水合牛血清白蛋白热稳定性的热力学研究

蛋白质的初级结合水对于蛋白质分子的构象及热稳定性有着重要影响。将不同吸附水量的牛血清白蛋白样品密封入铝制挥发型样品盘中,用P/E DSC_(-2)型差示扫描量热计对蛋白样品的变性温度、变性焓及变性前后的比热变化等热力学参数进行测量。实验证明,随水含量增多变性温度T_D下降,当含水量R(克H_2O/克BSA)>0.24时T_D的下降渐微,当R=0.5时T_D通过最小值后又略有增大。变性焓ΔH_D也与水含量密切相关,当R<0.5时ΔH_D渐趋恒值,为200千卡/克分子。本实验还观察到在低含水量范围内0.02相似文献   

水合溶菌酶及其热稳定性的NMR研究

本文用90MHz脉冲NMB波谱仪记录了具不同含水量的溶菌酶的质子宽谱线NMR谱图及自由感应衰减曲线,并记录了水合度为0.10及0.19克水/克溶菌酶的溶菌酶样品从室温到热变性温度范围内的谱图.用线宽参量随水合度及温度的变化讨论溶菌酶在水合及热变性过程中溶菌酶分子及水分子运动性的变化.结果表明,溶菌酶分子及水分子的运动性与水合溶菌酶中的水含量密切相关;低水含量的水合溶菌酶在热变性过程中酶分子运动性的变化经历了两个转变,分别对应于酶分子间的解缔合及分子内的解旋.     

水合溶菌酶的H-D交换与分子活动性

应用红外吸收光谱研究不同含水量的溶菌酶膜的H-D交换动力学,以期阐明水合对溶菌酶分子活动性的影响。 酰胺Ⅱ带与酰胺Ⅰ带峰高之比A_Ⅱ/A_Ⅰ随交换时间延长成指数下降,它反映处于不同状态的质子交换速率。在一定水合范围内交换速率与水合溶菌酶水含量有关。分别在交换10分钟及5小时后的A_Ⅱ/A_Ⅰ测量表明,在R<0.2范围内,交换的质子数随水含量同步增大。R≥0.2交换质子数不再进一步增大。最初10分钟内交换的反应速度常数K_f随水含量的改变亦遵循定律。R≈0.2为一临界水含量,是溶菌酶分子维持其晶体和溶液中的空间结构及表现其活性所必需。低于此值时,溶菌酶分子的活动性随水含量而改变。     

胶原纤维内吸附水熔融热的差示扫描量热法的研究

用DSC法研究了天然与变性牛蹄跟腱胶原蛋白内吸附水的熔融热.在含水量(R)为0.33克(水)/克(蛋白)时观察到冰的熔融峰.在0.57克/克以下,水的积分熔融热(Q_f)与水含量呈非线性关系,由此得到出现熔融峰的临界水含量为0.26克/克.在0.57—1.05克/克范围内Q_f同R接近线性关系,其表观微分熔融热dQ/dR=69.5卡/克(水).R>1.05克/克时,dQ/dR=79.2卡/克.     

牛胰RNaseA的热转变的差示扫描量热研究

用差示扫描量热法对含水量为0.05-3.15克/克的牛胰核糖核酸酶A的热转变进行了研究.当R<0.40克/克时,在315-345和400-450K左右,分别观察到峰Ⅰ和峰Ⅲ.文中对峰Ⅰ的“恢复”进行了讨论.在R<1.1克/克时,通常被认为热变性峰的峰Ⅱ的峰温,随R的增加而降低,变性热随R的增加而减少,但在 R≥1.1克/克时,二者均取稳定值,Ttr=335.5K和Qtr=7.38CaⅠ/go峰Ⅱ的半宽在R=0.40克/克处取极小值,在R≥1.65时取稳定值,△T1/2=7.34K,文中首次给出了水合状态下热变性峰的转变热和峰温的关系曲水线.对水合球状蛋白的热变性的一种可能解释为,变性焓是温度的函数,而转变温度直接受含水量影响.     

