京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20710号 / 工博第4407号 / 新制||工||1685(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 古賀 毅, 教授 吉崎 武尚, 教授 竹中 幹人 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

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Capturing protein dynamics in biological crowded environments is essential for under- standing cellular function. In this project, we have explored the dynamic properties of apoferritin in aqueous solutions under varying conditions, including different temper- ature, solvent viscosity and protein concentrations. Dynamic light scattering (DLS) was applied here at various scattering angles from 90 to 150 degrees and at temperatures 295 K and 263 K on three different samples, including one with 19.5 mg/ml apo- ferritin, 6 mg/ml NaCl and 50% glycerol in volume fraction, one with 20 mg/ml apoferritin and 6 mg/ml NaCl, and one with 196.7 mg/ml apoferritin, 6 mg/ml NaCl and 50% glycerol in volume fraction. With the intensity autocorrelation func- tions from DLS measurements, the corresponding diffusion coefficients, hydrodynamic radii and relaxation time constants for each sample under varying conditions were ex- tracted. By comparing with the previous studies, unexpectedly large hydrodynamic radii were noticed and were attributed to undissolved protein crystallites. Still, it can be indicated from our experiment that applying smaller momentum transfer, decreas- ing temperature, increasing solvent viscosity and increasing protein concentration in the solutions can slow down the diffusion dynamics of protein molecules and clusters. Especially by increasing protein concentration, the slowing down of dynamics may be due to crowding effects, as well as increased size of the crystallites. In addition, the data indicate that in all cases, larger solution viscosity can lead to slower diffusivity of proteins.

Lösligheten av xylaner utvunna från trä undersöktes med Dynamisk Ljusspridning i syfte att observera eventuella förändringar i hydrodynamisk volym och ζ-potential. Xylaner extraherade från bok, björk och gran användes, där alkalisk extraherad bok erhölls kommersiellt och prover från björk och gran extraherades i förhand med subkritiskt vatten och alkalisk extraktion. Proverna analyserades vid koncentrationerna 1 g/L respektive 20 g/L. Förändringar i pH observerades stegvis med titrering av 1 g/L prover och förändringar över tid vid konstant pH observerades upp till en vecka efter upplösning. 20 g/L proverna undersöktes över tid vid bestämda pH enligt samma process för 1 g/L för både filtrerade och ofiltrerade prover. En undersökning av utvalda filter genomfördes där stora variationer observerades mellan det olika arterna men även mellan filtrens porstorlek. Med dynamisk ljusspridning observerades att populationer av högsta molekylvikt avlägsnades, där inverkan var störst för proven med lägst total koncentration och för högre koncentration efter en vecka. SEC visade att även populationer med mindre molekylvikt avlägsnades som medför att aggregering av proven kan ha förekommit, vilket kan ha förhindrat passingering genom filtren. Med några få undantag, samtliga provers hydrodynamiska volym visades vara heterogena, med endast små ökningar vid ökning i koncentration och icke-signifikanta förändringar över tid och med varierande pH. ζ-potentialvärdena visade markanta förändringar och tydliga trender med ändringar i pH och koncentration, där minskande pH och ökande koncentrationer resulterade i minskad stabilitet i lösning. Över tid ökades absolut ζ-potentialen för de prover med högre koncentration för alla pH miljöer. Filtrering av 20 g/L proverna resulterade i högre initial ζ-potential men efter en vecka ökade stabiliteten för de ofiltrerade till något högre slut ζ-potential för ofiltrerade. / The solubility of wood xylans was investigated using Dynamic Light Scattering to calculate number-average size and ζ-potential of samples in aqueous solution. The sources of xylan were a commercial alkaline extracted beech glucuronoxylan as well as pre-prepared samples of birch glucuronoxylan and spruce arabinoxylan, extracted using subcritical water extraction and alkaline extraction. The samples were analysed at concentrations of 1 g/L and 20 g/L, where the lower concentration samples were analysed at varying pH using titration and set pH over the span of hours and finally up to a week after preparation. The higher concentration samples were observed at set pH following the same procedure as the lower concentration for both filtered and unfiltered samples. An analysis of the filtration of 1 g/L samples was conducted by freeze-drying and weighing the samples before and after using 0.2 μm and 1.2 μm filters. These were then compared to unfiltered samples to evaluate the loss of total mass after filtration. The results of this investigation found significant variance between the samples and filters selected. Comparisons of filtered and unfiltered samples consistently showed that the filtration removed higher molecular weight portions of the samples, the most significant differences being observed at lower concentrations and higher concentration after 1 week. SEC analysis showed that not only larger molecular weight fraction but all the observed populations were removed during filtration, indicating that aggregates could be present, inhibiting the smaller molecular weight fractions from passing through. The size measurements showed that the vast majority of samples were heterogeneous in nature and with a slight increase in number-average size with increasing concentration and little change when varying both pH and over time. ζ-potential measurements showed substantial changes in stability when varying pH, coinciding well with predicted pKa values and generally decreased stability at higher concentrations. For higher concentrations lower initial absolute ζ-potential values were observed for both filtered and unfiltered however over time the unfiltered samples showed slightly greater increases for all measured pH values.

The number of protein containing therapeutic drugs is growing day by day. Lack of proper storage conditions can cause protein degradation or aggregation. The osmotic second virial coefficient, B22, is a thermodynamic parameter, which can predict protein interaction with other proteins and solvent molecules. B22 has been successfully used as predictor of crystallization conditions for a protein in the solution, and in this study an attempt has been made to relate B22 as a predictor of stability of the protein. Static light scattering was used to measure B22 in our studies. B22 and the solubility of three proteins were measured in several excipient solutions. George et al. in 1997 related the osmotic second virial coefficient with the solubility of protein in a solution. In this study we have attempted to relate solubility with B22 and stability of lysozyme, human serum albumin, and ovalbumin in buffer solutions containing various excipients.

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