Photophysics of ANS. V. Decay modes of ANS in proteins: the IFABP-ANS complex

The fluorescence properties of ANS as bound to proteins are treated. Several points of view concerning the origin of these properties are reviewed and synthesized into one framework. On proteins where the quantum yield (QY) is appreciable, as in organic solvents, the preferred conformation of ANS is often with the phenyl ring nearly (65 degrees -85 degrees ) orthogonal to the naphthalene. The major consequence of this geometry is water exclusion from the critical zone of ANS at which the largest amount of solvent dipolar relaxation originates. This, in turn, leads to a depression of the rate of electron transfer to the surroundings, together with other effects, as noted in the literature and in our lab. Alternative quenching pathways for ANS on the protein vs in water are also elucidated.     

BOUND WATER IN MUSCLE

1. The amount of free unfrozen water, i.e. water acting as normal solvent, in frog''s muscle at temperatures below the initial freezing-point has been calculated from the vapour pressure isotherm of the muscle. 2. Significant amounts of free water are present at –20°C. The total amount of unfrozen water at –20°C. cannot, therefore, be taken as a measure of the bound water in muscle. 3. The calculated values of free water, when compared with experimentally determined values of total unfrozen water, indicate that the amount of bound water in muscle at various temperatures is small. 4. A temperature considerably below –20°C., roughly between –40° and –60°C., is required to freeze completely the free water in muscle.     

Radiation-induced DNA damage as a function of hydration. I. Release of unaltered bases.

The release of unaltered bases from irradiated DNA, hydrated between 2.5 and 32.7 mol of water per mole of nucleotide (gamma), was investigated using HPLC. The objective of this study was to elucidate the yield of the four DNA bases as a function of dose, extent of hydration, and the presence or absence of oxygen. The increase in the yield of radiation-induced free bases was linear with dose up to 90 kGy, except for the DNA with gamma = 2.5, for which the increase was linear only to 10 kGy. The yield of free bases as a function of gamma was not constant in either the absence or the presence of oxygen over the range of hydration examined. For DNA with gamma between 2.5 and 15, the yield of free bases was nearly constant under nitrogen, but decreased under oxygen. However, for DNA with gamma greater than 15, the yield increased rapidly under both nitrogen and oxygen. The yield of free bases was described by a model that depended on two factors: 1) a change in the DNA conformation from a mixture of the A and C conformers in vacuum-dried DNA to predominantly the B conformer in the fully hydrated DNA, and 2) the proximity of the water molecules to the DNA. Irradiation of the inner water molecules (gamma less than 15) was less efficient than irradiation of the outer water molecules (gamma greater than 15), by a factor of approximately 3.3, in forming DNA lesions that resulted in the release of an unaltered base. This factor is similar to the previously published relative efficiency of 2.8 with which hydroxyl radicals and base cations induce DNA strand breaks. Our irradiation results are consistent with the hypothesis that the G value for the first 12-15 water molecules of the DNA hydration layer is the same as the G value for the form of DNA to which it is bound (i.e., the pseudo-C or the B form). Thus we suggest that the release of bases originating from irradiation of the hydration water is obtained predominantly: (1) by charge transfer from the direct ionization of the first 12-15 water molecules of the primary hydration layer and (2) by the attack of hydroxyl radicals generated in the outer, more loosely bound water molecules.     

A dual role for intracellular trehalose in the resistance of yeast cells to water stress.

It is well known that yeast cells survive environmental stresses such as desiccation and freezing and there is evidence that these phenomena may be related to the presence of trehalose in the cells. However, the molecular mechanism by which trehalose might exert an influence on cell functions remains unknown. In this report, thermogravimetry and differential thermal analysis were used to estimate the amount of bound water in yeast cells. It is shown that when the trehalose content is greater than 2-3% of the cell dry weight, the amount of bound water is drastically decreased and the viability of the dried cells is increased. This implies that a major portion of the bound water is replaced by trehalose. In addition, measurements of the NMR spin-lattice relaxation time of the intracellular water protons show that trehalose acts as a water-structuring agent in hydrated yeast cells. This dual role is essential for high resistance to water stress in yeast cells.     

