L-乳酸脱氢酶热变性的热力学研究

用差示扫描量热法对L-乳酸脱氢酶的热变性进行了研究(温度扫描范围为290—390K,酶蛋白溶液浓度为0.28—0.72mg蛋白/mg溶液)。实验观察到当酶溶液浓度在0.62—0.72mg蛋白/mg溶液范围内有一个吸热转变,酶溶液浓度小于0.62mg蛋白/mg溶液时有两个未完全分开的吸热转变。 这个酶的量热焓与范德霍夫焓的比远大于1,而接近于2,这表明乳酸脱氢酶的变性过程不是一个简单的两态转变,从热力学和吸热峰的形状、大小分析,可以推断乳酸脱氢酶分子是由两个以弱相互作用相连结的合作结构区组成,而每一个结构区是由两个相互作用很强的亚基组成。也就是说乳酸脱氨酶的变性过程包括两个半独立的合作结构区的转变,每一个结构区的转变都近似一个两态转变,ΔHeal与ΔHvh的比值是随着两个半独立部分相互作用的增强,即蛋白浓度的增加而减小。随着蛋白浓度的减小,蛋白质周围水分子增多,酶分子中两个半独立部分的相对独立性增强,这可由热谱图上一个吸热转变变成两个半独立的转变得到证实。     

天冬氨酸酶在盐酸胍变性时的酶活性与构象变化

 本文应用荧光光谱法和CD光谱法测定了天冬氨酸酶在不同浓度盐酸胍中变性时的构象与活力变化,并测定了天冬氨酸酶在不同浓度盐酸胍中变性时的巯基暴露速度。发现一部分色氨酸残基位于分子疏水核内部,另一部分位于分子表面;至少一部分酪氨酸残基与其相邻近基团形成氢键。该酶的大部分巯基位于分子内部结构比较稳定的区域而不在分子表面。低浓度盐酸胍作用下,构象发生明显变化,而活力维持原水平;盐酸胍达到一定浓度后,活力才发生骤然下降。CD谱表明,α-螺旋构象维持整个分子构象,因而对于维持活性中心构象是重要的。     

豆壳过氧化物酶的盐酸胍变性与化学修饰研究

研究了盐酸胍对豆壳过氧化物酶(soybeanhullperoxidase,SHP,EC1.11.1.7)构象与活力的影响,发现去辅基SHP的盐酸胍变(复)性及荧光变化关系与SHP全酶分子的盐酸胍变(复)性及荧光变化关系明显不同。应用过碘酸氧化法去除SHP分子表面糖链,研究糖链去除对酶性质的影响,则证实了SHP分子表面的糖链去除导致酶热稳定性下降。应用不同的蛋白质侧链修饰剂对SHP进行化学修饰则表明,巯基、酪氨酸和色氨酸残基为酶活力非必需,而羧基、组氨酸和精氨酸残基为酶活力所必需。     

酵母乙醇脱氢酶胍变性时的失活与去折叠的比较研究

应用荧光发射光谱,圆二色光谱,二阶导数光谱和紫外差吸收光谱等监测手段,研究了酵母乙醇脱氢酶在胍溶液中的去折叠。比较不同盐酸胍浓度下酵母乙醇脱氢酶的失活与构象变化,实验表明酶的失活先于构象变化：在低浓度胍溶液中,构象尚未发生明显变化时,酶活几乎已经完全丧失。由上述结果可见,含有辅基金属离子Ｚｎ~（２＋）酶的活性部位较酶分子的整体结构也具有柔性。     

Effects of proanthocyanidins on porcine pancreatic lipase: Conformation,activity, kinetics and thermodynamics

The interactions between proanthocyanidins (PC) and porcine pancreatic lipase (PL) were investigated from variant aspects of lipase conformation, activity, kinetics, and thermodynamics. Results show that 34% inhibitory rate of PC on PL is achieved after about 30-min incubation, and the inhibitory rate increases with the increase of PC concentration and then plateaus at the PC/PL ratio of 200. Circular dichroism and fluorescence spectroscopic analyses demonstrate that PC decreases the α-helix content while increases the β-sheet content of PL, but does not change the microenvironment of Trp, and PC quenches the fluorescence of PL both dynamically and statically through the formation of PL–PC complex. PC induces PL aggregation and then stabilizes the lipase aggregates. Kinetic studies reveal that PC does not change the K_m value while decreases the V_max value, implying that PC non-competitively inhibits the PL activity.     

