聚乙二醇修饰的单克隆抗体的某些生物活性

 不久前我们报道了PEG-1900修饰的McAb的某些理化性质,本文主要报道经PEG-1900修饰的McAb在某些生物活性方面的变化。与天然的McAb相比,修饰的McAb有以下的变化:(1)抗原性与免疫原性下降;(2)抗体活性下降;(3)在体内存活的时间延长;(4)对温度及pH的抵抗力增强。修饰的酶与天然的酶相比,酶活性有所下降,但下降的程度要比修饰的McAb小得多。     

双链聚乙二醇与超氧化物歧化酶共价结合物的理化和免疫学性质

用三聚氯氰活化单甲氧基聚乙二醇,得到了双链聚乙二醇(PEGm₂).以PEGm₂修饰牛血Cu,Zn-SOD,得到了氨基修饰率为30％,残余活力为80％的纯PEGm₂-SOD.修饰酶的荧光光谱及圆二色谱均有改变;盐酸胍变性及胃蛋白酶水解实验结果表明,修饰酶的稳定性高于天然酶;免疫学实验表明,与天然酶比较,修饰酶的免疫原性及抗原抗体反应性大为降低.     

血清乳酸脱氢酶同功酶的分离及测定

乳酸脱氢酶(LDH)是葡萄糖酵解过程中相当关键的酶,其主要作用在于催化丙酮酸与乳酸相互转换的化学反应。实验证明,LDH实际上包含着几种具有同样催化效能的蛋白质,但它们的分子组成、理化性质与免疫学特性却有明显差异,这些蛋白质统称为LDH同功酶。用电泳方法从血液中可分离出的LDH同功酶有:泳动速度最快的LDH-1,其次为LDH-2,LDH-3,LDH-4,和泳动速度最慢的LDH-5。     

聚乙二醇对菠萝蛋白酶的化学修饰

方法:用琥珀酸酐法活化的聚乙二醇对菠萝蛋白酶进行化学修饰,得到菠萝蛋白酶的修饰酶,对比研究三种菠萝蛋白酶:修饰酶、混合酶、天然酶的热稳定性及酸碱稳定性,考察金属离子对三种菠萝蛋白酶的影响。结果:当在55℃水浴保温100min后天然酶活力只保留20%,混合酶活力保留37%,修饰酶活力保留58%;在pH3.0-4.5及pH6.0-7.0的条件下,修饰酶活力高于天然酶活力。当Ca2 的浓度达到0.05mg/mL时,修饰酶的活力高达257.66%;当Mg2 的浓度达到0.035mg/mL时,修饰酶的活力高达147.25%。一价离子Na 对三种菠萝蛋白酶无明显影响。结论:修饰的菠萝蛋白酶对温度和pH值的稳定性均比天然酶有很大程度的提高。混合酶的活力介于天然酶和修饰酶之间说明聚乙二醇对菠萝蛋白酶有一定的保护作用。二价离子Ca2 、Mg2 对三种菠萝蛋白酶活力均有不同程度的激活作用。     

六种鼠组织中过氧化物酶和酯酶变异的比较

本文研究姬鼠属（Ａｐｏｄｅｍｕｓ）２种鼠和鼠属（Ｒａｔｕｓ）４种鼠的心组织过氧化物酶和肝组织酯酶同工酶等位基因的变异。结果表明,６种鼠心组织过氧化物酶皆为４条区带,其中社鼠和白腹巨鼠的酶带泳动速度略快于褐家鼠和黄毛鼠的酶带;鼠属４种鼠的酶带间距大致相同,而姬鼠属的酶带间距略大于鼠属,这种谱型的区别就所分析的６种鼠而言,可以认为是该同工酶表现的属间区别。肝组织酯酶区带较多,其酶带数和泳动速度的差异具有明显的种间特征;泳动最快的酶带在姬鼠中是２条,在鼠属中是１条,可以认为是属间特征的区别。从９项生化指标的异同作动物配对比较,表明褐家鼠与黄毛鼠亲缘上较近,社鼠与白腹巨鼠亲缘上也较近,而且白腹巨鼠在进化上可能要比其余３种鼠属动物更接近于姬鼠属     

山莨若碱通过膜脂影响兔肾(Na~++K~+)-ATP酶的构象及活性

研究了山莨菪碱对处于不同脂双层的兔肾外髓质(Na~++K~+)-ATP酶活性的影响,结果表明山莨菪碱对(Na~++K~+)-ATP酶的抑制作用与该酶所处的脂环境密切相关,如对去脂后的酶活性无明显影响,而对重组于酸性磷脂脂质体的酶比对重组于中性磷脂脂质体的酶有更大的抑制作用。园二色性实验表明,山莨菪碱使带349个界面脂分子的(Na~++K~+)-ATP酶二级结构发生明显变化,而对带189个界面脂分子的酶无明显作用。另外利用差示量热扫描研究表明山莨菪碱对酸性磷脂和中性磷脂脂质体或脂酶体相变行为有不同的影响。     

