Dissertations / Theses on the topic 'Α-crystallin'

The lens of the eye needs to be completely transparent in order to allow all light entering the eye to reach the retina. This transparency is maintained by the highly ordered structure of the lens proteins the crystallins. Any disruption to the lens proteins can cause an opacity to develop which is known as cataract. During cortical cataract formation there is increased truncation of the lens crystallins. It is believed that overactivation of calcium-dependent cysteine proteases, the calpains, is responsible for the increased proteolysis of the crystallins seen during cataractogenesis. Within the ovine lens there are three calpains, calpain 1, 2 and the lens specific calpain Lp82. The aim of this thesis was to determine the changes in the lens proteins during ageing and cataractogenesis, and to establish the role of the calpains in these processes. Calpain 1 and 2 were purified from ovine lung and Lp82 was purified from lamb lenses using chromatography. Activity and presence of the calpains was determined by using the BODIPY-FL casein assay, gel electrophoresis, Western blot and casein zymography. Changes in the lens proteins, specifically the crystallins, were visualised using two-dimensional electrophoresis (2DE). Lenses from fetal, 6 month old and 8 year old sheep were collected, as well as stage 0, 1, 3 and 6 cataractous ovine lenses. The proteins from the lenses were separated into the water soluble and urea soluble fractions and analysed by 2DE. Mass spectrometry was used to determine the masses and therefore modifications of the crystallins. Finally, the individual crystallins were separated using gel filtration chromatography and incubated with the purified calpains in the presence of calcium. The extent of the proteolysis was visualised using 2DE and truncation sites determined by mass spectrometry. Purification of the calpains resulted in samples that were specific for each calpain and could be used in further experiments. 2DE analysis showed that there were changes to the crystallins during maturation of the lens. The α-crystallins become increasingly phosphorylated as the lens ages and a small amount becomes truncated. The β-crystallins were also modified during ageing by truncation and deamidation. When crystallins from cataractous lenses were compared using 2DE there were changes to both the α- and β-crystallins. The α-crystallins were found to be extensively truncated at their C-terminal tail. Four of the seven β-crystallins, βB1, βB3, βB2 and βA3, showed increased truncation of their N-terminal extensions during cataract formation. All three calpains truncated αA and αB-crystallin at their C-terminal ends after incubation. Calpain 2 and Lp82 each produced unique αA-crystallin truncations. All three calpains truncated βB1 and βA3 and calpain 2 also truncated βB3. When the truncations from the calpain incubations were compared to those seen during cataract formation, many of the truncations were found to be similar. Both the unique truncations from calpain 2 and Lp82 were found in cataractous lenses, with the Lp82 more obvious in the 2DE. The β-crystallin truncations found after incubation with the calpains were similar to those found during cataractogenesis. In conclusion this study documents the changes to the ovine lens during maturation and cataractogenesis and indicates a role for the calpain family in the increased proteolysis observed in the ovine cataract.

Mycobacterium tuberculosis remains a major human health problem killing millions of people around the world. Therefore, the need for an in depth understanding of its pathogenicity is very important to enable rational development of new control strategies.  Not all M. tuberculosis infected individuals progress to active disease at the time of primary infection as some carry an asymptomatic persistent infection that may reactivate later in life to cause disease. Despite a great deal of M. tuberculosis research, the ability of M. tuberculosis to cause long-term persistent infection in immune-competent hosts is poorly understood. When M. tuberculosis is inside the body it faces many environmental stress conditions including hypoxia, starvation and oxidative stress that damage proteins, DNA and other molecules. Gene expression studies have identified many bacterial genes that are differentially regulated when M. tuberculosis is subjected to such environmental stresses. This thesis focusses on three of these genes, the ssrA gene, which encodes the tmRNA molecule involved in ribosome recycling and degradation of denatured proteins, and two genes encoding -crystallin molecular chaperones (acr1 and acr2). This study was designed to achieve two aims: (1) to examine the role of tmRNA in translational control and protein homeostasis in stressed mycobacteria; and (2) to understand the roles of Acr1 and Acr2 in the stress response of M. tuberculosis and to reveal the degree of redundancy between them. In this thesis it was shown that ssrA/tmRNA is essential for bacterial viability as it was not possible to delete the gene unless a second fully functional copy was introduced elsewhere in the genome. The results suggested that the protease tagging function of tmRNA is essential alongside its role in ribosome recycling. A recombinant His-tagged tmRNA was expressed in the mycobacteria in an attempt to identify if tmRNA is directly involved in the translation of stress proteins. Expression of the His-tagged tmRNA was detrimental to the cell and appeared to preclude successful tagging of tmRNA substrate polypeptides. Thus there was insufficient evidence to support the hypothesis. Ultrastructural localisation of Acr1 and Acr2 by immuno-electron microscopy and Western blotting of subcellular fractions of mycobacteria showed that Acr1 and Acr2 were localised in different parts of the cell. Assay of the phenotypes of single and double deletion mutants of Acr1 and Acr2 in different in vitro conditions failed to show any evidence that the two chaperones are functionally redundant. Indeed, experiments on intracellular infection of macrophages showed no phenotypic consequences resulted from loss of Acr1 but deletion of Acr2 resulted in an altered cytotoxic effect on the host cell.