Cationic effect of imidazolium-based ionic liquid on the stability of myoglobin

The conformational change of myoglobin (Mb) during guanidine hydrochloride (GuHCl)-induced protein unfolding in the presence of various ionic liquids (ILs) in phosphate buffer was investigated using both the Soret band absorption and the fluorescence of tryptophan measurements. The GuHCl-induced denaturation midpoints of Mb derived from the absorption and fluorescence spectra were almost similar in the presence of 150 mM ILs with the same cation 1-butyl-3-methylimidazolium (Bmim⁺) but different anions (BF₄⁻, NO₃⁻, Cl⁻, and Br⁻) in phosphate buffer. In addition, the denaturation midpoints of Mb in the presence of ILs were little lower than those in the absence of ILs in phosphate buffer. For the sake of clarity and comparison, we also measured the GuHCl-induced denaturation midpoints of Mb in the presence of 150 mM sodium salts with different anions (BF₄⁻, NO₃⁻, Cl⁻, and Br⁻) in phosphate buffer and found that their corresponding denaturation midpoints of Mb were almost similar to those observed in the absence of sodium salts in phosphate buffer. These experimental data indicate that Bmim⁺ cation can promote the unfolding of Mb. Further experiments revealed that the denaturation ability of ILs increases with increasing alkyl chain length of imidazolium cation of ILs and that hydroxyl-substituted imidazolium cation could also promote the unfolding of Mb.     

Denaturation process of laccase in various media by refractive index measurements

In this work, we are interested in the denaturation process of a laccase from Tramates versicolor via the determination of the refractive index, the refractive index increment and the specific volume in various media. The measurements were carried out using an Abbe refractometer. We have shown that the refractive index increment values obtained from the slope of the variation of the refractive index vs. Concentration are outside the range refractive index increments of proteins. To correct the results, we have followed the theoretical predictions based on the knowledge of the protein refractive index from its amino acids composition. The denaturation process was studied by calculating the specific volume variation where its determination was related to the Gladstone-Dale and the Lorentz-Lorentz models.     

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。     

Distribution and biotransformation of arsenic species in chicken cardiac and muscle tissues

This study evaluated the bioaccumulation and biotransformation of arsenic species in chicken heart and meat tissues. The experimental study was carried out using two sets of samples. In the first one, 10-d-old chickens were exposed to sodium arsenate, using spiked drinking water. These chickens grew normally and were killed after 50 d of arsenic exposure. The second set were edible chickens used as blanks for a parallel study. The total arsenic and arsenic species content in the exposed samples were at least twice those in the normal edible chicken. It has been demonstrated that sodium arsenate is biotransformed to arsenite and an unknown species and its distribution varies among the different cardiac and meat tissues. One important aspect is the capability of the auricle to preconcentrate the most toxic species, arsenite, in the exposed chicken. A nonidentified arsenic species from the edible chicken was detected. Arsenobetaine was also detected in several tissues. This article shows that chicken can be used as a representative animal when considering inorganic arsenic exposure in humans.     

菓菠萝蛋白酶的分子构象与活力变化的研究

发现CBZ-Lys·pNP能有效地被菓菠萝蛋白酶(Fruit Bromelain E.C.3.4.22.5)作用,测得Km为4.167×10~(-4)mol/L,k_(cat)为742min~(-1)。以荧光和紫外差示光谱为监测手段,对酶分子构象变化进行研究。酶的荧光强度随胍浓度增大而逐渐下降,4mol/L胍变性时,发射峰自332nm红移到353nm,并在310nm处出现新的发射峰。酶的荧光强度都因SDS存在而下降,SDS浓度大于3.47mmol/L有所回升,并出现红移,同时在315nm处出现新的发射肩;紫外差示光谱显示在236nm有一个较显著的员峰,此峰与β-螺旋结构变化有关,278、286和295nm出现三个负峰,260nm有较小正峰,说明酶分子中Tyr、Trp和Phe的微环境发生了明显的变化。测定酶在不同浓度胍和SDS中的变性和失活速度常数,对酶构象变化及催化活力的关系作了比较研究,酶的失活速度均大于变性速度。     

底物对变性酶的修复作用

 兎肌肌酸激酶被LDS变性后,底物能够诱导变性酶使其活力和构象得到部分恢复。变性程度不同的酶,构象和活力的恢复程度也不同:低浓度LDS变性酶,恢复程度较高;反之亦然。活力的恢复与构象的恢复两者呈对应关系。底物修复作用的pH以8.2为好。底物修复作用受其它蛋白质(例如BSA)存在的影响。等速电泳结果表明,BSA能竞争性结合LDS-酶复合物的LDS,使酶成为游离酶。变性酶先与BSA保温再加底物所得的活力恢复,大约是变性酶与含BSA的底物保温所得活力的10倍。这一结果似表明LDS变性酶仍能结合底物;被结合的底物还能使变性酶的构象发生变化。     

Stability of Collagen During Denaturation

The stability of calf skin collagen (CSC) type I during thermal and chemical denaturation in the presence of glycerol was investigated. Thermal denaturation of type I collagen was performed in the presence of glycerol or in combination with urea and sodium chloride. The denaturation curves obtained in the presence of urea or sodium chloride retained their original shape without glycerol. These curves were shifted upward proportionally to the glycerol concentration in the reaction medium. This means that glycerol and the denaturants act independently. The explanation is based on the difference in the mechanism of their action on the collagen molecule.     