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。     

牛血清白蛋白对超氧化物歧化酶的化学修饰

目的：通过化学修饰提高超氧化歧化酶（SOD）的稳定性,考察金属离子在不同浓度下对SOD活性的影响。方法：用戊二醛作为交联剂,用牛血清白蛋白（BSA）将牛红细胞超氧化物歧化酶进行化学修饰,得到SOD的修饰酶。对比研究三种SOD：修饰酶,混合酶及天然酶的理化性质。结果：修饰酶等电点降低,对温度、pH的稳定性较天然酶有很大提高,对胰蛋白酶和胃蛋白酶也表现出很强的耐水解性。二价离子Mg^2 、Mn^2 对天种SOD活力均有不同程度的抵制作用,Ca^2 、Zn^2 、Cu^2 对修饰酶活力有激活作用,一价离子K^ 对三种OSD活力均无明显影响.结论:修饰酶较天然酶的稳定性有很大的提高,加入Ca^2 、Zn^2 、Cu^2 可提高修饰酶的活力。     

山莨若碱通过膜脂影响兔肾(Na~++K~+)-ATP酶的构象及活性

研究了山莨菪碱对处于不同脂双层的兔肾外髓质(Na~++K~+)-ATP酶活性的影响,结果表明山莨菪碱对(Na~++K~+)-ATP酶的抑制作用与该酶所处的脂环境密切相关,如对去脂后的酶活性无明显影响,而对重组于酸性磷脂脂质体的酶比对重组于中性磷脂脂质体的酶有更大的抑制作用。园二色性实验表明,山莨菪碱使带349个界面脂分子的(Na~++K~+)-ATP酶二级结构发生明显变化,而对带189个界面脂分子的酶无明显作用。另外利用差示量热扫描研究表明山莨菪碱对酸性磷脂和中性磷脂脂质体或脂酶体相变行为有不同的影响。     

溶液介电常数对铜锌超氧化物歧化酶活性的影响

溶液介电常数对天然酶和修饰酶的活性影响不同,天然酶随介电常数增加而酶活性下降,修饰酶则反之,这表明静电相互作用在铜锌超氧化物歧化酶(Cu·Zn-SOD)与超氧阴离子(-O_2~(·-))反应过程中起着重要作用,酶分子活性中心附近ε-NH_3~+为O_2~(·-)进入活性中心提供静电吸引力。在有机溶剂中,SOD的构象会发生变化,从而导致酶活性降低。实验还表明,Cl~-对SOD有明显的抑制作用。     

Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase.

Methoxypolyethylene glycols of 1900 daltons (PEG-1900) or 5000 daltons (PEG-5000) were covalently attached to bovine liver catalase using 2,4,6-trichloro-s-triazine as the coupling agent. Rabbits were immunized by the intravenous and intramuscular routes with catalase modified by covalent attachment of PEG-1900 to 43% of the amino groups (PEG-1900-catalase). The intravenous antiserum did not yield detectable antibodies against PEG-1900-catalase or native catalase, as determined by Ouchterlony and complement fixation methods, whereas the intramuscular antiserum contained antibodies to both PEG-1900-catalase and catalase. PEG-1900 did not react with either antiserum. Catalase was prepared in which PEG-5000 was attached to 40% of the amino groups (PEG-5000-catalase). This catalase preparation did not react with either antiserum. PEG-1900-catalase retained 93% of its enzymatic activity; PEG-5000-catalase retained 95%. PEG-5000-catalase resisted digestion by trypsin, chymotrypsin, and a protease from Streptomyces griseus. PEG-1900-catalase and PEG-5000-catalase exhibited enhanced circulating lives in the blood of acatalasemic mice during repetitive intravenous injections. No evidence was seen of an immune response to injections of the modified enzymes. Mice injected repetitively with PEG-5000-catalase remained immune competent for unmodieied catalase, and no evidence of tissue or organ damage was seen.     

