无花果蛋白酶在胍溶液中的分子折叠与活力变化研究

本文研究无花果蛋白酶（ＥＣ．３．４．４．１２）在不同浓度盐酸胍溶液中分子构象与活力变化关系。酶的内源荧光光谱,圆二色光谱与酶活力的变化表明：荧光光谱呈现二个明显的变化区域,低浓度胍（低于２ｍｏｌ／Ｌ）中,荧光发射峰基本不变,但荧光强度随胍浓度上升,随胍浓度断续增大（高于２ｍｏｌ／Ｌ）,酶的最大发射波长明显红移。当胍浓度低于１ｍｏｌ／Ｌ时,不仅不会使酶失活,反而使酶激活,当胍浓度高于１ｍｏｌ／Ｌ以上时,酶逐渐失活,使酶完全失活的胍浓度为６ｍｏｌ／Ｌ酶的圆二色光谱也随着胍浓度的改变而发生复杂的变化。将荧光变化,ＣＤ谱变化及活力改变结合起来,表明活力的激活与构象的明显变化似是同步发生的,从另一角度进一步说明酶活性部位柔性是充分表现酶活力所必需。     

无花果蛋白酶在胍溶液中的分子折叠与活力变化研究

本文研究无花果蛋白酶（ＥＣ．３．４．４．１２）在不同浓度盐酸胍溶液中分子构象与活力变化关系。酶的内源荧光光谱,圆二色光谱与酶活力的变化表明：荧光光谱呈现二个明显的变化区域,低浓度胍（低于２ｍｏｌ／Ｌ）中,荧光发射峰基本不变,但荧光强度随胍浓度上升,随胍浓度断续增大（高于２ｍｏｌ／Ｌ）,酶的最大发射波长明显红移。当胍浓度低于１ｍｏｌ／Ｌ时,不仅不会使酶失活,反而使酶激活,当胍浓度高于１ｍｏｌ／Ｌ以上     

超氧化物歧化酶全酶和脱辅基酶胍变性时失活与去折叠的比较研究

利用紫外差吸收光谱和荧光发射光谱等监测手段研究天然铜锌ＳＯＤ（ｈｏｌｏ－ＳＯＤ）和脱铜锌ＳＯＤ（ａｐｏ－ＳＯＤ）在不同浓度胍溶液中的去折叠及活力变化．结果表明ｈｏｌｏ－ＳＯＤ和ａｐｏ－ＳＯＤ分别在４．０和２．０ｍｏｌ／Ｌ胍溶液中去折叠,而分别在２．０和０．５ｍｏｌ／Ｌ胍溶液中其构象尚未发生明显改变时活性几乎完全丧失．提示金属离子对维持酶的整体及活性部位构象具有重要作用,脱去金属离子的酶分子的构象特别是活性部位的构象更易受到变性剂的破坏．     

钙离子对辣根过氧化物酶同工酶C的溶液构象的影响

运用荧光光谱方法研究了脱辅基的辣根过氧化物酶同工酶Ｃ。结果表明：（１）ａｐｏ－ＨＲＰ（Ｃ）与其他“Ｂ”类蛋白质不同,有明显的Ｔｙｒ荧光;（２）低浓度变性剂（〈２ｍｏｌ／Ｌ脲或０．２ｍｏｌ／Ｌ盐酸胍）能增强酶的Ｔｒｐ荧光,但并不改变光谱特性;（３）进一步增加变性剂浓度则使Ｔｒｐ残基暴露于水溶液中,荧光强度略有降低,荧光谱红移;（４）Ｃａ＾２＋络合剂ＥＤＴＡ对色氨酸荧光的影响与低浓度变性剂相同。有意思     

