Biological electron transfer in copper proteins

Nature has used a variety of protein systems to mediate electron transfer. In this thesis I examine aspects of the control of biological electron transfer by two copper proteins that act as natural electron carriers.

In the first study, I have made a mutation to one of the ligand residues in the azurin blue copper center, methionine 121 changed to a glutamic acid. Studies of intramolecular electron transfer rates from that mutated center to covalently attached ruthenium complexes indicate that the weak axial methionine ligand is important not only for tuning the reduction potential of the blue copper site but also for maintaining the low reorganization energy that is important for fast electron transfer at long distances.

In the second study, I begin to examine the reorganization energy of the purple copper center in the CuA domain of subunit II of cytochrome c oxidase. In this copper center, the unpaired electron is delocalized over the entire binuclear site. Because long-range electron transfer into and out of this center occurs over long distances with very small driving forces, the reorganization energy of the CuA center has been predicted to be extremely low. I describe a strategy for measuring this reorganization energy starting with the construction of a series of mutations introducing surface histidines. These histidines can then be labeled with a series of ruthenium compounds that differ primarily in their reduction potentials. The electron transfer rates to these ruthenium compounds can then be used to determine the reorganization energy of the CuA site.

Hydrothermal experiments on the thermal stability of amino substances in sediments

Aspartic acid, threonine, serine and other thermally unstable amino acids have been found in fine-grained elastic sediments of advanced geologic age. The presence of these compounds in ancient sediments conflicts with experimental data determined for their simple thermal decomposition.

Recent and Late Miocene sediments and their humic acid extracts, known to contain essentially complete suites of amino acids, were heated with H₂O in a bomb at temperatures up to 500°C in order to compare the thermal decomposition characteristics of the sedimentary amino compounds.

Most of the amino acids found in protein hydrolyzates are obtained from the Miocene rock in amounts 10 to 100 times less than from the Recent sediment. The two unheated humic acids are rather similar despite their great age difference. The Miocene rock appears uncontaminated by Recent carbon.

Yields of amino acids generally decline in the heated Recent sediment. Some amino compounds apparently increase with heating time in the Miocene rock.

Relative thermal stabilities of the amino acids in sediments are generally similar to those determined using pure aqueous solutions. The relative thermal stabilities of glutamic acid, glycine, and phenylalanine vary in the Recent sediment but are uniform in the Miocene rock.

Amino acids may occur in both proteins and humic complexes in the Recent sediment, while they are probably only present in stabilized organic substances in the Miocene rock. Thermal decomposition of protein amino acids may be affected by surface catalysis in the Recent sediment. The apparent activation energy for the decomposition of alanine in this sediment is 8400 calories per mole. Yields of amino compounds from the heated sediments are not affected by thermal decomposition only.

Amino acids in sediments may only be useful for geothermometry in a very general way.

A better picture of the amino acid content of older sedimentary rocks may be obtained if these sediments are heated in a bomb with H₂O at temperatures around 150°C prior to HCl hydrolysis.

Leucine-isoleucine ratios may prove to be useful as indicators of amino acid sources or for evaluating the fractionation of these substances during diagenesis. Leucine-isoleucine ratios of the Recent and Miocene sediments and humic acids are identical. The humic acids may have a continental source.

The carbon-nitrogen and carbon-hydrogen ratios of sediments and humic acids increase with heating time and temperature. Ratios comparable to those in some kerogens are found in the severely heated Miocene sediment and humic acid.

Structural studies of the hipersaline adaptation of proteins belonging to halophilic archaea

183 p.

Molecular modeling on some human interleukins sharing gamma c of interleukin-2 receptor, and structure-function relationships

Five models for human interleukin-7 (HIL-7), HIL-9, HIL-13, HIL-15 and HIL-17 have been generated by SYBYL software package. The primary models were optimized using molecular dynamics and molecular mechanics methods. The final models were optimized using a steepest descent algorithm and a subsequent conjugate gradient method. The complexes with these interleukins and the common gamma chain of interleukin-2 receptor (IL-2R) were constructed and subjected to energy minimization. We found residues, such as Gln127 and Tyr103, of the common gamma chain of IL-2R are very important. Other residues, e.g. Lys70, Asn128 and Glu162, are also significant. Four hydrophobic grooves and two hydrophilic sites converge at the active site triad of the gamma chain. The binding sites of these interleukins interaction with the common gamma chain exist in the first helical and/or the fourth helical domains. Therefore, we conclude that these interleukins binds to the common gamma chain of IL-2R by the first and the fourth helix domain. Especially at the binding sites of some residues (lysine, arginine, asparagine, glutamic acid and aspartic acid), with a discontinuous region of the common gamma chain of IL-2R, termed the interleukins binding sites (103-210). The study of these sites can be important for the development of new drugs. (C) 2000 Elsevier Science B.V. All rights reserved.

