金属硫蛋白分离纯化过程中简便监测方法

动物组织用 pH8.6,0.01mol/L Tris-HCl 缓冲液匀浆,经加热处理后,离心上清经凝胶过滤分离,洗脱组分用简便、快速的线扫极谱监测,富含半胱氨酸残基的金属硫蛋白或其类似物在钴氨溶液中产生良好的极谱波,从而达到准确、简便的监测目的.此法特别适用于从新的动植物体提取由复杂因素诱导合成的金属硫蛋白或其类似蛋白.     

金属螯合亲和层析法纯化抗乙肝核心抗原单克隆抗体

采用金属螯合亲和层析法,纯化了小鼠腹水来源的抗乙肝核心抗原单克隆抗体,对上样缓冲液的pH和离子强度、洗脱液种类和洗脱方式进行优化。结果表明,采用降低pH分步洗脱时,最佳上样缓冲液为pH8.0,20mmol/LPB+0.5mol/LNaCl,抗体在pH5.0被洗脱下来,抗体回收率80%,纯度85%。采用咪唑浓度梯度洗脱时,最佳的上样缓冲液为pH8.0,20mmol/LPB+5mmol/L咪唑,抗体纯度大于95%,回收率65%;在上样缓冲液中不添加NaCl而添加少量的咪唑,更有利于抗体分离。以上洗脱方式都能较好地保持mAb的生物学活性,为该抗体的应用提供了必要的实验基础。     

灌注色谱法快速分离纯化茶薪菇金属硫蛋白

为建立一种快速高效分离Cd诱导茶薪菇金属硫蛋白（MT）的方法,以灌注色谱技术为平台,采用BioCAD 700E灌注色谱系统分离纯化Cd诱导茶薪菇金属硫蛋白,并对色谱条件进行了优化,结果表明：经优化相关参数（缓冲液pH值8．2,流速5ml/min,洗脱体积10CV）建立的快速灌注色谱法能高效地进行茶薪菇Cd-MT分离,且具有快速、高效、自动化操作、易放大等优点。     

提取SOD的关键之一是控制离子强度

从动物血中提取SOD时,磷酸盐缓冲液的离子强度大小是分离成功与否的关键因素。pH 7.4的磷酸盐缓冲液的离子强度低于6.000时,SOD不能从DEAE-纤维素柱上洗脱下来。故磷酸盐缓冲液的梯度洗脱浓度应为0.0025mol/L～0.25mol/L。并介绍了计算离子强度的公式。     

棕尾别麻蝇金属硫蛋白的分离纯化及性质分析

将棕尾别麻蝇Boettcherisca peregrina幼虫置于含800 μg/g CdCl₂的食物中取食48 h后,可诱导金属硫蛋白(MT)的合成。诱导处理后的幼虫匀浆上清液经Sephadex G-50分子筛柱、UNO^TM Q1阴离子交换柱和Bio-Gel P-6脱盐柱层析,纯化得到2个亚型,即MT-Ⅰ和MT-Ⅱ。MT-Ⅰ和MT-Ⅱ的分子量均为9 kD,每蛋白分子均含7个Cd和20个巯基,且具254 nm的Cd-SH特征吸收肩。两者的氨基酸组成中,以半胱氨酸含量最高,分别为36.6%和31.8%;而芳香族氨基酸和组氨酸含量甚少,约1%～2%。     

金属螯合亲和层析分离蛋白质的研究

金属螯合亲和层析是近20年发展起来的一项新型分离技术。它以配基简单、吸附量大、分离条件温和、通用性强等特点,逐渐成为分离纯化蛋白质等生物工程产品最有效的技术之一。本文从单组分蛋白质入手,考查了pH值、铵离子浓度、不同铵盐等对蛋白质洗脱的影响,并进行了分析。还对不同的金属螯合柱和不同性质蛋白质的洗脱性能进行了研究,比较了不同金属离子与蛋白质亲和力的区别,为实际体系的分离研究打下了基础。     