牛胰RNaseA的热转变的差示扫描量热研究

用差示扫描量热法对含水量为0.05-3.15克/克的牛胰核糖核酸酶A的热转变进行了研究.当R<0.40克/克时,在315-345和400-450K左右,分别观察到峰Ⅰ和峰Ⅲ.文中对峰Ⅰ的“恢复”进行了讨论.在R<1.1克/克时,通常被认为热变性峰的峰Ⅱ的峰温,随R的增加而降低,变性热随R的增加而减少,但在 R≥1.1克/克时,二者均取稳定值,Ttr=335.5K和Qtr=7.38CaⅠ/go峰Ⅱ的半宽在R=0.40克/克处取极小值,在R≥1.65时取稳定值,△T1/2=7.34K,文中首次给出了水合状态下热变性峰的转变热和峰温的关系曲水线.对水合球状蛋白的热变性的一种可能解释为,变性焓是温度的函数,而转变温度直接受含水量影响.     

用差示扫描量热计对低水含量蛋白质热变性前峰的研究

本文用P/EDSC-2型差示扫描量热计检测了11个具低水含量的高纯蛋白质的DSC曲线.发现它们无一例外地在变性峰前都具有小的吸热峰,即热变性前峰.实验表明变性峰和前峰的峰温、峰面积都强烈现依赖于蛋白质的含水量,而且样品被第一次扫描后在室温放置一定时间后前峰仍可以再现,再现前峰的峰温和峰面积取决于样品的水合度、第一次被扫描的终止温度以及第一次扫描后在室温放置的时间.本文还检测了一些多聚物、氨基酸、多肽以及热变性后蛋白质的DSC曲线.发现热变性后的蛋白质仍可出现前峰,但变性峰不再出现,显然两个峰的出现机制不同.本文并就前峰出现的可性能机制进行了初步讨论.     

不同入流条件下植被过滤带对坡面径流氮、磷的拦截效果

植被过滤带能有效拦截坡面径流泥沙,在防治水土流失和农业面源污染等方面具有重大潜力.为探讨不同入流条件下植被过滤带对坡面径流氮、磷的拦截效果及其水动力学机理,本研究通过模拟上方来水冲刷试验,定量分析了上方来水流量、氮磷含量浓度等因素影响下植被过滤带对径流氮、磷的拦截规律及其与径流水动力学参数之间的关系.结果表明: 植被过滤带能有效拦截上方来水中氮、磷等污染物,入流流量分别为200、400、600 L·h^-1时,模拟植被过滤带对总氮的拦截率分别为74.9%、62.0%、58.3%,对总磷拦截率分别为85.0%、75.6%、72.0%,在上方来水流量较低时拦截效果最优;上方来水入流氮、磷浓度变化对植被过滤带拦截效率的影响不显著.不同入流条件处理下,植被过滤带对氮、磷的拦截率随弗洛德数增大而增大,呈显著线性正相关关系;与阻力系数、水流剪切力、水流功率呈线性负相关关系;其中,氮、磷拦截率与水流剪切力的相关关系最优,可以用含水流剪切力的公式表征植被过滤带对氮、磷的拦截效率.     

镉污染对水稻土微生物量、酶活性及水稻生理指标的影响

水稻盆栽条件下,研究了外源Cd不同处理对土壤微生物学指标、土壤酶活性及部分水稻生理指标的影响.结果表明,土壤微生物量C和N开始随Cd浓度增加而上升,到一定浓度时则随Cd浓度增加而下降,其转折点因土壤性质有所差异.同时土壤酶活性变化规律与土壤微生物量C、N变化规律相似,但其转折点浓度因土壤类型及土壤酶种类不同而有差异.Cd污染后的变异系数依次为:脱氢酶＞酸性磷酸酶＞脲酶.土壤呼吸作用强度和代谢熵都随Cd浓度增大而缓慢增加.水稻叶绿素含量随Cd处理浓度增加表现出先上升后下降,其转折点受供试土壤性质不同而不同;脯氨酸含量与过氧化物酶活性随着Cd处理浓度增大而增加.Cd污染后水稻生理指标的变异系数在黄松田水稻土中依次为过氧化物酶活性＞叶绿素含量＞脯氨酸含量;黄红壤性水稻土中依次为过氧化物酶活性＞脯氨酸含量＞叶绿素含量.相关分析表明,种植水稻条件下Cd污染对土壤微生物量、酶活性及水稻生理指标的影响是相辅相成的.     