超氧化物歧化酶全酶和脱辅基酶胍变性时失活与去折叠的比较研究

利用紫外差吸收光谱和荧光发射光谱等监测手段研究天然铜锌ＳＯＤ和脱铜锌ＳＯＤ在不同浓度胍溶液中的去折叠及活力变化。结果表明ｈｏｌｏ－ＳＯＤ和ａｐｏ－ＳＯＤ分别在４．０和２．０ｍｏｌ／Ｌ胍溶液中去折叠,而分别在２．０和０．５ｍｏｌ／Ｌ胍溶液中其构象尚未发生明显改变时活性几乎完全丧失。提示金属离子对维持酶的整体及活性部位构象具有重要作用,脱去金属离子的酶分子的构象特别是活性部位的构象更易受到变性剂的破坏     

钙调神经磷酸酶在胍变性过程中活力及构象变化的比较

钙调神经磷酸酶（ＣａＮ）在盐酸胍溶液中的内源荧光、远紫外ＣＤ谱及剩余活力的变化提示：ＣａＮ的酶活力在胍浓度为０．５ｍｏｌ／Ｌ左右可完全丧失,同时伴有内源荧光强度的下降,３３３ｎｍ最大发射峰的红移（提示了色氨酸和酪氨酸残基的暴露）。比较不同胍浓度下牛脑ＣａＮ的失活与整体构象变化,表明酶的失活先于整体构象变化。在０．６ｍｏｌ／Ｌ胍溶液中,内源荧光变化的动力学过程只能测出一相,而酶失活的动力学过程为快、慢两相,快相动力学速度常数比整体构象变化速度常数大１－２个数量级,慢相失活速度常数与整体构象变化速度常数相近。提示低浓度胍可引起该酶的完全失活,活性部位的空间构象比整个酶分子的构象更易受到变性剂的扰乱。     

岩豆凝集素的圆二色性与生物学活性关系的研究

岩豆凝集素（ＭＤＬ）的远紫外圆二色性谱（ＣＤ谱）显示２１６－２１７ｎｍ处的单一负峰。此时ＭＤＬ分子含有１６．２％的α螺旋,４６．３％的β折叠和３７．５％的无规卷曲。ｐＨ９．０时负峰红移至２２０ｎｍ,且在２１７－２２２ｎｍ处的峰值几乎相同;在２０－４０℃范围内,ＣＤ谱的变化甚微;６０℃时谱峰蓝移;在８０℃或１００℃时,２１２ｎｍ处出现一大负峰。１ｍｏｌ／Ｌ或２ｍｏｌ／Ｌ脲时,ＭＤＬ的ＣＤ谱已发生明显变化,二级结构单元也有变化,凝集兔红细胞的活性也随之减弱;随脲浓度的增加,ＭＤＬ的谱峰蓝移,最终在２１２ｎｍ处出现大负峰。当胍浓度为０．７５ｍｏｌ／Ｌ时,ＭＤＬ的ＣＤ谱即有明显变化和活性丧失;胍浓度继续增加,ＣＤ谱逐渐成为特征的无规卷曲的谱形。在ｐＨ９．０、温度超过８０℃、脲或胍浓度分别高于２ｍｏｌ／Ｌ和０．７５ｍｏｌ／Ｌ时,ＭＤＬ的ＣＤ谱发生显著变化的同时,其凝集兔红细胞的生物学活性全部丧失,分子的二级结构单元也发生很大改变。     

缢蛏碱性磷酸酶胍溶液中分子折叠与活力变化研究

报道了缢蛏碱性磷酸酶（简称ＡＬＰ）经不同浓度盐酸胍处理时酶的分子构象发生的变化以及酶变化和失活的动力学过程。在胍中酶荧光发谢峰强度下降,紫外差光谱在２４６ｎｍ和２８５ｎｍ处出现２个负峰,ＣＤ谱中酶的α螺旋度下降,且随浓度增大,变化程度也加大。动力学研究表明,酶在０．５ｍｏｌ／Ｌ、１．０ｍｏｌ／Ｌ、２．０ｍｏｌ／Ｌ、３．０ｍｏｌ／Ｌ、４．０ｍｏｌ／Ｌ盐酸胍中的变性速度常数分别为３．２１×１０＾－４ｓ     