镉诱导家兔肝脏含锌金属硫蛋白的纯化及鉴定

预先给家兔皮下注射CdCl_2四次,取肝脏经匀浆、离心、乙醇沉淀后,通过Sephadex G-50凝胶过滤层析,DEAE Sepharose Fast Flow离子交换层析和Sephadex G-25层析柱脱盐,得到MT的两个“亚型”—MT-1和MT-2,对其进行鉴定,其紫外扫描图谱示在280和250nm处无吸收峰,而最大吸收在220nm处。MT-1和MT-2均不含镉和铜,而每分子结合6个锌。用简化巯基试剂测定,MT-1含18.8个巯基,MT-2含19.1个巯基;HPLC分析MT主要以单体存在,也有少量二聚体。氨基酸组成与Cd-MT一致。整个纯化过程采用示波极谱法和火焰原子吸收光谱法测锌含量。结果表明前者可代替后者。     

豌豆球蛋白及其亚基的分离提纯

本文详细地叙述了提取、分离、提纯豌豆球蛋白及其亚基的方法:用硼酸钠缓冲液提取豌豆球蛋白,用85%饱和度硫酸铵沉淀除去其它蛋白质杂质,再用凝胶柱进一步提纯;提纯的豌豆球蛋白通过DE_(52)-纤维素阴离子树脂柱(8M尿素作为变性分离试剂)和S_(200)凝胶柱(70%甲酸作为变性分离试剂)则可分离出豌豆球蛋白的各个亚基;试验结果还表明:在pH8.5时,33,000的亚基携带少量负电荷;而12,000的亚基携带大量负电荷。     

用离心逆流分配色谱仪分离POLY I:C

用两相溶剂系统的离心逆流分配色谱仪,分离人工合成聚肌胞Poly I:C.采用步进洗脱和梯度洗脱.在梯度洗脱中对Ito氏的缓冲液进行改进,得到了很好的分离结果,分成四个组分,其沉降系数分别为12s,7.0s,6.3s,和3.8s.此分离结果优于普通柱色的结果.     

小鼠肝脏金属硫蛋白Metallothioneins的分离纯化与鉴定

由健康小鼠通过皮下注射一定量的氯化镉,取肝脏匀浆,离心,经SephadexG-50和DEAE-Sephadex A-50柱层析分离,即可获得MT-Ⅰ和MT-Ⅱ两种金属硫蛋白。经鉴定:两个分子各含18个巯基,结合4个Cd原子和3个Zn原子,无金属蛋白质Thionein的分子量约为6kD,半胱氨酸含量约占氨基酸总量的28％,所提取到的MT-Ⅰ和MT-Ⅱ经氨基酸组成,HPLC和PAGE分析皆证明为高度均一的,且与有关文献报道基本相符。其中MT-Ⅱ已获得晶体。     

N-acetyl-beta-D-hexosaminidase A from rat urine: partial purification and characterization

N-Acetyl-beta-D-hexosaminidase A was purified from rat urine by ion-exchange chromatography on DEAE-cellulose, followed by concanavalin A chromatography, and finally by chromatography on 2-acetamido-N-(epsilon-aminocaproyl)-2-deoxy-beta-glucosylamine-Se pharose 4B. The enzyme was purified 482-fold with a yield of about 7%. The optimal pH was 4.5 for N-acetyl-glucosaminidase activity and 4.0-4.5 for N-acetylgalactosaminidase activity. The enzyme was heat-labile and stable from pH 4.5 to pH 7.0 but it was very unstable at lower pH values. Km values were 0.55 mM and 0.059 mM, respectively. The glycoprotein nature of the enzyme was deduced from its behavior on concanavalin A. The effect of some carbohydrates and ionic compounds on the activities of the enzyme was studied. When N-acetyl-D-glucosaminolactone and N-acetyl-D-galactosaminolactone were used as inhibitors, Ki values were also calculated.     

A simple high-yield procedure for isolation of human urinary kallikreins.

Two human urinary kallikreins (fractions A-1 and A-2) were purified to apparently homogeneous forms. The two kallikreins were separated by affinity chromatography using Trasylol (aprotinin) covalently bound to Sepharose. The kallikreins were eluted with a pH gradient (pH 9.5-3.0). Fraction A-1 was eluted between pH 6.2 and 4.2 and fraction A-2 was eluted between pH 4.2 and 3.1. Final purification was obtained by chromatography on Sephacryl SS-200. Antibodies prepared against fraction A-2 were also reactive with fraction A-1; thus it may be possible to measure both by radioimmunoassay using the same antibody preparation.     