Binding thermodynamics of substituted diaminopyrimidine renin inhibitors

Renin is an aspartyl protease involved in the production of angiotensin II, a potent vasoconstrictor. Renin inhibitors can prevent blood vessel constriction and therefore could be useful for the treatment of hypertension. High-throughput screening efforts identified a small molecule renin inhibitor with a core substituted diaminopyrimidine ring. Parallel medicinal chemistry efforts based on this lead resulted in compound 1. A complex of 1 bound to renin was crystallized, and structural data were obtained by X-ray diffraction. The structure indicated that there were adjacent unoccupied binding pockets. Synthetic efforts were initiated to extend functionality into these pockets so as to improve affinity and adjust pharmacokinetic parameters. Thermodynamics data for inhibitor binding to renin were also collected using isothermal titration calorimetry. These data were used to help guide inhibitor optimization by suggesting molecular alterations to improve binding affinity from both thermodynamic and structural perspectives. The addition of a methoxypropyl group extending into the S3 subpocket improved inhibitor affinity and resulted in greater binding enthalpy. Initial additions to the pyrimidine ring template that extended into the large hydrophobic S2 pocket did not improve affinity and dramatically altered the thermodynamic driving force for the binding interaction. Binding of the core template was enthalpically driven, whereas binding of initial inhibitors with S2 extensions was both enthalpically and entropically driven but lost significant binding enthalpy. Additional electrostatic interactions were then incorporated into the S2 extension to improve binding enthalpy while taking advantage of the favorable entropy.     

降胆固醇乳酸菌对肉鸡胆固醇的影响研究

目的探讨经体外实验筛选出的降胆固醇乳酸菌对肉鸡血清胆固醇及胸肌组织、腿肌组织和肝脏组织胆固醇含量的影响。方法将肉鸡随机分为2组,对照组喂食普通饲料,实验组喂食含有DM86056菌的饲料;喂食56d后分离其血清及肝脏等组织,并应用硫酸铁铵法测定其胆固醇含量,观察对照组与实验组结果差异是否有显著性。结果对肉鸡血清、胸肌组织、腿肌组织胆固醇含量测定后,对照组与实验组结果差异有显著性（P〈0．05）,而对肝脏组织胆固醇含量测定后,对照组与实验组结果差异无显著性（P〉0．1）。结论菌株DM86056可明显降低肉鸡血清、胸肌组织、腿肌组织中胆固醇含量,而对肝脏组织胆固醇含量没有明显改变。     

Effects of pressure on the structure of metmyoglobin: molecular dynamics predictions for pressure unfolding through a molten globule intermediate.

We investigated the pathway for pressure unfolding of metmyoglobin using molecular dynamics (MD) for a range of pressures (0.1 MPa to 1.2 GPa) and a temperature of 300 K. We find that the unfolding of metmyoglobin proceeds via a two-step mechanism native --> molten globule intermediate --> unfolded, where the molten globule forms at 700 MPa. The simulation describes qualitatively the experimental behavior of metmyoglobin under pressure. We find that unfolding of the alpha-helices follows the sequence of migrating hydrogen bonds (i,i + 4) --> (i,i + 2).     

Immunomorphologic characterization of chicken thrombocytes

Summary Chicken thrombocytes were enriched for immunization by utilizing their strong capacity to adhere to plastic surfaces. The produced rabbit anti-thrombocyte serum ATS 3 reacted by means of the unlabeled antibody enzyme method (PAP) specifically with thrombocytes of fixed chicken-blood smears, but not with lymphocytes or other blood cells. When ATS 3 (substrate diaminobencidinetetrahydrochloride = DAB) and a 11 mixture of an anti-bursa serum and anti-thymus serum (ABS/ATS; substrate 4-chloro-1-naphthol = 4-Cl-1-N) were used simultaneously, thrombocytes revealed the brown color typical for DAB, whereas lymphocytes showed the blue stain of 4-Cl-1-N. The finding of a thrombocyte surface antigen not shared by lymphocytes is regarded as a further proof of the diversity of both cell systems, i.e., for the existence of a genuine thrombocyte system in chickens.     