Kinetic approach to the interaction of sodium n-dodecyl sulphate with heme enzymes

The kinetics of interaction of sodium n-dodecyl sulphate (SDS) with catalase has been studied by absorbance and fluorescence changes. The results have been compared with circular dichroism spectra and activity measurements. The tertiary structure of catalase is modified by SDS in the monomeric and micellar form. The secondary structure of catalase is altered only in the presence of SDS micelles. On the other hand, neither spectroscopic properties nor activity of horseradish peroxidase change in the presence of SDS below micellar concentration. In the presence of SDS micelles, however, changes of secondary and tertiary structure of this protein are detected. The reason for relatively high stability of horseradish peroxidase in the presence of SDS is discussed.     

Elucidating the interaction of clofazimine with bovine liver catalase; a comprehensive spectroscopic and molecular docking approach

Nowadays, understanding of interface between protein and drugs has become an active research area of interest. These types of interactions provide structural guidelines in drug design with greater clinical efficacy. Thus, structural changes in catalase induced by clofazimine were monitored by various biophysical techniques including UV‐visible spectrometer, fluorescence spectroscopy, circular dichroism, and dynamic light scattering techniques. Increase in absorption spectra (UV‐visible spectrum) confers the complex formation between drug and protein. Fluorescence quenching with a binding constants of 2.47 × 10⁴ M⁻¹ revealed that clofazimine binds with protein. Using fluorescence resonance energy transfer, the distance (r ) between the protein (donor) and drug (acceptor) was found to be 2.89 nm. Negative Gibbs free energy change (ΔG °) revealed that binding process is spontaneous. In addition, an increase in α‐helicity was observed by far‐UV circular dichroism spectra by adding clofazimine to protein. Dynamic light scattering results indicate that topology of bovine liver catalase was slightly altered in the presence of clofazimine. Hydrophobic interactions are the main forces between clofazimine and catalase interaction as depicted by molecular docking studies. Apart from hydrophobic interactions, some hydrogen bonding was also observed during docking method. The results obtained from the present study may establish abundant in optimizing the properties of ligand‐protein mixtures relevant for numerous formulations.     

Characterization of the coral allene oxide synthase active site with UV-visible absorption, magnetic circular dichroism, and electron paramagnetic resonance spectroscopy: evidence for tyrosinate ligation to the ferric enzyme heme iron

Coral allene oxide synthase (AOS), a hemoprotein with weak sequence homology to catalase, is the N-terminal domain of a naturally occurring fusion protein with an 8R-lipoxygenase. AOS converts 8R-hydroperoxyeicosatetraenoic acid to the corresponding allene oxide. The UV--visible absorption and magnetic circular dichroism spectra of ferric AOS and of its cyanide and azide complexes, and the electron paramagnetic resonance spectra of native AOS (high-spin, g = 6.56, 5.22, 2.00) and of its cyanide adduct (low-spin, g = 2.86, 2.24, 1.60) closely resemble the corresponding spectra of bovine liver catalase (BLC). These results provide strong evidence for tyrosinate ligation to the heme iron of AOS as has been established for catalases. On the other hand, the positive circular dichroism bands in the Soret region for all three derivatives of ferric AOS are almost the mirror image of those in catalase. In addition, the cyanide affinity of native AOS (K(d) = 10 mM at pH 7) is about 3 orders of magnitude lower than that of BLC. Thus, while these results conclusively support a common tyrosinate-ligated heme in AOS as in catalase, significant differences exist in the interaction between their respective heme prosthetic groups and protein environments, and in the access of small molecules to the heme iron.     

Improvement of proteolytic resistance of immunoadsorbents by chemical modification with polyethylene glycol

Immunoadsorbents were modified with monomethoxy-polyethylene glycol (PEG; average molecular weights of 5000 (PEG-5000) and 1900 (PEG-1900)) activated with cyanuric acid (activated PEG) by four different methods. In the two methods, anti-BSA antibodies were modified with activated PEG with and without protection of antigen binding sites with BSA and then were coupled to CNBr-activated Sepharose 4B. In the other two methods, Immunoadsorbents, which were prepared by coupling anti-BSA antibodies to CNBr-activated Sepharose 4B, were modified with activated PEG with and without the protection. The effects of PEG modification by these four methods on the binding ratio (the ratio of the numbers of moles of antigen adsorbed to the numbers of moles of binding sites of antibody coupled), the antigen binding property and the resistance to proteolytic digestion of immunoadsorbents were studied. The decrease in the binding ratio by the modification with activated PEG was small enough to use modified immunoadsorbents for industrial purification processes. The resistance to proteolytic digestion of immunoadsorbents was improved by modification with activated PEG. The modification without protection of antigen binding sites gave higher resistance to proteolytic digestion than that with protection, while the former caused larger decrease in the binding ratio of modification. The immunoadsorbents modified with activated PEG-5000 showed higher resistance to proteolytic digestion than those modified with activated PEG-1900.     

