniques are in agreement with each other and coincide with the secondary structure computed from the amino acid sequence. In solution, calexcitin is one-third in ␣-helix and one-fifth in ␤-sheet. The conformation of the protein in solid state depends on the concentration of the starting solution, suggesting the occurrence of surface aggregation. The secondary structure also depends on the binding of calcium, which causes an increase in ␣-helix and a decrease in ␤-sheet, as estimated by circular dichroism. The conformation of calexcitin is independent of ionic strength, and the calciuminduced structural transition is slightly inhibited by Mg 2؉ and low pH, while favored by high pH. The switch of calexcitin's secondary structure upon calcium binding, which was confirmed by intrinsic fluorescence spectroscopy and nondenaturing gel electrophoresis, is reversible and occurs in a physiologically meaningful range of Ca 2؉ concentration. The calcium-bound form is more globular than the apoprotein. Unlike other EFhand proteins, calexcitin's overall lipophilicity is not affected by calcium binding, as assessed by hydrophobic liquid chromatography. Preliminary results from patchclamp experiments indicated that calcium is necessary for calexcitin to inhibit potassium channels and thus to increase membrane excitability. Therefore the calciumdependent conformational equilibrium of calexcitin could serve as a molecular switch for the short term modulation of neuronal activity following associative conditioning.Pavlovian conditioning in animal models has been used to study associative learning and memory on a cellular and molecular basis. Following this strategy, a 22-kDa, low abundance

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“…3B). This phenomenon is characteristic of aggregating proteins (36,37). The deconvolution into Gaussian components yielded reconstructed spectra indistinguishable from the original ones.…”

Section: Resultsmentioning

confidence: 99%

Secondary Structure and Ca2+-induced Conformational Change of Calexcitin, a Learning-associated Protein

Ascoli¹,

Luu²,

Olds³

et al. 1997

Journal of Biological Chemistry

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“…21,22 The denatured lysozyme has different aggregation properties. During denaturation the disulfide bonds are disrupted and new bonds are formed.…”

Section: Resultsmentioning

confidence: 99%

Hydration of Thermally Denatured Lysozyme Studied by Sorption Calorimetry and Differential Scanning Calorimetry

Kocherbitov

Arnebrant

2006

J. Phys. Chem. B

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We have studied hydration (and dehydration) of thermally denatured hen egg lysozyme using sorption calorimetry. Two different procedures of thermal denaturation of lysozyme were used. In the first procedure the protein was denatured in an aqueous solution at 90°C, in the other procedure a sample that contained 20% of water was denatured at 150°C. The protein denatured at 90°C showed very similar sorption behavior to that of the native protein. The lysozyme samples denatured at 150°C were studied at several temperatures in the range of 25-60°C. In the beginning of sorption, the sorption isotherms of native and denatured lysozyme are almost identical. At higher water contents, however, the denatured lysozyme can absorb a greater amount of water than the native protein due to the larger number of available sorption sites. Desorption experiments did not reveal a pronounced hysteresis in the sorption isotherm of denatured lysozyme (such hysteresis is typical for native lysozyme). Despite the unfolded structure, the denatured lysozyme binds less water than does the native lysozyme in the desorption experiments at water contents up to 34 wt %. Glass transitions in the denatured lysozyme were observed using both differential scanning calorimetry and sorption calorimetry. Partial molar enthalpy of mixing of water in the glassy state is strongly exothermic, which gives rise to a positive temperature dependence of the water activity. The changes of the free energy of the protein induced by the hydration stabilize the denatured form of lysozyme with respect to the native form.

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“…A number of texts (Franks, 1985;Baffi and Garnick, 1991;Constantino et al, 1995;Anchordoquy and Carpenter, 1996;Chang et al, 1996a,b;Carpenter et al, 1997;Colaco et al, 1997) are available which outline the current thinking in this area.…”

Section: The Effects Of Water On Stabilitymentioning

confidence: 99%

The relevance of the amorphous state to pharmaceutical dosage forms: glassy drugs and freeze dried systems

Craig

1999

International Journal of Pharmaceutics

412

204

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Fourier-transform infrared spectroscopic investigation of protein stability in the lyophilized form

Cited by 115 publications

References 15 publications

Secondary Structure and Ca2+-induced Conformational Change of Calexcitin, a Learning-associated Protein

Secondary Structure and Ca2+-induced Conformational Change of Calexcitin, a Learning-associated Protein

Hydration of Thermally Denatured Lysozyme Studied by Sorption Calorimetry and Differential Scanning Calorimetry

The relevance of the amorphous state to pharmaceutical dosage forms: glassy drugs and freeze dried systems

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