重组水蛭素HV2的稳定性

重组水蛭素ＨＶ２是凝血酶的特异性抑制剂,是一种非常稳定的蛋白质,温度的升高（１００℃水浴）和ＰＨ（１－１３）的改变不影响其活力,在某些变性剂（８ｍｏｌ／Ｌ素、１％ＳＤＳ和６ｍｏｌ／Ｌ盐酸胍）存在的条件下也非常稳定,０．１ｍｏｌ／Ｌ的ＤＴＴ在７０℃时使其部分失活,只有ＰＨ和温度同时升高其活力才开始下降,ＰＨ１３、８０℃处理１５ｍｉｎ即完全失活,氨基酸组成和活性分析发现失活样品的Ｃｙｓ和Ｌｙｓ被破坏。     

缢蛏碱性磷酸酶胍溶液中分子折叠与活力变化研究

报道了缢蛏碱性磷酸酶（简称ＡＬＰ）经不同浓度盐酸胍处理时酶的分子构象所发生的变化以及酶变化和失活的动力学过程。在胍中酶荧光发射峰强度下降,紫外差光谱在２４６ｎｍ和２８５ｎｍ处出现２个负峰,ＣＤ谱中酶的α螺旋度下降,且随浓度增大,变化程度也加大。动力学研究表明,酶在０．５ｍｏｌ／Ｌ、１．０ｍｏｌ／Ｌ、２．０ｍｏｌ／Ｌ３．０ｍｏｌ／Ｌ、４．０ｍｏｌ／Ｌ盐酸胍中的变性速度常数分别为３．２１×１０~（－４）ｓ~（－１）、６．３８×１０~（－４）ｓ~（－１）、２．１７×１０~（－３）ｓ~（－１）、２．３３×１０~（－３）ｓ~９－１）、５．１７×１０~（－３）ｓ~（－１）;而酶在相应盐酸胍中的失活速度常数分别为２．３３×１０~（－４）ｓ~（－１）、３．５７×１０~（－４）ｓ~（－１）、５．８６×１０~（－４）ｓ~（－１）、１．１４×１０~（－３）ｓ~（－１）、３．４５×１０~（－３）ｓ~（－１）;表现为失活与构象伸展变化基本平行。     

溶剂极性及盐酸胍对碳酸酐酶构象的影响

以紫外荧光光谱为检测手段,比较研究了溶剂极性及盐酸胍对碳酸酐酶构象的影响,结果表明,当盐酸胍浓度小于１ｍｏｌ／Ｌ时酶呈天然构象,１．５ｍｏｌ／Ｌ－２．０ｍｏｌ／Ｌ的盐酸胍导致该酶呈伸展构象;而乙醇不以导致该酶产生稳定的中间态构象,１ｍｏｌ／Ｌ和３ｍｏｌ／Ｌ的甲醇以及３ｍｏｌ／Ｌ的乙醇对盐酸胍致碳酸酐酶中间态的影响不大,甲醇略使致中间态的盐酸胍浓度下降,乙醇略使之上升。     

邻苯二甲醛修饰法探测中华猕猴桃蛋白酶在胍溶液中活性部位的构象变化

Palczewsski等［1］以邻苯二甲醛修饰醛缩酶活性部位的氨基和流基以形成一异蚓噪环,利用该基团的荧光特性来探测醛缩酶的活性部位构象,Weq［2］,Le［3］并成功地运用这一方法研究肌酸激酶和酵母乙醇脱氢酶的活性部位构象变化．中华猕猴桃蛋白酶的唯一游离流基（CyS-25）是催化功能团【'」,而且氨基也是活性部位的必需基因【到,符合邻苯二甲醛的反应性,所以我们借鉴Pal＿ski等的方法【1］将这一荧光基因引人中华猕猴桃蛋白酶,用以探测该酶在抓溶液中活性部位的构象变化,并与相应的活力变化以及酶的内源荧光及CD谱变化作比较．1材…     