Measurement of 5-day biochemical oxygen demand without sample dilution or bacterial and nutrient enhancement

A direct method for measuring the 5-day biochemical oxygen demand (BODS) of aquaculture samples that does not require sample dilution or bacterial and nutrient enrichment was evaluated. The regression coefficient (R-2) between the direct method and the standard method for the analyses of 32 samples from catfish ponds was 0.996. The slope of the regression line did not differ from 1.0 or the Y-intercept from 0.0 at P = 0.05. Thus, there was almost perfect agreement between the two methods. The control limits (three standard deviations of the mean) for a standard solution containing 15 mg/L each of glutamic acid and glucose were 17.4 and 20.4 mg/L. The precision of the two methods, based on eight replicate analyses of four pond water samples did not differ at P = 0.05. (c) 2005 Elsevier B.V All rights reserved.

CYCLIC PEPTIDE HEPATOTOXINS FROM FRESH-WATER CYANOBACTERIAL (BLUE-GREEN-ALGAE) WATERBLOOMS COLLECTED IN CENTRAL CHINA

Toxic cyanobacteria (blue-green algae) waterblooms have been found in several Chinese water bodies since studies began there in 1984. Waterbloom samples for this study contained Anabaena circinalis, Microcystis aeruginosa and Oscillatoria sp. Only those waterblooms dominated by Microcystis aeruginosa were toxic by the intraperitoneal (i.p.) mouse bioassay. Signs of poisoning were the same as with known hepatotoxic cyclic peptide microcystins. One toxic fraction was isolated from each Microcystis aeruginosa sample. Two hepatotoxic peptides were purified from each of the fractions by high-performance liquid chromatography and identified by amino acid analysis followed by low and high resolution fast-atom bombardment mass spectrometry (FAB-MS). LD50 i.p. mouse values for the two toxins were 245-mu-g/kg (Toxin A) and 53-mu-g/g (Toxin B). Toxin content in the cells was 0.03 to 3.95 mg/g (Toxin A) and 0.18 to 3.33 mg/kg (Toxin B). The amino acid composition of Toxin A was alanine [1], arginine [2], glutamic acid [1] and beta-methylaspartic acid [1]; for Toxin B it was the same, except one of the arginines was replaced with a leucine. Low- and high-resolution FAB-MS showed that the molecular weights were 1,037 m/z (Toxin A) and 994 m/z (Toxin B), with formulas of C49H76O12N13 (Toxin A) and C49H75O12N10 (Toxin B). It was concluded that Toxin A is microcystin-RR and Toxin B is microcystin-LR, both known cyclic heptapeptide hepatotoxins isolated from cyanobacteria in other parts of the world. Sodium borohydride reduction of microcystin-RR yielded dihydro-microcystin-RR (m/z = 1,039), an important intermediate in the preparation of tritium-labeled toxin for metabolism and fate studies.