碩士
國防醫學院
生物化學研究所
102
The lenticular α-crystallin consisits of two 20-kDa polypeptide chains, αA andαB, which belongs to the small heat shock protein family. They have 57% sequence homology among themselves. In this study, the change in structure, chaperone activity, complex size and thermostability of αA and αB- homooligomer and their heterooligomer in different ratio incubated at 37℃ were investigated. The result showed that heterooligomer of αA and αB-crystallin have about 29% smaller than αB-homooligomer in exposed hydrophobic region as measured by ANS binding. Thermalstability analysis showed the Tm values of heterooligomer in the ratio of 1:3,3:1 and 1:1 were about 83.5℃,91.6℃ and 92.6℃,respectively which were between the Tm values of αA and αB-homooligomer, 70.8℃ and 92.8℃ respectively.The CD data showed about 4% increased and 6% decreased in α-helix and β-sheet content respectively,as comparing the heterooligomer in ratio of 3:1 with αA-crystallin homooligomer and showed about 4% increased and 4% decreased in the contents as compared with αB-homooligomer.The heterooligomer in ratio of 3:1,1:1 and 1:3 showed about 90%, 84% and 74%,respectively,protection of hASL from thermodenaturation. The protection ability of αA-crystallin homooligomer was about 92.3% higher than the αB- homooligomer(34.5%).The heterooligomer that incubated for 24h then through freezing-thawing cycle showed higher stability than that incubated for 2 hours.The results showed that slow freezing and thawing rate induces damage in protein stability.The protein concentration remained after freez-thaw forαA and αB- homooligomer and heteroligomer in ratio of 1:1,1:3,1:7,3:1and 7:1 were 79.8%、88.4%、96.5%、88.2%、97.9%、91.2% and 93.5%,respectively.The heterooligomer showed a better stability than αA and αB- homooligomer.

碩士
國防醫學院
生物化學研究所
96
Taxon-specific -crystallin, the major soluble protein component of the avian and reptilian eye lens, is considered to play a structural role in maintaining a high refractory index while ensuring transparency. However, the most astounding relationship is an evolutionary strategy of gene sharing that the taxon specific crystallins seems to be present in which the same gene product is utilized in dual function as a lens crystallin and as an enzyme in non-lens tissues. These sequence similarities such as the -crystallins are closely related to argininosuccinate lyase. -Crystallin and ASL are a superfamily of metabolic enzymes that catalyze the reversible cleavage of argininosuccinate to arginine and fumarate. ASL is one of the cytosolic enzymes of the urea cycle in ureagenic animals. -Crystallin is the member of the small heat shock protein family and function as a molecular chaperone-like activity in preventing the non specific thermal aggregation of lens proteins. In addition, -Crystallin consists of two polypeptide chains, A and B. In the present study, we used the temperature-dependent to investigate the effect of recombinant goose A- and B-crystallins on recombinant goose -crystallin and HASL. At 25°C, the addition of A and B led to 103% and 130% increase in the specific activity of HASL, respectively. It is found that the optimal temperature of HASL was at 40°C brought about 149% in the specific activity, whereas the specific activity was about 7% at 60°C. In contrast, HASL is stabled up to approximately 50°C in the presence of A and B, and even has about 17% and 31% at 60°C, respectively. Conditions for incubation of -crystallin and HASL were set at room temperature for 1 hr that is determined through time and temperature dependent assays of ASL activity in the presence of A and B. The tryptophan fluorescence experiments of HASLWT on A show significantly decreased at about 30% ~ 50%. Under the temperature-dependent conditions, the protection effect of B on -crystallin is more remarkable than A. In addition, calibrated S-200 size exclusion chromatography revealed that -crystallin and HASL in the presence of B form a stable complex.