Sequential conformation change and activation of chicken liver dihydrofolate reductase in low concentration of guanidine hydrochloride

The conformation changes of dihydrofolate reductase (DHFR) from chicken liver in guanidine hy-drochloride were monitored by protein intrinsic fluorescence, hydrophobic fluorescence probe TNS and limited proteol-ysis by proteinase K. The kinetics of the enzyme denaturation were also studied and compared with its activity changes. It was indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene (TNS) that a subtle conforma-tional change of the enzyme in dilute GuHCl parallels GuHCl-induced activation. At GuHCl concentration higher than 0.75 mol/L, the conformational change can be detected by increased susceptibility of the enzyme to proteinase K, but no significant gross conformational change of the enzyme molecule is observed by intrinsic fluorescence up to a GuHCl concentration of 1.2 mol/L. The results suggest that the denaturation of DHFR by GuHCl does not follow strictly the two-state model. The enzyme seems to open up sequentially with increasing concentrations of denaturants, mainly at th     

Inactivation Kinetics of Guanidinium Chloride on <Emphasis Type="Italic">Penaeus vannamei</Emphasis>β - <Emphasis Type="Italic">N</Emphasis>-Acetyl-D-Glucosaminidase and the Relationship of Enzyme Activity and its Conformation

The effects of guanidinium chloride (GuHCl) on the activity of Penaeus vannamei β-N-acetyl-d-glucosaminidase (NAGase) have been studied. The results show that GuHCl, at appropriate concentrations, can lead to reversible inactivation of the enzyme, and the IC₅₀ is estimated to be 0.6 M. Changes of activity and conformation of the enzyme in different concentrations of GuHCl have been studied by measuring the fluorescence spectra and its relative activity after denaturation. The fluorescence intensity of the enzyme decreases distinctly with increasing GuHCl concentrations, and the emission peaks appear red-shifted (from 339.4 to 360 nm). Changes in the conformation and catalytic activity of the enzyme are compared. The extent of inactivation is greater than that of conformational changes, indicating that the active site of the enzyme is more flexible than the whole enzyme molecule. The kinetics of inactivation has been studied using the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The value of k₊₀ is larger than that of k₊₀^′ which suggests that the enzyme is protected by substrate to a certain extent during guanidine denaturation.     

氨基酰化酶在LDS溶液中的失活与去折叠的比较研究

氨基酰化酶在阴离子去圬剂十二烷基硫酸锂（ＬＤＳ）溶液中的失活与去折叠的研究结果表明,在低浓度的ＬＤＳ溶液（０．６ｍｍｏｌ／Ｌ）中变性时,以荧光和紫外差吸收方法监测的酶分子构象尚未发生明显变化。而酶的活力已经大部分或几乎全部丧失。当ＬＤＳ浓度达１．６ｍｍｏｌ／Ｌ时,此时酶分子的构象变化才达到最大程度,在实验使用的ＬＤＳ的浓度范围内,用远紫外ＣＤ光谱监测的二级结构没有发生明显的变化。从上述研究结果,可以认为含锌氨基酰化酶的活性部位也具有相对的柔性。     

Intermediate studies on refolding of arginine kinase denatured by guanidine hydrochloride.