Anomalous temperature dependence of the thermal expansion of proteins

The temperature dependence of the apparent expansibility of lysozyme and ovalbumin in solution has been measured as a function of pH. This temperature dependence is explained in terms of suppressed fluctuations in bound water due to the protein. It is shown that the thermal expansion coefficient of bound water is different from bulk water. The pH dependence can be explained by increased hydration of side chains at lower pH. The amount in volume of hydration water in a typical protein-water system varies from 0.16 to 0.7. How the intrinsic thermal expansion coefficient of proteins can be derived from the apparent quantity is discussed. Intrinsic values of the thermal expansion coefficient for lysozyme at room temperature are between 1.7 and 4.4 x 10(-4) K-1 for a 10% solution.     

Water structure in modified bovine plasma albumin (BPA) gel and bovine mercaptalbumin solution. 1H-n.m.r. studies

Bovine plasma albumin Fr. V (BPA) has been known to contain small amounts of proteolytic enzyme. Wilson & Foster (1971) found a very limited and specific cleavage of BPA catalyzed by the enzyme with BPA in the F-form near pH 3.8, resulting in the formation of partially hydrolyzed BPA (BPA*). BPA* had a tendency to form a transparent gel at pD 4.0 (pD range of the F-form) above 8%, though proteolytic enzyme-free bovine mercaptalbumin (BMA) was in a transparent solution at pD 4.0 even at 12%. Water structures of the F-form of BMA in the solution state and of BPA* in the gel state were studied by measuring 1H-n.m.r. spectra, spin-lattice relaxation time (T1) and cross relaxation time (TIS) between irradiated and observed protons. Protein concentration-dependent changes of T1 of water protons indicated that the amount of hydrated water of BPA* in the gel state is far greater than that of the F-form of BMA in the solution state. TIS values from protein protons to water protons also indicated a large amount of hydration of BPA*, strong interaction between water and BPA* and rapid exchange between bound and bulk water in the gel state.     

High-affinity binding of water by proteins is similar in air and in organic solvents

Published data for water adsorption by proteins suspended in organic solvents (of interest as enzyme reaction mixtures) have been converted to a basis of thermodynamic water activity (aw). The resulting adsorption isotherms have been compared with those known for proteins equilibrated with water from a gas phase. This comparison can show any effects of the solvent on the interaction between the protein and water at the molecular level. At lower water contents (aw less than about 0.4), similar adsorption isotherms are found in each solvent and in the gas phase; differences are probably less than the likely errors. Hence, it may be concluded that the presence of an organic solvent has little effect on the interaction between proteins and tightly bound water; on a molecular scale there is probably little penetration of the primary hydration layer by solvent molecules, even fairly polar ones such as EtOH. At higher aw values, there are differences between the isotherms which probably are significant. Nonpolar solvents increase the amount of water bound by the enzyme (at fixed aw), while polar solvents (mainly EtOH) may reduce the amount of water bound by the enzyme, presumably by occupying part of the secondary hydration layers in place of water.     

The effect of low molecular weight ligands on relaxation of water protons in protein solutions

Interaction of low-molecular ligands (LML) isolated from blood serum albumin (SA) and serum proteins leads to higher T1 values for water protons compared with those observed in LML-free solutions, although the total amount of bound water increases. The latter was revealed by low-temperature NMR spectroscopy, as well as by the amount of water sorbed on SA + LML at a relative humidity P/Ps greater than 0.7. In the region 0.2 less than P/Ps less than 0.6 the amount of SA + LML-sorbed water decreased, as compared with that in SA indicating that the oppositely charged groups of LML screen some charged groups of the protein. A decrease of charge-to-charge interactions in solution, or with a high water content, leads to the hydration of those groups. The increase of the T1 value for water protons in solution is, probably, due to a hindered exchange between the sorbed water and bulk water. It is outlined that charge interactions between macromolecules may significantly affect water sorption by proteins.     