脲对果菠萝蛋白酶活力与构象的影响

本文用荧光光谱,紫外差示光谱和CD谱研究果菠萝蛋白酶在不同浓度的脲溶液中的构象及酶活力的变化情况。酶的荧光强度随脲浓度增大而明显增加,8mol/L脲使荧光强度增强65％,发射峰出现红移。差示谱表明在232nm和288nm出现二个正峰,它们均随脲浓度增大而加剧,前者与主链构象变化有关,而后者与生色基团(Trp、Tyr)的微环境变化相关。CD谱表明:天然酶在208nm和225nm处有二个负峰,脲变性后,225nm的负峰基本上不随脲浓度增大而变化,但208nm峰则明显发生变化并逐渐出现红移,6mol/L以上此峰则完全消失。     

Hydration-dependent far-infrared absorption in lysozyme detected using synchrotron radiation.

Using the National Synchrotron Light Source (NSLS) at Brookhaven far-infrared absorption in the frequency range 15-45 cm-1 was detected in samples of lysozyme at different hydrations and in water. The absorption is due to the presence of low-frequency (picosecond timescale) motion in the samples, such as are calculated in molecular dynamics simulations. The form of the transmission profile is temperature independent but varies significantly with the degree of hydration of the protein. At higher hydrations the profile resembles closely that of pure water in the region 20-45 cm-1. At a low hydration marked differences are seen with, in particular, the appearance of a transmission minimum at 19 cm-1. The possible origins of the hydration dependence are discussed. The results demonstrate the usefulness of long-wavelength synchrotron radiation for the characterisation of biologically-important low-frequency motions in protein samples.     

EXAFS analysis of the pH dependence of the blue-copper site in amicyanin from Thiobacillus versutus

The room temperature Cu K-edge EXAFS (extended X-ray absorption fine structure) spectrum of reduced and oxidized amicyanin, the blue copper protein from Thiobacillus versutus, was measured at low and high pH. The data interpretation was partly based on independent NMR evidence for the occurrence of a ligand histidine protonation at low pH (pKa = 6.9) in the reduced protein. In the oxidized protein two nitrogen-donors (from two histidines; Cu-N distances 1.95-2.01 A and 1.86-1.89 A) and a sulfur-donor (from a cysteine; Cu-S distance 2.11-2.13 A) were identified and the coordination appears independent of pH. Upon reduction at high pH the Cu-S bond and one of the Cu-N bonds lengthen slightly (from 2.11 to 2.19 A and from 2.01 to 2.18 A, respectively). Upon lowering of the pH one of the N-donors of the Cu in reduced amicyanin disappears from the Cu EXAFS and a second S-donor (from a methionine) becomes visible at 2.41 A from the Cu. The Debye-Waller factors are compatible with a Cu-N vibrational stretch frequency in the range of 150-250 cm-1 and one greater than 285 cm-1, and a Cu-S vibrational stretch frequency of about 150 cm-1 (Cu-Smet; reduced amicyanin at low pH) and one in the range of 230-800 cm-1 (Cu-Scys).     

Conformation of oxytocin studied by laser Raman spectroscopy

The peptide backbone conformation and salient structural details of oxytocin were examined by laser Raman spectroscopy. Spectra were obtained in the solid phase, water, 2H2O, and dimethyl sulfoxide solutions. A distinct Amide I band was obtained at 1663 cm-1 for aqueous and deuterated samples and 1666 cm-1 for the solid sample. A relatively high frequency Amide III band at 1260 cm-1 was obtained. It is concluded that these Amide I and III bands arise from the "beta-turn"-like conformation of oxytocin. The tyrosine side chain, according to the I850 cm-1/I830 cm-1 intensity ratio, is exposed to the solvent. The S-S stretching vibration at 512 cm-1 indicates the conformation of C-C-S-S-C-C in the disulfide bridge of oxytocin in the ring is gauche-gauche-gauche.     