博士
長庚大學
臨床醫學研究所
86
ABSTRACT: Recently, α-crystallin is regarded as a member of small heat shock proteins. Joseph Horwitz also demonstrated in 1992 that it can act as a molecular chaperone to prevent thermal aggregation of other crystallins and enzymes. The main purpose of this thesis is to explore the relationship between the conformational states of α-crystallin and its chaperone activity. We have first determined the cDNA sequence of αB-crystallin from porcine lenses by cDNA cloning technique. It contains one complete full-length reading frame of 525 base pairs, covering a protein sequence of 175 amino acids. Further expression of this αB subunit chain in E.coli generated a polypeptide which can cross-react with the antiserum against the native αB-crystallin, and also possesses chaperone activity. The next, a comparative study was conducted to analyze the chaperone activity of α-crystallin from bovine (mammal), duck (bird), caiman (reptile), and shark (fish), respectively. Our results show that the difference in the chaperone activity of α-crystallins among different species is relatively small, indicative of the evolutionary conservation in function similar to that revealed by their conserved protein structure. α-Crystallins of different species also show no substrate specificity in their chaperone activity. In addition to its anti-heat shock property, α-crystallin can also act as a chaperone against UV-irradiation, H2O2-oxidation, and other stress. However, in terms of stoichiometry, such protective ability is not so efficient as that under thermal stress. Part of the reason is the photochemical susceptibility of α-crystallin to UV-irradiation. The UV-induced destruction was found to correlate with its loss of chaperone activity. Similarly, there is some age-dependent change in the chaperone activity of α-crystallin obtained from a young and normal lens as compared to an old and cataractous lens. Thus, the gradual loss of chaperone activity of α-crystallin upon aging is probably through an accumulative event of long term exposure to UV light, which may also shed light on human cataract formation. The mechanism underlying the chaperone activity of α-crystallin involves preferential binding of the partially denatured substrate protein to α-crystallin, and the formation of a stable complex. We have demonstrated that the binding of substrate proteins to α-crystallin by short-term pre-incubation may mimic the in vivo conditions of crystallin association. Under such conditions, the chaperone activity of α-crystallin to inhibit ultraviolet-, or oxidation-induced protein aggregation can be greatly enhanced. Thus, the presence ofα-β and α-γ complex in vivo may be relevant to the process of maintaining lens transparency. From the result of circular dichroism and other studies, it is generally accepted that the secondary structure of α-crystallin consists predominantly of β-sheets. A minor but detectable perturbation in the tertiary structure of α-crystallin occurs at above 30℃, which correlates with the extent of exposure of its hydrophobic surfaces. Such increase in surface hydrophobicity also correlates with its increased chaperone activity. These results indicate that hydrophobic interaction play a major role in the chaperone action of α-crystallin. Another thermotropic transition in the secondary structure of α-crystallin occurs at a range between 50 and 70℃, which is largely reversible. However, the heat-induced changes in secondary structure shows a relatively little effect on the chaperone activity of α-crystallin. In summary, α-crystallin is not only a major structural protein of the lens but may also play an important "house-keeping" role as a molecular chaperone. Both the conformational state of α-crystallin and the association complex with its substrate may contribute to a generalized mechanism of its chaperone function. Such results may provide us a new direction in the study of cataractogenesis and its treatment in the future.

α-Crystallin B (cryAB) is the most abundant small heat shock protein in cardiomyocytes (CMs), where it has been shown to have potent anti-apoptotic properties. The mechanism by which cryAB prevents apoptosis has not been fully characterized. Therefore, I was interested in elucidating its protective mechanism in CMs. I identified its sub-cellular localization and its binding interactors following H2O2 exposure. I found that cryAB is found in the cytosol under control conditions and that following H2O2 exposure it becomes phosphorylated and translocates to the mitochondria. CryAB silencing resulted in increased apoptosis levels in CMs. Co-immunoprecipitation revealed an apparent increased interaction of cryAB and PcryAB with mitochondrial VDAC, caspase 12 and uncleaved caspase 3 in stressed hearts relative to controls. These results suggest that the cardio-protective effects of cryAB are mediated by its translocation to the mitochondria and its interaction with VDAC, caspase 12 and caspase 3 following exposure to H2O2.