Evidence against bicarbonate bound in the O2-evolving complex of photosystem II

The oxidation of water to molecular oxygen by photosystem II (PSII) is inhibited in bicarbonate-depleted media. One contribution to the inhibition is the binding of bicarbonate to the non-heme iron, which is required for efficient electron transfer on the electron-acceptor side of PSII. There are also proposals that bicarbonate is required for formation of O 2 by the manganese-containing O 2-evolving complex (OEC). Previous work indicates that a bicarbonate ion does not bind reversibly close to the OEC, but it remains possible that bicarbonate is bound sufficiently tightly to the OEC that it cannot readily exchange with bicarbonate in solution. In this study, we have used NH 2OH to destroy the OEC, which would release any tightly bound bicarbonate ions from the active site, and mass spectrometry to detect any released bicarbonate as CO 2. The amount of CO 2 per PSII released by the NH 2OH treatment is observed to be comparable to the background level, although N 2O, a product of the reaction of NH 2OH with the OEC, is detected in good yield. These results strongly argue against tightly bound bicarbonate ions in the OEC.     

Water proton magnetic resonance studies of normal and sickle erythrocytes. Temperature and volume dependence.

The temperature and cell volume dependence of the NMR water proton line-width, spin-lattice, and spin-spin relaxation times have been studied for normal and sickle erythrocytes as well as hemoglobin A and hemoglobin S solutions. Upon deoxygenation, the spin-spin relaxation time (T2) decreases by a factor of 2 for sickle cells and hemoglobin S solutions but remains relatively constant for normal cells and hemoglobin A solutions. The spin-lattice relaxation time (T1) shows no significant change upon deoxygenation for normal or sickle packed red cells. Studies of the change in the NMR linewidth, T1 and T2 as the cell hydration is changed indicate that these parameters are affected only slightly by a 10-20% cell dehydration. This result suggests that the reported 10% cell dehydration observed with sickling is not important in the altered NMR properties. Low temperature studies of the linewidth and T1 for oxy and deoxy hemoglobin A and hemoglobin S solutions suggest that the "bound" water possesses similar properties for all four species. The low temperature linewidth ranges from about 250 Hz at -15 degrees C to 500 Hz at -36 degrees C and analysis of the NMR curves yield hydration values near 0.4 g water/g hemoglobin for all four species. The low temperature T1 data go through a minimum at -35 degrees C for measurements at 44.4 MHz and -50 degrees C for measurements at 17.1 MHz and are similar for oxy and deoxy hemoglobin A and hemoglobin S. These similarities in the low temperature NMR data for oxy and deoxy hemoglobin A and hemoglobin S suggest a hydrophobically driven sickling mechanism. The room temperature and low temperature relaxation time data for normal and sickle cells are interpreted in terms of a three-state model for intracellular water. In the context of this model the relaxation time data imply that type III, or irrotationally bound water, is altered during the sickling process.     

高产条件下不同小麦品种耗水特性和水分利用效率的差异

设置不灌水(W0)、底墒水+拔节水(W1)、底墒水+拔节水+开花水(W2)3个灌水处理,采用6个冬小麦(Triticum aestivum.L.)品种,研究了不同品种耗水特性和水分利用效率的差异.结果表明:(1)依据籽粒产量和水分利用效率2个因子,采用聚类分析的方法,将供试品种分为高水分利用效率组(Ⅰ组)、中水分利用效率组(Ⅱ组)和低水分利用效率组(Ⅲ组).同一灌水条件下的籽粒产量,Ⅰ组显著高于Ⅱ组和Ⅲ组;Ⅱ组和Ⅲ组在W0条件下无显著差异,在W1和W2条件下Ⅱ组显著高于Ⅲ组.(2)从Ⅰ组、Ⅱ组、Ⅲ组中分别取1个品种,泰山23、潍麦8号、山农12进一步分析表明,在W0 和W1条件下,泰山23和潍麦8号的阶段耗水量和耗水模系数为开花至成熟>播种至拔节>拔节至开花,山农12为播种至拔节>开花至成熟>拔节至开花.W2条件下,3个品种的阶段耗水量和耗水模系数为开花至成熟>播种至拔节>拔节至开花;播种至拔节和拔节至开花的耗水模系数为泰山23>山农12>潍麦8号,此阶段的耗水量和耗水强度为泰山23品种最高;开花至成熟的耗水模系数为潍麦8号>山农12 >泰山23,此阶段的耗水量和耗水强度为泰山23品种最低.(3) 在W0 和W1条件下,总耗水量和灌水量、降水量及土壤耗水量占总耗水量的百分率为泰山23品种居中;W2条件下,灌水量和降水量占总耗水量的百分率为泰山23>潍麦8号>山农12,土壤耗水量及其占总耗水量的百分率反之,但泰山23的总耗水量最低.(4) 同一灌水条件下,泰山23品种100～200cm土层的土壤耗水量高于潍麦8号,表明该品种能充分利用深层土壤水;山农12品种在W0和W2条件下,100～200 cm土层的土壤耗水量高于泰山23和潍麦8号,但其籽粒产量和水分利用效率显著低于上述两品种.     