Fractionation of the naringinase complex from Aspergillus terreus by dye affinity chromatography

Affinity chromatography with immobilised triazine dyes was used to separate the main enzymes present in the naringinase complex produced by Aspergillus terreus CECT 2663. One alpha-L-rhamnosidase and two beta-D-glucosidases (beta G1 and beta G2) were separated by a simple two-step procedure involving chromatography with Red HE-3B immobilised on Sepharose 4B first at pH 5.5 and then at pH 4.7. Maximum activity of the beta-D-glucosidases was from pH 4 to 6 and at 65 degrees C. Both glucosidases were active on p -nitrophenol glucoside and prunin with respective Km values of 1.9 mm and 1.6 mm for beta G1 and 2.1 mm and 0.25 mm for beta G2. Only beta G1 hydrolysed cellobiose (Km = 5.7 mm).     

Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis

Phosphotransbutyrylase (phosphate butyryltransferase [EC 2.3.1.19]) from Clostridium acetobutylicum ATCC 824 was purified approximately 200-fold to homogeneity with a yield of 13%. Steps used in the purification procedure were fractional precipitation with (NH4)2SO4, Phenyl Sepharose CL-4B chromatography, DEAE-Sephacel chromatography, high-pressure liquid chromatography with an anion-exchange column, and high-pressure liquid chromatography with a hydrophobic-interaction column. Gel filtration and denaturing gel electrophoresis data were consistent with a native enzyme having eight 31,000-molecular-weight subunits. Within the physiological range of pH 5.5 to 7, the enzyme was very sensitive to pH change in the butyryl phosphate-forming direction and showed virtually no activity below pH 6. This finding indicates that a change in internal pH may be one important factor in the regulation of the enzyme. The enzyme was less sensitive to pH change in the reverse direction. The enzyme could use a number of substrates in addition to butyryl coenzyme A (butyryl-CoA) but had the highest relative activity with butyryl-CoA, isovaleryl-CoA, and valeryl-CoA. The Km values at 30 degrees C and pH 8.0 for butyryl-CoA, phosphate, butyryl phosphate, and CoASH (reduced form of CoA) were 0.11, 14, 0.26, and 0.077 mM, respectively. Results of product inhibition studies were consistent with a random Bi Bi binding mechanism in which phosphate binds at more than one site.     

Resolution of myocardial phospholipase C into several forms with distinct properties.

Phospholipase C activity capable of hydrolysing phosphatidylinositol in bovine heart was resolved into four forms (I-IV) by ion-exchange chromatography. Some of these forms could only be detected if the assay was performed at acidic pH (I and IV) or in the presence of deoxycholate (II). Gel-filtration chromatography indicated that the four forms had different molecular weights in the range 40000-120000. I, II and III all had pH optima in the range 4.5-5.5. However, the major form (III) also had substantial activity at pH 7.0 and above. The activities of I, II and III at pH 7.0 were stimulated by deoxycholate; this effect was most marked with I and II, which had very low activity at this pH. All forms of the enzyme were inhibited by EGTA and required 2-5 mM-CaCl2 for maximal activity. When the fractions eluted from the ion-exchange and gel-filtration columns were assayed with polyphosphoinositides as substrates there was a close correspondence to the elution profile obtained with phosphatidylinositol as substrate; there was no evidence for the existence in heart of phospholipase C activities specific for individual phosphoinositides.     

Purification and Some Properties of Acid-stable α-Amylases from Shochu koji (Aspergillus kawachii)

Aspergillus kawachii α-amylase [EC 3.2.1.1] I and II were purified from shochu koji extract by DEAE Bio-Gel A ion exchange chromatography, Sephacryl S-300 gel chromatography (pH 3.6), coamino dodecyl agarose column chromatography and Sephacryl S-200 gel chromatography. By gel chromatography on a Sephacryl S-300 column, the molecular weights of the purified α-amylase I and II were estimated to be 104,000 and 66,000, respectively. The isoelectric points of α-amylase I and II were 4.25 and 4.20, respectively. The optimal pH range of α-amylase I was 4.0 to 5.0, and the optimum pH of α-amylase II was 5.0. The optimum temperatures of both α-amylases were around 70°C at pH 5.0. Both α-amylases were stable from pH 2.5 to 6.0 and up to 55°C, retaining more than 90% of the original activities. Heavy metal ions such as Hg^{2 +} and Pb^{2 +} were potent inhibitors for both α-amylases.     