固定化红酵母细胞合成L-苯丙氨酸研究

In this paper, bioconversion of trans-cinnamic acid(t-Ca)to L-phenylalanine (L-phe) has been investigated by using immobilized yeast cells with induced L-phe Ammonia-lyase(PAL, EC.4.3.1.5) as biocatalysts. The contents are the following. (1) Thirty strains of yeasts, including two genera (Rhodotorula, Sporobolomyces), six species (R. glutinis R. minuta,R.rubra,R.sineses,R.roseus and S.salmonicolor)were screened for their ability to converse the substrates, t-Ca and ammonia, to the product, L-phe, by using yeast cells as biocatalyst, and primary evaluation for PAL activity of the selected strains was investigated. From the results of the screening experiments, it was found that 22 strains were able to produce L-phe from t-Ca with the range of conversion yield from 2% to 67%. Studies on PAL formation time course during cultivation show that the maximum PAL activity of several different strains ranges from 2.3 to 14.4×10-3U/mg cell dry weight. The biomass of tested strains at their maximum enzyme activity is also greatly varied. (2)One of the selected strains, R. rubra as 2.166, was used for immobilized cells as biocatalysts to produce L-phe. The optimum conversion conditions and effective stablization agents were investigated. The results shown that polyacrylamide gel was chosen as a suitable matrix for immobilization of the yeast cells, and it can retain 88% of the PAL activity in the reverse direction at the following reactive conditions: [t-Ca]: 34mM. [NH4OH]: 6.OM.PH10.00, temperature: 30℃. (3) The effects of various kinds of effectors on the production of L-phe were also examined. Membrane permeabilizing agents can stimulate L-phe synthesis, but make the stability of PAL decline greatly. Polyalchoholic agents and glutamic acid were very effective for the stabilization of PAL. At the presence of glutamic acid (5%), the half life of L-phe productivity with the immobilized cells was extended to 192 hours, which was much higher than most of that having been reproted, while the half life of resting cells was only about 15 hours. (4) Use of initial velocity studies on the kinetics of enzyme-catalized reaction indicated that the apparent Km value was 13.0mM for the immobilized cells, and 4.8mM for the resting cells. Thermostability of the immobilized cells was better than the resting cells. Fluid bed bioreactor is more effective than batch bioreator in prolonging the thermostability of the biocatalysts. (5) CGA- 688 resin column chromatographic procedure was employed in the isolation and purification of L-phe, t-Ca and other substances from the reactire mixture. (6) Preparative-scale production of L-phe on a level of gram amount by immobilized cells from the culture broth of R. rubra AS2.166 allowed for the conversion yield with 30%. The characteristic physico-chemical criteria (including melting point, optical activity, elements analysis, IR, NMR) are the same with the standard L-phe. 本文报告了利用诱导的苯丙氨酸解氨酶 (PAL.EC.4.3.1.5)催化反式肉桂酸(t-Ca)氨加 成制备L-苯丙氨酸(L-phe)的研究，主要内容为：(1) 我们搜集了三十株酵母菌株，利用全细胞转化t-Ca生成L-phe的能力进行了直 接筛选，并对其PAL活性水平进行了初步评估研究。研究结果表明，其中22株酵母具有转化t-Ca生产L-phe的能力，它们包括 Rhodotorula glutinis,R.rubra, R.sineses 和Sporobolomyces roseus 的菌株，转化率在2-67%。细胞生长和PAL形成过程的研究 表明，不同菌株PAL最大活力在2.3-14.4×10-3U/mg 细胞干重，达到最大PAL活性时各株酵母的生长情况也极不一致。(2) 利用筛 选出的一株深红酵母R.rubra AS2.166 作为供试菌株，研究了细胞固定化条件下生物转化的最适条件及PAL在固定化条件下的稳定 性。结果表明以聚丙烯酰胺凝胶包埋法较为理想，能使细胞合成L-phe活力保持88%，最适t-Ca浓度为34mM，最适NH4OH浓度为6M,最 适PH10.0，最适温度45℃。(3) 多种效应物对L-phe 合成的影响研究表明：表面活性剂能刺激L-phe的合成，但使PAL稳定性下降。 多羟基化合物及Glu对PAL的稳定十分有效在有Glu存在下，能使固定化细胞合成L-phe的半寿期达192小时左右，高于大部分现已报 导的固定化结果。(4) 用初速度法研究了深红酵母AS2.166中PAL的酶促反应特征，测得固定化细胞对t-Ca的表观米氏常数Km为 13.0mM，全细胞为4.8mM，细胞固定后热稳定性提高。(5) 建立了适合低浓度分离纯化产物与底物的聚苯乙烯大孔树脂柱层析技术 ，能使L-phe与t-Ca及产物混合物中其它成分有效分开。(6) 利用固定化的R.rubra AS2.166细胞所做的制备实验能够使L-phe的产 率达到30%左右，其主要的理化指标(包括熔点、比旋光度、元素分析、IR、NMR等)与标准L-phe一致。

Novel pH- and temperature-responsive block copolymers with tunable pH-responsive range

A series of novel pH- and temperature-responsive diblock copolymers composed of poly(N-isopropylacrylamide) (PNIPAM) and poly[(L-glutamic acid)-co-(gamma-benzyl L-glutamate)] [P(GA-co-BLG)] were prepared. The influence of hydrophobic benzyl groups on the phase transition of the copolymers was studied for the first time. With increasing BLG content in P(GA-co-BLG) block, the thermal phase transition of the diblock copolymer became sharper at a designated pH and the critical curve of phase diagram of the diblock copolymer shifted to a higher pH region.