碩士
國立成功大學
化學系碩博士班
91
α-Crystallin and high molecular weight aggregate (HMWA) were isolated from four weeks old Rat lenses. α-Crystallin was further heated at various temperatures (50-60℃) to induce thermal aggregation of HMWA, which was used to make a compare with in vivo HMWA. Spectroscopic measurement were performed to study the structure and functionality of both HMWA. Conformation differences of native HMWA were suggested based on the data of increased trytophan (Trp), non- tryptophan (non-Trp), 1-anilino- naphthalene-8-sulfonic acid (ANS), and 2-(4’-maleimidylanilino) naphthalene-6-sulfonic acid (MIANS) fluorescence intensity as well as the increased far-UV and near-UV circular dichroism (CD). These results indicated that native HMWA was more hydrophobic than α-crystallin, possibly resulting from the partial unfolding of native α-crystallin. Gel filtration chromatography showed that α-crystallin heat-induced HMWA prepared by preheating at 60℃ for an hour with the same molecular weight as that of in vivo HMWA. Trp, ANS, and MIANS fluorescence as well as far-UV CD measurements indicated that heat-induced HMWA and in vivo HMWA shared structural similarity, which further suggested the same aggregation mechanism. Chaperone-like activity was observed toward the aggregation of dithiothreitol (DTT)-induced insulin B-chain show that α-crystallin preheated at 50℃ has better activity than α-crystallin and native HMWA. With the increase of preheating temperature, the activity of α-crystallin decreased and it was observed that under 60℃, it was less active than native HMWA. The correlation between the ANS fluorescence and the chaperone-like activity suggests that surface hydrophobicity was not the sole determinant of the chaperone function of the α-crystallin. Cysteine modification of α-crystallin was carried out using 10 mM and 50 mM β-mercaptoethanol followed by heating at 60℃ for an hour, then was subjected to gel filtration and SDS-PAGE. Heat-induced HMWA remained formed from 50 mM modification of α-crystallin, whereas there was no disulfide bond was observed, indicated that disulfide bond formation was not the (main) factor leading to the formation of HMWA, at least in heat-induced HMWA formation. Our study suggests that heat-induced HMWA proceed similar mechanism as that of in vivo HMWA via partial unfolding, however, the unfolding process may differ as to show different non-Trp fluorescence, near-UV CD and chaperone-like activity as well.

碩士
國立成功大學
化學系
87
Fluoroscence spectroscopy, circular dichroism spectroscopy (CD), and fast protein liquid chromatography (FPLC) have been used to study the aggregation of rat lens α-crystallin under the effects of Mg2+, trifluoroethanol and pH. The chaperone activity of α-crystallin toward ditiothreitol-induced insulin aggregation was measured under various concentration of Mg2+ and the activity was correlated with the structural change of α-crystallin. It was observed that ANS and tryptophan fluorescence emissions increased as the concentration of Mg2+ increased, indicating α-crystallin underwent a structural change with more hydrophobic region exposed. However, the change of chaperone activity did not correlate with the observed increase of fluorescence, suggesting that surface hydrophobicity and microenvironment of tryptophan are not necessarily relate to the chaperone function of α-crystallin under the influence of Mg2+. The aggregation study using FPLC showed a 20 kDa and a 540 kDa fractions, and the ratio of 20 kDa to 540 kDa was Mg2+ concentration dependent. Interestingly, re-chromatography of the 540 kDa fraction also gave a 20 kDa and a 540 kDa fraction, whereas the 20 kDa fraction remained no change after re-chromatographed. The aggregation also showed pH dependent. It was found that only 20 kDa fraction was observed at pH 4 and 5, while at pH 9.0 two fractions at 60 kDa and 690 kDa were observed. In the presence of trifluoroethanol, α-crystallin solution started to go opaque as the concentration of trifluoroethanol reached 10%. As trifluoroethanol concentration increased (less that 10 %), spectroscopic studies showedα-crystallin underwent a strnctural change, including loss of secondary structure, enhancement of tryptophan fluorescence and decrease of surface hydrophobicity.