Hydration dependence of conformational dielectric relaxation of lysozyme

Dielectric response of hen egg white lysozyme is measured in the far infrared (5-65 cm-1, 0.15-1.95 THz, 0.6-8.1 meV) as a function of hydration. The frequency range is associated with collective vibrational modes of protein tertiary structure. The observed frequency dependence of the absorbance is broad and glass-like. For the entire frequency range, there is a slight increase in both the absorbance and index of refraction with increasing hydration for <0.27 h (mass of H2O per unit mass protein). At 0.27 h, the absorbance and index begin to increase more rapidly. This transition corresponds to the point where the first hydration shell is filled. The abrupt increase in dielectric response cannot be fully accounted for by the additional contribution to the dielectric response due to bulk water, suggesting that the protein has not yet achieved its fully hydrated state. The broad, glass-like response suggests that at low hydrations, the low frequency conformational hen egg white lysozyme dynamics can be described by a dielectric relaxation model where the protein relaxes to different local minima in the conformational energy landscape. However, the low frequency complex permittivity does not allow for a pure relaxational mechanism. The data can best be modeled with a single low frequency resonance (nu approximately 120 GHz=4 cm-1) and a single Debye relaxation process (tau approximately .03-.04 ps). Terahertz dielectric response is currently being considered as a possible biosensing technique and the results demonstrate the required hydration control necessary for reliable biosensor applications.     

An FT-IR study of DNA and RNA conformational transitions at low temperatures

Fourier Transform Infrared (FT-IR) spectra of solid samples of DNA and RNA obtained from freeze-drying at solid CO2 and liquid nitrogen temperatures, have been recorded and correlation between the conformational transitions and spectral changes is proposed. It is concluded that an equilibrium exists between A, B and Z conformations at low temperatures for the DNA molecule, which is temperature dependent, whereas the RNA molecule exhibits only the A conformation. The results have been compared with the metal-adducts of DNA and RNA, where one of the conformations is predominant. Marker infrared bands for the B conformer have been found to be the strong band at 825 cm-1 (sugar conformer mode) and a band with medium intensity at 690 cm-1 (guanine breathing mode). The A conformation showed characteristic bands at 810 and 675 cm-1. The B to Z conformational transition was characterized by the strong absorption bands near 820-810 cm-1 and at 665-600 cm-1.     

Infrared spectroscopic discrimination between alpha- and 3(10)-helices in globular proteins. Reexamination of Amide I infrared bands of alpha-lactalbumin and their assignment to secondary structures

We have undertaken a new and more detailed Fourier-transform infrared (FTIR) spectroscopic study of alpha-lactalbumin (in D2O solution) aimed at correlating its secondary structures to observed Amide I' infrared bands. The spectra reported here were interpreted in light of the recently determined crystal structure of alpha-lactalbumin and by comparison with the spectra and structure of the homologous protein lysozyme. Of particular importance is the new evidence supporting the assignment of the band at 1639 cm-1 to 3(10)-helices. This assignment is in excellent agreement with one based on theoretical and experimental studies of 3(10)-helical polypeptides. The frequency observed for 3(10)-helices is distinctly different from that at which alpha-helices are typically found (viz., around 1655 cm-1). In the present study, two bands are clearly resolved in the latter region at 1651 and 1659 cm-1. Both are apparently associated with alpha-helices. These results suggest that for D2O solutions of globular proteins. FTIR spectroscopy can be a facile method for detecting the presence of these two different types of helical conformation and distinguishing between them. This provides a distinct advantage over ultraviolet circular dichroism spectroscopy (UV-CD). This work also provides a basis for future studies of alpha-lactalbumin which examine the effects of environment (e.g., pH, temperature) and ligands (e.g., Ca2+, Mn2+) on its conformation.     

Thermal denaturation of globular proteins. Fourier transform-infrared studies of the amide III spectral region.