Synthesis and self-assembly of a novel Y-shaped copolymer with a helical polypeptide arm

novel biodegradable Y-shaped copolymer, poly(L-lactide)(2)-b-poly(gamma-benzyl-L-glutamic acid) (PLLA(2)-b-PBLG), was synthesized by the ring-opening polymerization (ROP) of N-carboxyanhydride of gamma-benzyl-L-glutamate (BLG-NCA) with centrally amino-functionalized poly(L-lactide), PLLA(2)-NH2, as a macroinitiator in a convenient way. The Y-shaped copolymer and its precursors were characterized by H-1 NMR, FT-IR, GPC, WAXD and DSC measurements. The self-assembly of the PLLA(2)-b-PBLG copolymer in toluene and benzyl alcohol was examined. It was found that the self-assembly of the copolymer was dependent on solvent and on relative length of the PBLG block. For a copolymer with PLLA blocks of 26 in total degree of polymerization (DP), if the PBLG block was long enough (e.g., DP = 54 or more), the copolymer/toluene solution became a transparent gel at room temperature. In benzyl alcohol Solution, only PLLA(2)-b-PBLG containing ca. 190 BLG residues could form a gel: those with shorter PBLG blocks (e.g., DP = 54) became nano-scale fibrous aggregates and these aggregates were dispersed in benzyl alcohol homogeneously.

Synthesis of biodegradable thermo- and pH-responsive hydrogels for controlled drug release

Novel intelligent hydrogels composed of biodegradable and pH-sensitive poly(L-glutamic acid) (PGA) and temperature sensitive poly(N-isopropylacrylamide-co-2-hydroxyethyl methacrylate) (PNH) were synthesized and characterized for controlled release of hydrophilic drug. The influence of pH on the equilibrium swelling ratios of the hydrogels was investigated. A higher PNH content resulted in lower equilibrium swelling ratios. Although temperature had little influence on the swelling behaviors of the hydrogels, the changes of optical transmittance of hydrogels as a function of temperature were marked, which showed that the PNH part of hydrogel exhibited hydrophobic property at temperature above the lower critical solution temperature (LCST). The biodegradation rate of the stimuli-sensitive hydrogels in the presence of enzyme was directly proportional to the PGA content. Lysozyme was chosen as a model drug and loaded into the hydrogels.

Biodegradable amphiphilic polymer-drug conjugate micelles

The coupling of drugs to macromolecular carriers received an important impetus from Ringsdorf's notion of polymer-drug conjugates. Several water-soluble polymers, poly(ethylene glycol), poly[N-(2-hydroxypropyl) methacrylamidel, poly(L-glutamic acid) and dextran, are studied intensively and have been utilized successfully in clinical research. The promising results arising from clinical trials with polymer-drug conjugates (e.g., paclitaxel, doxorubicin, camptothecins) have provided a firm foundation for other synthetic polymers, especially biodegradable polymers, used as drug delivery vehicles. This review discusses biodegradable polymeric micelles as an alternative drug-conjugate system. Particular focus is on A-B or B-A-B type biodegradable amphiphilic block copolymer such as polylactide, morpholine-2,5-dione derivatives and cyclic carbonates, which can form a core-shell micellar structure, with the hydrophobic drug-binding segment forming the hydrophobic core and the hydrophilic segment as a hydrated outer shell. Polymeric micelles can be designed to avoid uptake by cells of reticuloendothelial system and thus enhance their blood lifetime via the enhanced permeability and retention effect.