碩士
國立成功大學
化學系
87
In this study, circular (CD) and ANS fluorescence emission have been used to study the structural change of both the oxidized rat lens a-crystallin and the dialyzed oxidatively insulted a-crystallin by Ascorbate-FeCl3-EDTA-H2O2 oxidation system. The study of the relationship between secondary structure of a-crystallin and its chaperone activity under various times of oxidation then followed by dialysis was also studied. It was found that ANS fluorescence emission intensity increased as oxidation time increased. And the intensity was higher than normal a-crystallin when oxidation time was longer than 10 hours. Dialysis can result in the recovery of ANS fluorescence emission intensity. It was found that after dialysis of oxidized a-crystallin for 10 hours the intensity is almost the same as that of normal one. The study of chaperone activity of a-crystallin toward dithiothreitol-induced insulin-B aggregation showed the chaperone activity decreased for oxidized a-crystallin, however, dialysis resulted in the recovery of its activity. CD study shows that for normal a-crystallin there is a negative band centering at 217 nm, indicating that b-sheet form makes a dominant contribution to the total conformation. Oxidation caused the change of its secondary structure with the 217 nm band blue shifted and intensity decreased. With the increased of dialysis time, the secondary structure was gradually recovered, however, it did not recover completely to its original structure. With the analysis using SELCON 3 program, it is able to estimate the percentage of its secondary components. It was found that the percentage of a-helix increased 20﹪, whiles b-sheet decreased 10﹪for 5-hours and 10-hours oxidized a-crystallins. With the increase of dialysis time, it was observed that secondary components gradually recovered, with a-helix recovering back to 18﹪and 12﹪(18﹪for normal) and b-sheet recovering back to 32﹪and 33﹪(35﹪for normal) for 10-hour dialysis of oxidized a-crystallin. Our study has showed that the decreased of chaperone activity is due to the structural change caused by oxidation and dialysis is a way to regain its structure and chape

As the major protein of the lens, α-crystallin is a molecular chaperone that stabilises lens proteins to prevent their precipitation into solution. In this role it is vital in maintaining lens transparency. The chaperone ability of α-crystallin and its individual subunits, αΑ- and αB-crystallin, has been shown to be sensitive to a variety of environmental and intrinsic factors, including temperature, denaturation and post-translational modification. The effect of pH on α-crystallin chaperone ability, however, has not been thoroughly investigated. There is limited evidence to suggest that the chaperone ability of α-crystallin is pH-sensitive such that α- crystallin is a significantly worse chaperone at pH 6.0 than at pH 8.0. This is of physiological significance since in the lens there is a measurable pH gradient of pH 7.2 in outer lens cells, compared to pH 6.7 in the lens nucleus. A loss of α-crystallin chaperone function in the lens nucleus, as a consequence of decreased pH, may compromise lens transparency. Similarly, extra-lenticular fibrillar aggregation of some disease-related target proteins (Aβ-peptide, for example) is promoted by acidic pH. This study investigates the effect of pH on the chaperone ability of α-crystallin and its subunits. Further, this study characterises the structural changes to α-crystallin accompanying pH variation in an attempt to explain the structural basis for the observed pH sensitivity. In addition, this study examines the chaperone function of cyclodextrins, a class of chemical chaperones that may act in conjunction with α-crystallin as part of a two-step protein refolding pathway. This study demonstrated that the chaperone activity of α-crystallin is pH sensitive between pH 6.0 and 8.0; the ability of α-crystallin to protect against temperature- and reduction-stress induced amorphous aggregation is significantly reduced at pH 6.0 and 6.5 compared to pH 7.0 and above. The decreased chaperone ability of α-crystallin at pH 6.0 and 6.5 was accompanied by partial unfolding of the protein, and a loss of secondary structure, while α-crystallin quaternary structure remained unchanged. Interestingly, α-crystallin was found to have significant chaperone ability below pH 4.0, conditions under which α-crystallin is largely unfolded. The unfolding of α-crystallin at pH 6.0 and 6.5 is comparatively minor, and it is difficult to say whether this unfolding is directly responsible for the observed pH sensitivity of α-crystallin chaperone ability. The thermal stability of α-crystallin was compromised at pH 6.0 and 6.5, which may partially explain its decreased chaperone ability at these pH values in heat-stress assays conducted at temperatures above 50oC. However, α-crystallin chaperone activity remained pH sensitive at 37°C and 45°C, at which temperatures it is thermally stable. Blocking exposed αB-crystallin histidine residues by chemical modification removed, to a large extent, the pH-sensitivity of its chaperone activity. This suggests that the protonation of an exposed histidine residue(s) at pH 6.0 and 6.5 is responsible for the observed pH sensitivity of α-crystallin chaperone ability. Inhibiting the protonation of a specific histidine residue, H83, by site-directed mutagenesis (H83A) did not remove the pH sensitivity of αB-crystallin chaperone activity, and suggests that protonation of this residue alone does not explain the decreased chaperone ability of α-crystallin at mildly acidic pH. This residue lies within the putative chaperone-binding region of αB-crystallin, and is highly conserved between species and between the human small heat shock proteins. It appears that the protonation of several histidine residues, or residues other than H83, is primarily responsible for the influence of pH on α-crystallin chaperone ability observed in this study. The observed decrease in α-crystallin chaperone function below pH 7.0 partially explains the preferential formation of age-related cataract in the lens nucleus, as the chaperone ability of α-crystallin would be compromised under the mildly acidic conditions characteristic of the nucleus. Additionally, the pH sensitivity of α-crystallin chaperone ability may be significant in the ability of extra-lenticular αB-crystallin to inhibit amyloid-related disease at sites of localised acidosis. Cyclodextrins are a family of cyclic oligosaccharides that have been shown to function as chemical chaperones under specific protein aggregation conditions. Cyclodextrins have been demonstrated to facilitate the refolding of chemicallystressed target proteins that have already bound to synthetic nanogels, which act in a manner reminiscent of small heat shock proteins. In this study, cyclodextrins were unable to act in conjunction with α-crystallin to facilitate the refolding of thermallystressed target proteins. β-Cyclodextrin (βCD) demonstrated little or no ability to inhibit the amorphous aggregation of target proteins, but was able to significantly inhibit the fibrillar aggregation of a number of target proteins, including the diseaserelated A53T α-synuclein mutant. Characterisation of the binding of βCD to target proteins during fibrillar aggregation via circular dichroism, intrinsic and extrinsic fluorescence and competitive chaperone assays provided a model of the cyclodextrin chaperone mechanism. In this model, cyclodextrins interact with already partially unfolded, pre-fibrillar protein intermediates via the insertion of aromatic residues into the cyclodextrin anulus, and by doing so inhibit intra-fibrillar π-bonding and protofilament assembly. This suggests the potential for cyclodextrins as therapeutic molecular chaperones in vivo that may be able to inhibit the pathogenic aggregation of target proteins.
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Thesis (Ph.D.) - University of Adelaide, School of Chemistry and Physics, 2009