Fourier transform-infrared (FT-IR) spectra are reported for the amide III spectral region of the native and thermally denatured forms of chymotrypsinogen, ribonuclease, bovine serum albumin, and lysozyme. Chymotrypsinogen denatures into structures containing substantial contributions from beta-sheets, while lysozyme and bovine serum albumin show increased amounts of random-coil forms. The changes observed for ribonuclease are quite small. Bovine serum albumin shows at least six bands in the 1,260-1,320 cm-1 region which undergo large intensity changes upon thermal denaturation, and hence are assignable to alpha-helical amide III modes. The large number of observed bands suggests that slight variations in helical geometry, symmetry, or interactions result in changed amide III frequencies, so that simple correlations between narrow frequency ranges and secondary structures may not be applicable for this mode. A widened frequency range is suggested as diagnostic for helical structures.     

Raman spectral studies of poly(1-methylinosinic acid).

The synthesis and characterization of poly(1-methylinosinic acid) are described. Laser Raman spectra of poly (mII) were obtained as a function of temperature in D2O solution. Thermal melting profiles derived from the intensity variations of the 712, 795, 814, 986, 1333, 1509, 1550 and 1680 cm-1 bands indicate a cooperative melting temperature of 9 +/- 1 degree C. The low temperature form of poly(mII) exhibits a carbonyl frequency at 1710 cm-I which is decreased to 1680 cm-I upon melting. The Raman hypochromism in the bands reported are equal to or much larger than any reported for other nucleic acids. The data are consistent with the low temperature form of poly(mII) being an ordered single stranded unit with a high degree of basestacking. The melting profiles obtained from the uv and cd spectra are consistent with and support the Raman data. This single stranded RNA exhibits an uncharacteristic behavior in that it melts cooperatively.     

An infrared spectroscopic study of the interactions of carbohydrates with dried proteins

Fourier-transform infrared spectroscopy was used to characterize the interaction of stabilizing carbohydrates with dried proteins. Freeze-drying of trehalose, lactose, and myo-inositol with lysozyme resulted in substantial alterations of the infrared spectra of the dried carbohydrates. In the fingerprint region (900-1500 cm-1), there were large shifts in the frequencies of bands, a decrease in absorbance, and a loss of band splitting. These effects mimic those of water on hydrated trehalose. Bands assigned to hydroxyl stretching modes (around 3350 cm-1) were decreased in intensity and shifted to higher frequencies in the presence of the protein. In complementary experiments, it was found that dehydration-induced shifts in the positions of amide I and amide II bands for lysozyme could be partially and fully reversed, respectively, when the protein was freeze-dried in the presence of either trehalose or lactose. In addition, the carboxylate band, which was not detectable in the protein dried without the sugar, was apparent when these sugars were present. myo-Inositol was less effective at shifting the amide bands, and the carboxylate band was not detected in the presence of this carbohydrate. Also tested was the concentration dependency of the carbohydrates' influence on the position of the amide II band for dried lysozyme. The results showed that the ability of a given concentration of a carbohydrate to shift this band back toward the position noted with the hydrated protein coincided, at least in the extreme cases, with the capacity of that same level of carbohydrate to preserve the activity of rabbit skeletal muscle phosphofructokinase during freeze-drying.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct measurement of hydration-related dynamic changes in lysozyme using inelastic neutron scattering spectroscopy

Inelastic neutron scattering spectroscopy is used to investigate dynamic changes in lysozyme powder at two different low D2O hydrations (0.07g D2O/g protein and 0.20 g D2O/g protein). In the higher hydration sample, the inelastic scattering between 0.8 and 4.0 cm-1 energy transfer is increased and the elastic scattering is decreased. The decreased elastic scattering suggests increased atomic amplitudes of motion and the increased 0.8 to 4.0 cm-1 scattering suggests increased motions in this frequency range. Comparison with normal mode models of lysozyme dynamics shows that the inelastic difference occurs in the frequency region predicted for the lowest frequency, largest amplitude, global modes of the molecular [M. Levitt, C. Sander and P.S. Stern, J. Mol. Biol. 181, 423 (1985). B. Brooks and M. Karplus, Proc. Natl. Acad. Sci (U.S.A) 82, 4995 (1985), R.E. Bruccoleri, M. Karplus and J.A. McCammon, Biopolymers 25 1767 (1986)]. Our results are consistent with a model in which an increased number of low frequency global modes are present in the higher hydrated sample.