疏水相互作用层析分离重组人干扰素α2b

目的采用疏水相互作用层析分离重组人干扰素α2b,去除干扰素样品中的二聚体,得到高纯度的干扰素用于进一步的研究。方法首先采用阳离子交换层析纯化复性重组人干扰素α2b,去除了大部分的杂蛋白,然后采用疏水相互作用层析纯化重组人干扰素α2b,去除复性过程中产生的错误折叠体和二聚体,并考察盐浓度、pH值、流速和洗脱液中尿素对疏水相互作用层析纯化效果的影响。结果硫酸铵初始浓度1.2 mol/L、缓冲液pH值6.0、流速2.5 mL/min、洗脱液中添加尿素浓度为2 mol/L时疏水相互作用层析纯化效果最佳。最终得到的重组人干扰素α2b非还原型SDS-PAGE电泳均呈单一条带。结论确定了疏水层析纯化重组人干扰素α2b的最优条件,成功提取到具有高活性、高纯度的重组人干扰素α2b纯品。     

Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis.

Phosphotransbutyrylase (phosphate butyryltransferase [EC 2.3.1.19]) from Clostridium acetobutylicum ATCC 824 was purified approximately 200-fold to homogeneity with a yield of 13%. Steps used in the purification procedure were fractional precipitation with (NH4)2SO4, Phenyl Sepharose CL-4B chromatography, DEAE-Sephacel chromatography, high-pressure liquid chromatography with an anion-exchange column, and high-pressure liquid chromatography with a hydrophobic-interaction column. Gel filtration and denaturing gel electrophoresis data were consistent with a native enzyme having eight 31,000-molecular-weight subunits. Within the physiological range of pH 5.5 to 7, the enzyme was very sensitive to pH change in the butyryl phosphate-forming direction and showed virtually no activity below pH 6. This finding indicates that a change in internal pH may be one important factor in the regulation of the enzyme. The enzyme was less sensitive to pH change in the reverse direction. The enzyme could use a number of substrates in addition to butyryl coenzyme A (butyryl-CoA) but had the highest relative activity with butyryl-CoA, isovaleryl-CoA, and valeryl-CoA. The Km values at 30 degrees C and pH 8.0 for butyryl-CoA, phosphate, butyryl phosphate, and CoASH (reduced form of CoA) were 0.11, 14, 0.26, and 0.077 mM, respectively. Results of product inhibition studies were consistent with a random Bi Bi binding mechanism in which phosphate binds at more than one site.     

Purification and characterization of a collagenase from the mackerel,Scomber japonicus

Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and 55 degrees, respectively. The K(m) and V(max) of the enzyme for collagen Type I were approximately 1.1mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by Hg2+, Zn2+, PMSF, TLCK, and the soybean-trypsin inhibitor.     

Preparation and properties of a lysozyme derivative in which two domains are cross-linked intramolecularly between Trp62 and Asp101.

A lysozyme derivative in which two domains were cross-linked intramolecularly was newly prepared by means of a two-step reaction. First, the beta-carboxyl group of Asp101 in lysozyme was selectively modified with 2-(2-pyridyldithio)ethylamine in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride. After reduction of the pyridyldithio moiety of Asp101 modified lysozyme at pH 4.5 with dithiothreitol, the derivative was allowed to cross-link intramolecularly by reaction with 1,3-dichloroacetone at pH 7. Intramolecularly cross-linked lysozyme thus formed was purified by gel chromatography followed by ion-exchange chromatography. Based on the results of 1H-NMR and peptide analyses, it was concluded that Asp101 was cross-linked to Trp62 with a -CH2COCH2SCH2CH2NH-bridge in this derivative. The derivative showed minor but distinct activity against Micrococcus lysodeikticus and glycol chitin. Its melting temperature for thermal denaturation was higher by 7.3 degrees than that of native lysozyme at pH 3.