Intramolecular esterification by lipase powder in microaqueous benzene: effect of moisture content

In view of the biochemical reaction catalyzed by enzyme powder suspended in a water-insoluble organic solvent, an equation was derived to estimate the amount of water bound to the enzyme powder. With this equation, an apparent adsorption isotherm between free water (water freely dissolved in benzene) and bound water (water bound to crude lipase powder of Pseudomonas fluorescens) was obtained. A direct lactonization reaction (synthesis of cyclopentadenolide from 15-hydroxypen-tadecanoic acid) catalyzed by crude lipase powder of Pseudomonas fluorescens was carried out batchwise in microaqueous benzene at 40oC. A kinetic model of the enzymatic reversible lactonization reaction was derived, from which the effect of moisture content on the initial reaction rate with a fully hydrated enzyme was mathematically expressed. The observed initial reaction rate first increased, then decreased with increasing moisture content, giving rise to the maximum rate at a certain level of the moisture content. The drop in the reaction rate at lower moisture content was due to a lesser hydration of the enzyme molecule (hydration-limited) and the decrease in the reaction rate at higher moisture content was attributed to the dependence of the true initial rate of the reversible reaction on the moisture content (true reversible reaction limited), and could be simulated by the kinetic model. The equilibrium yield approached 100% at a lower moisture content.     

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.     

Enzymatic fatty acid exchange in digalactosyldiacylglycerol

Six different lipases were screened for their ability of acidolysis between digalactosyldiacylglycerol (DGDG) and heptadecanoic acid in toluene. Lipases from Geotrichum candidum, Alcaligenes sp. and Penicillium camembertii did not catalyse the acidolysis reaction. Rhizopus arrhizus and Rhizomucor miehei (Lipozyme) catalysed the acidolysis but produced a mixture of DGMG, DGDG, acyl-DGMG and acyl-DGDG. The extra acyl group is bound to the primary hydroxyl of the digalactosyl moiety. Candida antarctica also catalysed the acidolysis but the TLC analysis showed bands with higher Rf values than acyl-DGDG, these probably being different tetra and higher esters. R. arrhizus lipase was the most promising enzyme under the conditions used, with no tetra esters being formed and giving the highest reaction rate of the enzymes investigated. Low water activity (0.06 or 0.11) and high fatty acid concentration (400 mM) increased the formation of acyl-DGDG whilst higher water activities (0.33 and 0.54) increased the amount of DGMG when R. arrhizus lipase was used as catalyst. At a water activity of 0.11 and a fatty acid concentration of 400 mM a yield of 24% modified DGDG was obtained. In this product the fatty acid originally present in the sn-1 position had been exchanged by heptadecanoic acid.     

堇菜叶片草酸钙晶体与水分维持的关系

随着全球气候变化加重,干旱强度和持续时间逐渐增加,严重影响植物生长和作物产量。喀斯特为典型的干旱和高钙生境,植物叶片富集大量的草酸钙晶体,而该晶体与植物耐旱性之间的关系并不清楚。该研究以喀斯特适生植物堇菜(Viola verecumda)为材料,土壤进行自然干旱,分析堇菜叶片的草酸钙晶体变化特征与水分之间的关系。结果表明:在土壤自然干旱条件下,堇菜主要通过细胞内束缚水的释放,维持细胞内水分平衡;而在干旱后期,叶片通过关闭气孔,将部分自由水转变为束缚水,防止水分流失。此外,草酸钙晶体的密度与束缚水含量具有极其显著的强正相关线性回归关系(r=0.825 3,P0.000 1),表明草酸钙晶体作为主要的束缚水物质。因此,堇菜植物在耐旱过程中可能协调草酸钙晶体和气孔的生理行为忍耐干旱胁迫。     