Antioxidative effect of melatonin on cryopreserved chicken semen

This is a unique study because is the first time we are adding melatonin into an extender in order to determine its influence on cryopreserved chicken semen. The primary focus of our present study was to evaluate the influence of different concentrations of Melatonin on cryopreserved chicken semen. Semen samples were allocated into four treatments, being one control and three different combinations of antioxidants and after the freeze-thaw operation, the sperm motility, plasma membrane integrity, acrosome integrity, endogenous enzymes (GSH-Px, CAT, SOD), MDA and ROS of chicken spermatozoa were all evaluated. The collection of the semen samples was from 40 Arbor Acre roosters and this procedure was repeated twice a week and then mixed in an extender that contained different MEL treatments as follows: a diluent without MEL (control, M 0), a diluent comprising 0.125 mg/mL (M 0.125) 0.25 mg/mL, (M 0.25) and 0.5 mg/mL (M 0.5). It was revealed that the supplementation of the base extender with an optimal 0.25 mg/mL MEL led to a higher significant difference in the motility of chicken sperm (P < 0.01), higher acrosome integrity (P < 0.05) and a higher plasma membrane integrity (P < 0.01) when compared to the control group at post-thaw. Furthermore, when compared to the control group, 0.25 mg/mL MEL addition into the extender significantly enhanced the activity of endogenous enzymes (GSH-Px, CAT, and SOD) in the chicken spermatozoa at post-thaw (P < 0.05). Moreover, 0.5 mg/mL MEL supplementation into the extender enhanced the GSH-Px activity in the chicken spermatozoa when compared with the control group (P < 0.05) at post-thaw. In contrast, the addition of 0.25 mg/mL MEL into the extender resulted in a significantly lower MDA in comparison to the 0.125 mg/mL, 0.5 mg/mL MEL treatment group and the control group (P < 0.05). Also, compared to the control group, MEL concentration of 0.125 mg/mL and 0.5 mg/mL MEL into the extender resulted in a significantly low ROS concentration (P < 0.05) but the addition of 0.25 mg/mL MEL concentration resulted in a significantly lower ROS level when compared to the control group (P < 0.01). In summary, MEL improved the quality of cryopreserved chicken sperm quality by decreasing oxidative stress level and the most optimal concentration was 0.25 mg/mL.     

Heat-induced Irreversible Denaturation of the Camelid Single Domain VHH Antibody Is Governed by Chemical Modifications

The variable domain of camelid heavy chain antibody (VHH) is highly heat-resistant and is therefore ideal for many applications. Although understanding the process of heat-induced irreversible denaturation is essential to improve the efficacy of VHH, its inactivation mechanism remains unclear. Here, we showed that chemical modifications predominantly governed the irreversible denaturation of VHH at high temperatures. After heat treatment, the activity of VHH was dependent only on the incubation time at 90 °C and was insensitive to the number of heating (90 °C)-cooling (20 °C) cycles, indicating a negligible role for folding/unfolding intermediates on permanent denaturation. The residual activity was independent of concentration; therefore, VHH lost its activity in a unimolecular manner, not by aggregation. A VHH mutant lacking Asn, which is susceptible to chemical modifications, had significantly higher heat resistance than did the wild-type protein, indicating the importance of chemical modifications to VHH denaturation.     

Purification and isoelectric heterogeneity of chicken tyrosinase

Tyrosinase (EC 1.14.18.1) was purified from regenerating chicken feathers. Most of the enzyme activity was in the insoluble fraction, which was solubilized with 0.5% sodium cholate. Solubilized tyrosinase showed multiple forms on isoelectric focusing. The isoelectric points had the following pI values: 5.06, 4.83, 4.68, 4.56, 4.44, 4.32, 4.24, 4.14, 4.06 and 3.97. This tyrosinase fraction was subjected to trypsin (EC 3.4.21.4) cleavage, Sephacryl S-200, hydroxylapatite and DEAE-cellulose chromatography. Purified enzymatically active tyrosinase also showed multiple forms. Their isoelectric points were: 4.23, 4.14, 4.06, 3.99 and 3.91. Each active form had almost the same molecular weight, estimated at 66 000. Staining for 1,2-diol groups of glycoproteins and neuraminidase (EC 3.2.1.18) treatment suggested that chicken tyrosinase is a glycoprotein. The enzyme showed both dopa(L-3,4-dihydroxylphenylalanine) oxidase activity and tyrosine hydroxylase activity.