碩士
國立臺灣大學
生化科學研究所
89
Alpha-crystallins, the major components of lens proteins, contribute to lens proteins stability and confer UV resistance potency to lens epithelial cells. We introduced bullfrog αA and αB-crystallin genes to E.coli and induced their expression, these recombinant cells showed significant UV resistance up to more then 10 times survival ratios relative to control cells, which were consistent with those of previous reports in lens epithelial cells. UV resistance of E.coli transformed by truncated form of alpha-crystallins gene constructs showed that the N-terminal 60 residues also confer significant UV resistance to E.coli. The N-terminal domain showed no molecular chaperone activity in our classic chaperone activity assay. These results suggest that the UV resistance of E.coli was not due to the trivial chaperone activity but rather there might exist conserved cell death pathways in prokaryotics and mammalian cells such that similar experimental outcomes were obtained. Or α-crystallin might interrupted UV triggered E.coli cell death by unknown mechanisms awaiting further study. Our results demonstrated that α-crystallins might be promising tools to unveil the mysterious prokaryotics cell suicide pathways.

碩士
國防醫學院
生物化學研究所
98
英文摘要 Freezing is a very important operation in the biotechnological and biopharmaceutical industries, but it may result in freezing damage to the bioproducts. In this work, an attempt was made to examine and explain in qualitative and quantitative terms, the effect of freezing and thawing rate on protein damage and to investigate the minimization of protein damage and the additives of α-crystallin during freezing by optimizing the freezing protocols and solution conditions. The result showed that human argininosuccinate lyase (HASL) was highly inactivated during slow freezing and thawing. In the presence of α-crystalin the activity of HASL was protected. No subtle changes in the secondary and tertiary structure of HASL were observed after slow freezing and thawing, but dimers were dissociated from tetrameric HASL. Adding of α-crystalin before or after freezing was able to prevent the disassembly of HASL.