Measuring enzyme hydration in nonpolar organic solvents using NMR

A very sensitive NMR method has been developed for measuring deuterated water bound to proteins suspended in nonpolar solvents. This has been used to determine the amount of bound water as a function of water activity for subtilisin Carlsberg suspended in hexane, benzene, and toluene and for alpha-chymotrypsin in hexane. The adsorption isotherms for subtilisin in the three solvents are very similar showing that water activity can be usefully employed to predict the amount of water bound to proteins in nonpolar organic media. Comparison of the degree of enzyme hydration reached in nonpolar solvents with that obtained in air shows that adsorption of strongly bound water is hardly affected by the low dielectric medium, but adsorption of loosely bound water is significantly reduced. This suggests that the hydrophobic regions of the protein surface are preferentially solvated by solvent molecules, and that in a nonpolar environment formation of a complete monolayer of water over the protein surface is thermodynamically unfavorable. (c) 1995 John Wiley & Sons, Inc.     

1980-2016年三江源国家公园水源供给及水源涵养功能时空变化研究

为实现三江源国家公园水源供给及涵养功能评估,服务区域生态服务价值估算,基于InVEST模型,利用1980-2016年期间共7期土地利数据,结合气象数据,土壤数据,地形数据等,评估了三江源国家公园水源供给及水源涵养量的时间变化特征与空间分布状况。结果表明：1）1980-2016年三江源国家公园年降水呈不显著增加趋势;潜在蒸散、实际蒸散显著增加。在此影响下,园区产水量及水源涵养量总体呈不显著增加趋势。在不同年代,园区水资源总量经历了骤降-好转-略微降低的变化过程。降水量与实际蒸散量对园区产水量及水源涵养量影响最为显著。2）园区产水量及水源涵养量空间分布趋势一致,呈由北向南先减少后增加的变化趋势。这种空间差异主要由降水差异及地表覆盖特征引起的蒸散差异引起。3）在极端降水条件下,园区产水量及水源涵养量的数量和空间分布差异十分显著。长江源园区生态水源对降水变化的响应最为敏感。     

Surface viscosity measurements from large bilayer vesicle tether formation. II. Experiments.

A mechanical experiment has been developed that measures an upper bound for the viscosity of a lipid bilayer membrane. In this experiment, strands of membrane (tethers) are formed from phospholipid vesicles attached to micropipettes by subjecting the vesicles to fluid drag. The rate of tether formation is measured as a function of the velocity of the suspending fluid. The surface viscosity can be calculated from this data using a theoretical relationship derived in a companion paper. Because of the multilamellar character of the vesicles, these values provide an upper bound for the viscosity of a single bilayer. The smallest values obtained in these measurements fell in the range 5.0-13.0 x 10(-6) dyn s/cm. These values are in relatively good agreement with the values calculated from lateral and rotational mobility measurements.     

The relationship of bound water to the IR amide I bandwidth of albumin

Four different types of ir experiments, involving changes in pH, changes in pressure, and the use of nonaqueous solvents, and with either albumin molecules dissolved in saline or adsorbed albumin films, support the hypothesis that the bandwidth of the amide I vibration of albumin is directly related to the amount of bound water in this protein. From the amide I band narrowing and the amide I shift to higher frequencies, it is proposed that a more ordered helix structure results as the amount of bound water is decreased.     

Human plasma carboxypeptidase N. Isolation and characterization.

Human plasma carboxypeptidase N has been purified 2,600-fold from pooled, outdated plasma in a 30% yield. Isolation was accomplished by chromatography on DEAE-cellulose and on a p-aminobenzoyl-L-arginine-Sepharose 6B affinity column. Carbohydrate accounts for 17% of the weight calculated from its amino acid and carbohydrate composition. The enzyme appears to consist of three subunits of Mr = 83,000, 55,000, and 49,000 and contains a significant amount of bound zinc. Purified enzyme preparations are very sensitive to proteolytic degradation but are stable for at least 3 months at 4 degrees.