Compartmentation of ATP within renal proximal tubular cells

Temperature-dependent spin changes of the heme iron atom on cytochrome P-450scc were studied by optical absorption and circular dichroism measurements. The optical absorption and circular dichroism spectra of cholesterol-free cytochrome P-450scc did not change between 10 and 26 degrees C. In contrast, the absorbance at 390 nm and the ellipticity at 330 nm of cholesterol-bound cytochrome P-450scc decreased upon temperature elevation, and the absorbance at 424 nm correspondingly increased. These spectral changes were reversible in respect of temperature. The far-ultraviolet circular dichroism spectra of both cholesterol-bound and -free cytochrome P-450scc were not affected by temperature. In addition, bound cholesterol molecule is not released from the cytochrome molecule by increasing temperature. From these results, we propose that temperature modulates specific interactions between the heme protein and bound cholesterol rather than the gross secondary structural changes of the protein.     

Magnetic circular dichroism studies of bovine liver catalase.

Absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of beef liver catalase at pH 5.0 and 6.9, and its complexes with NaF, KCNO, NaCNS, NaN3 and NaCN, have been measured between 250 nm and 700 nm at room temperature. The pH 6.9 native catalase MCD shows the presence of several additional transitions not resolved in the absorption spectrum. While these bands can be seen in the spectra of all the derivatives, with the exception of the cyanide, their relative intensities changes considerably between complexes. Of special interest in the MCD of ferric hemes is the signal intensity at about 400 nm and 620 nm. The data indicate that the MCD intensity at 620 nm increases as the high spin iron porphyrin fraction increases, reaching a maximum with the fluoride complex. The 430 nm band intensity increases as the proportion of low spin iron increases, reaching a maximum with the cyanide complex. The MCD spectra also indicate clearly the existence of spin mixtures in the complexes with CNO-, CNS-, and N3-, where both the 430 nm and 620 nm bands have appreciable intensity. It is significant that despite almost identical absorption spectra the CNS- complex has higher fraction of low spin iron than either the CNO- or the N3- species. The differences between the pH 5 and 6.9 MCD spectra of the native catalase suggest that the environment of the heme centre is sensitive to protonation.     

Peroxidase improves the activity of catalase by preventing aggregation during TFE-induced denaturation

Catalase, a ubiquitous enzyme of the free radical scavenging machinery unfolds and aggregates in the presence of 2,2,2-triflouroethanol (TFE). Catalase molecule aggregates at 50% TFE as evident by high thioflavin T fluorescence, shifted congo red absorbance, change in circular dichroism and soret spectra. TEM images confirmed the nature of catalase aggregates to be oligomers. Organic solvent-induced aggregation of catalase is prevented by the presence of peroxidase (another enzyme of the free radical scavenging machinery). To alter the progress of aggregation in presence of increasing concentration of TFE, we determined the effect of peroxidase on catalase oligomerization by several different techniques, including turbidity measurement, activity assay, thioflavin T fluorescence, circular dichroism, shift in congo red absorbance, transmission electron microscopy (TEM), Rayleigh scattering, soret absorption spectra, and ANS fluorescence. The presence of peroxidase in the vicinity of folded catalase helps it to remain functionally active and inhibited aggregation in the presence of TFE, suggesting that proteins are stable in crowded environments. Moreover, this catalase-peroxidase interaction is biologically significant as it provides insights into how the aggregation process may be altered.     

Alkalin titrations of human somatotropin, human choriomammotropin and ovine prolactin by circular dichroism and fluorescence.

Structural transitions occurring during the alkalin titration of human somatotropin, human choriomammotropin, and ovine prolactin have been investigated by means of circular dichroism and fluorescence emission spectra. Human somatotropin exhibited an isodichroic point at 287 nm, with all spectral changes being reversed upon back titration from pH 12.50 to pH 8.0. Fluorescence quenching as a function of pH produced a simple sigmoidal curve. Human choriomammotropin exhibited an isodichroic point at 288 nm. The fluorescence and circular dichroism spectra of this protein were found to be reversible between pH 8.0 and 11.0. However, on titration above pH 11, the isodichroic point and the reversibility of the circular dichroism spectra were lost. This conformational transition was accompanied by a sharp increase in fluorescence quantum yield. The circular dichroism spectra of ovine prolactin showed essentially no change on titration to pH 11.0. However, between pH 11.0 and 12.0, a sharp conformational transition was observed similar to that seen in human choriomammotropin, but not exhibiting the same increase in fluorescence quantum yield. The fluorescence titration of prolactin was found to be essentially reversible upon back titration from pH 12.5, although the circular dichroism spectra were not reversible from this pH.