博士
國立成功大學
化學工程學系
89
The chemical structures of all the synthesized compounds were characterized by 1H-NMR, 13C-NMR and elemental analysis. FTIR, DSC, POM, TGA and Wide Angle X-ray were used to study the mesomorphism and liquid crystal properties of the synthesized compounds. This research used α-methylstilbene as mesogen. A homologues series of the compounds containing various lengths of alkyl chain terminated with hydroxyl group as spacer, 4,4’-bis(hydroxyalkyloxy)-α-methylstilbenes, denoted HAMS-n, has been synthesized. Here n is the carbon atom number of the peripheral n-alkyl chain; in this work, n= 2 to 8 and 11. All the novel HAMS-n with the OH hydrogen bonding is mesomorphic compounds. The lengths of the peripheral n-alkyl chain influenced on the mesomorphism and liquid crystalline properties. The homologues with n=8 and 11 were enantiotropic monomorphic, CrG or CrH phase. The odd homologues showed monotropic trimorphism on cooling, while the even homologues exhibited enantiotropic trimorphism including the CrG or CrH, SmA and nematic phases. The homologues with n=7 presented the co-existence of multiple mesophases. Tm and Ti were decreased with increasing of the length of n-alkyl chain and an obvious even-odd effect was observed at Tm. The spectroscopic studied of HAMS-4 and HAMS-8 in the 3300-3600cm-1 region showed that the band half-width of overall band and the ratio of integrated intensity of the modes of hydrogen bonding of OH stretch occurred significant discontinuous at the crystalline-CrG(or CrH), CrG(or CrH)-SmA and SmA-N transitions. The free OH groups formed upon entering the SmA phase. These results indicate that the hydrogen bonding of terminal OH groups may be the important factor dominating to the morphology of liquid crystals. The spectra about the bending and rocking progression of CH2 moieties as a function of temperature suggest the onset change of the conformation of alkyl terminal chain at the temperature associated with phase transitions. The spectra on the benzene ring C=C stretch and the ring skeletal vibration as functions of temperature provide the information on the onset of motion of the phenyl mesogenic core. In order to study the influences of OH hydrogen bonding on mesomorphic properties, a homologues series of corresponding compounds with methyl group at the terminal of terminal chain, 4,4’-bis(alkyloxy)-α-methylstilbenes, denoted AMS-n, were prepared. Here n is the carbon atom numbers of the peripheral n-alkyl chain; in this work, n= 2 to 7 and 9 to 11. The results of comparison of HAMS-n with AMS-n indicated that the hydrogen bonding effect can promote to produce enantiotropic mesophase, can increase the isotropic temperature, can broaden the temperature range of mesophase, and can promote to produce the smectic phase with higher orientational ordered such as CrG or CrH phase. A series of novel polycarbonates with various lengths of methylene units as spacers, Poly(-4-oxy-alkyloxy-α-methylstilbene-oxy-alkyloxy-carbonyl), denoted HAMS-n-PC, were prepared from the HAMS-n and diphenyl carbonate by melt polycondensation. Here n is the numbers of methylene units in polymer chain; in this work, n= 2 to 4, 6, 8 and 11. The reaction steps and conditions used in the melt polycondensation could give good yields (71-83%) and high molecular weight polycarbonates with intrinsic viscosity in the range of 0.73-1.73 dl/g. These new polycarbonates soluble in organic solvents such as chloroform to obtain the flexible films. Except for HAMS-11-PC, all the others HAMS-n-PC exhibited enantiotropic monomorphic nematic phase. The results of TGA showed the HAMS-n-PC had good thermal stability up to 400℃, which similar to the polymer with fully aromatic in the main chains. This indicated that the introducing of flexible methylene units into the polymer chain do not depress the decomposition temperature. Tg, Tm(Tm= Tk-N) and Ti(Ti= TN-i) were decreased with increasing of the spacer length, however, the decomposition temperatures were increased with increasing of the spacer length. The spacer lengths were also influenced on the temperature range of mesophase of HAMS-n-PC and found that the relative length of spacer to mesogen played an important role in the mesophase formation. The increasing of the molecular weight and having a carbonate linkage in the chain lead the HAMS-n-PC have better mesogenity, more stable mesophase, and higher Tm and Ti than the low molecular weight liquid crystals (AMS-n). The non-directed type structure in the polymer chain of HAMS-n-PC, can promote to produce enantiotropic mesophase, can lower the Tm and Ti , and broaden the temperature range of mesophase.