Order-disorder transition in eicosylated polyethyleneimine comblike polymers

Order-disorder transition (ODT) behavior in eicosylated polyethyleneimine (PEI20C) comblike polymer obtained by grafting n-eicosyl group on polyethyleneimine backbone was systematically investigated by the combination of differential scanning calorimetry (DSC), wide-angle X-ray diffraction (WAXD), Fourier transform infrared (FTIR) spectroscopy as well as solid-state high resolution nuclear magnetic resonance (NMR) spectroscopy. DSC investigations showed two obvious transitions, assigned to the transitions (1) from orthorhombic to hexagonal and (2) from hexagonal to amorphous phase, respectively. These transitions are induced by the variations of alkyl side chain conformation and packing structure with temperature changing, which consequently lead to the destruction of original phase equilibrium. The ODT behavior can also be confirmed by spectroscopic methods like WAXD, FTIR and NMR. The ordered structure and the transition behavior of the alkyl side chains confined by the PEI backbone are obviously different from those of pristine normal alkanes. The transition mechanism of ODT and the origin of the phase transition behavior in PEI20C comblike polymer were discussed in detail in this paper.

Self-assembly of polypeptide-containing ABC-type triblock copolymers in aqueous solution and its pH dependence

Self-assembling of novel biodegradable ABC-type triblock copolymer poly(ethylene glycol)-poly(L-lactide)-poly(L-glutamic acid) (PEG-PLLA-PLGA) is studied. In aqueous media, it self-assembles into a spherical micelle with the hydrophobic PLLA segment in the core and the two hydrophilic segments PEG and PLGA in the shell. With the lengths of PEG and PLLA blocks fixed, the diameter of the micelles depends on the length of the PLGA block and on the volume ratio of H2O/dimethylformamide (DMF) in the media. When the PLGA block is long enough, morphology of the self-assembly is pH-dependent. It assembles into the spherical micelle in aqueous media at pH 4.5 and into the connected rod at or below pH 3.2. The critical micelle concentration (cmc) of the copolymer changes accordingly with decreasing solution pH. Both aggregation states can convert to each other at the proper pH value. This reversibility is ascribed to the dissociation and neutralization of the COOH groups in the LGA residues. When the PLGA block is short compared to the PEG or PLLA block, it assembles only into the spherical micelle at various pH values.

Phase transition and conformational variation of N-alkylated branched poly(ethyleneimine) comblike polymer

A series of branched poly(ethyleneimine) (PEI) derived polymers with different lengths of n-alkyl side chains, denoted as PEI(n)Cs (n = 12, 14, 16, 18, 20, number of carbon atoms in alkyl side group), have been prepared by a N-alkylation method, and systematically characterized by differential scanning calorimertry (DSC) and wide-angle X-ray diffraction (WARD) as well as Fourier transform infrared spectroscopy (FTIR). The side chains grafted on these comblike polymers are long enough to form crystalline phase composed of paraffin-like crystallites. The crystallization of the side chains forces the branched poly(ethyleneimine) molecules to pack into layered structure, between which the crystallites are located. The melting temperatures of the side chain crystallites increase from -12.36 to +51.49 degreesC with increasing the length of the side chains from n. = 12 to n = 20, which are a little bit lower than the corresponding pristine n-alkanes. PEI18C was taken as an example in this work for the investigation of phase transition and conformational variation of the side chains with temperature changing.

Biodegradable amphiphilic triblock copolymer bearing pendant glucose residues: Preparation and specific interaction with Concanavalin A molecules

A novel biodegradable amphiphilic block copolymer PLGG-PEG-PLGG bearing pendant glucose residues is successfully prepared by the coupling reaction of 3-(2-aminoethylthio) propyl-R-D-glucopyranoside with the pendant carboxyl groups of PLGG-PEG-PLGG in the presence of N,N'-carbonyldiimidazole. The polymer PLGG-PEG-PLGG, i.e., poly {(lactic acid)-co-[(glycolic acid)-alt-(L-glutamic acid)]}-block-poly(ethylene glycol)-block-poly{( lactic acid)-co-[( glycolic acid)-alt-(L-glutamic acid)]}, is prepared by ring-opening copolymerization of L-lactide (LLA) with (3s)-benzoxylcarbonylethylmorpholine-2,5-dione (BEMD) in the presence of dihydroxyl PEG with molecular weight of 2000 as macroinitiator and Sn(Oct)(2) as catalyst, and then by catalytic hydrogenation. The glucose-grafted copolymer shows a lower degree of cytotoxicity to ECV-304 cells and improved specific recognition and binding with Concanavalin A (Con A). Therefore, this kind of glucose-grafted copolymer may find biomedical applications.