The refolding course and intermediate of guanidine hydrochloride (GuHCl)-denatured arginine kinase (AK) were studied in terms of enzymatic activity, intrinsic fluorescence, 1-anilino-8-naphthalenesulfonte (ANS) fluorescence, and far-UV circular dichroism (CD). During AK refolding, the fluorescence intensity increased with a significantly blue shift of the emission maximum. The molar ellipticity of CD increased to close to that of native AK, as compared with the fully unfolded AK. In the AK refolding process, 2 refolding intermediates were observed at the concentration ranges of 0.8-1.0 mol/L and 0.3-0.5 mol GuHCl/L. The peak position of the fluorescence emission and the secondary structure of these conformation states remained roughly unchanged. The tryptophan fluorescence intensity increased a little. However, the ANS fluorescence intensity significantly increased, as compared with both the native and the fully unfolded states. The first refolding intermediate at the range of 0.8-1.0 mol GuHCl/L concentration represented a typical "pre-molten globule state structure" with inactivity. The second one, at the range of 0.3-0.5 mol GuHCl/L concentration, shared many structural characteristics of native AK, including its secondary and tertiary structure, and regained its catalytic function, although its activity was lower than that of native AK. The present results suggest that during the refolding of GuHCl-denatured AK there are at least 2 refolding intermediates; as well, the results provide direct evidence for the hierarchical mechanism of protein folding.     

糖基化3－磷酸甘油醛脱氢酶的含糖量及其构象变化

通过对ＧＡＰＤＨ及ｇＧＡＰＤＨ含糖量、ＣＤ、荧光及ＤＴＮＢ的修饰表明：用间氨基苯硼酸琼脂糖（ｍ－ＡＰＢＡ－ＳｅｐｈａｒｏｓｅＣＬ６Ｂ）亲和层析法分离的兔肌ｇＧＡＰＤＨ每分子含有１．８９个糖基。ｇＧＡＰＤＨ及ＧＡＰＤＨ的远紫外ＣＤ谱差别较小,但近紫外差别较明显。两者内源荧光在不同浓度的ＧｕＨＣｌ溶液中的变化亦有一定差异。ＤＴＮＢ对酶活性部位巯基的修饰表明,ｇＧＡＰＤＨ的ＤＴＮＢ修饰的快相一级动力学常数大于ＧＡＰＤＨ动力学常数一个数量级。以上结果提示：糖基化导致酶分子及活性部位的空间结构改变,糖基化位点可能发生在酶活性部位附近。     

糖基化3－磷酸甘油醛脱氢酶的含糖量及其构象变化

通过对ＧＡＰＤＨ及ｇＧＡＰＤＨ含糖量、ＣＤ、荧光及ＤＴＮＢ的修饰表明：用间氨基苯硼酸琼脂糖（ｍ－ＡＰＢＡ－ＳｅｐｈａｒｏｓｅＣＬ６Ｂ）亲和层析法分离的兔肌ｇＧＡＰＤＨ每分子含有１．８９个糖基。ｇＧＡＰＤＨ及ＧＡＰＤＨ的远紫外ＣＤ谱差别较小,但近紫外差别较明显。两者内源荧光在不同浓度的ＧｕＨＣｌ溶液中的变化亦有一定差异。ＤＴＮＢ对酶活性部位巯基的修饰表明,ｇＧＡＰＤＨ的ＤＴＮＢ修饰的快相一级动力学常数大于ＧＡＰＤＨ动力学常数一个数量级。以上结果提示：糖基化导致酶分子及活性部位的空间结构改变,糖基化位点可能发生在酶活性部位附近。     

Unfolding and inactivation of Ampullarium crossean beta-glucosidase during denaturation by guanidine hydrochloride

Changes of activity and conformation of Ampullarium crossean beta-glucosidase in different concentrations of guanidine hydrochloride (GuHCl) have been studied by measuring the fluorescence spectra and its relative activity after denaturation. The fluorescence intensity of the enzyme decreased distinctly with increasing guanidine concentrations, the emission peaks appeared red shifted (from 338.4 to 350.8 nm), whereas a new fluorescence emission peak appeared near 310 nm. Changes in the conformation and catalytic activity of the enzyme were compared. A corresponding rapid decrease in catalytic activity of the enzyme was also observed. The extent of inactivation was greater than that of conformational changes, indicating that the active site of the enzyme is more flexible than the whole enzyme molecule. k(+0)>k(+0)' also showed that the enzyme was protected by substrate to a certain extent during guanidine denaturation.