2.聚合物在蛋白质分离中的应用
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2.聚合物在蛋白质分离中的应用. 蛋白质是由氨基酸通过酰胺键连接而成的高分子,两性,存在等 电点,因其空间结构的复杂性,有亲水或疏水之分。. 蛋白质在低于等电点时会带上正电荷,在高于等电点时带负电荷. 若缓冲溶液的 pH 值低于蛋白质等电点,蛋白质则吸附 H + 而带正电荷,由于 静电相互作用,蛋白质与管壁发生 吸附。. ( a )碱性蛋白质( pI = 8 )所带电荷量随 pH 的变化曲线; ( b )在 pH = 4 时,碱性蛋白质( pI = 8 )与毛细管壁发生吸附. 1 . 极端 pH 值法.

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2.聚合物在蛋白质分离中的应用

蛋白质是由氨基酸通过酰胺键连接而成的高分子,两性,存在等

电点,因其空间结构的复杂性,有亲水或疏水之分。



蛋白质在低于等电点时会带上正电荷,在高于等电点时带负电荷.a)碱性蛋白质(pI=8)所带电荷量随pH的变化曲线;

(b)在pH=4时,碱性蛋白质(pI=8)与毛细管壁发生吸附


蛋白质在低于等电点时会带上正电荷,在高于等电点时带负电荷.. 极端pH值法

  • pH高于蛋白质的pI时,毛细管内壁对蛋白质产生静电排斥抑制吸附,但是,蛋白质存在的自然状态为pH4-10,高pH如11.0时,容易使蛋白质变性或者水解。

  • pH小于1.5(熔融硅或石英的等电点)时,蛋白质的质子化导致其质荷比差别较小,分辨率难以提高。

  • 对等电点相近的蛋白质不易实现分离。

  • 一般认为,使蛋白质混合物分离的首选pH值应和蛋白质混合物的pKa值基本一致。

McManigill D. Anal Chem,1986,58:166~170.


2. 蛋白质在低于等电点时会带上正电荷,在高于等电点时带负电荷.添加小分子添加剂

  • 表面活性剂:季铵盐和氟化的阳离子表面活性剂。

  • 中性盐:如磷酸盐,硫酸钾等。

  • 胺类:三乙醇胺、三乙胺、N-乙基二乙醇胺,N,N, N’,N’-四甲基-1,3 -丁二胺(TMBD)等。

  • 有机溶剂:醇类、乙腈、丙酮、四氢呋喃、二甲亚砜等,其中最常用的是甲醇和乙腈。

  • 选用极端pH值或添加小分子添加剂的方法虽然能从一定程度上降低蛋白质吸附,但是容易导致蛋白质聚集和变性。目前最常用的方法是用聚合物对毛细管内壁进行改性处理。

何金兰等.高效毛细管电泳. 北京:科学出版社,1996, 72~75.

Verzola B, Gelfi C, Righetti P G. J Chromatogr A,2000,868:85~99.

Corradini D, Cannarsa G, Fabbri E, et al. J Chromatogr A,1995,709:127~134.

Corradini D, Cannarsa G. Electrophoresis,1995,16:630~635.


蛋白质在低于等电点时会带上正电荷,在高于等电点时带负电荷..聚合物涂覆毛细管内壁抑制  蛋白质吸附

(1) 化学键合的毛细管涂层

化学键合的交联的PVA涂层对4种碱性蛋白质实现第2次和第902次分离的电泳图谱

样品浓度:50ug/mL的(1)细胞色素C,(2)溶解酵素,(3)胰蛋白酶原,(4)胰凝乳蛋白酶原。分离条件:毛细管,内径50μm,总长48.5cm(有效长度40cm);注入,3 kV, 5 s;缓冲液,40mM磷酸钠;分离,309 V/cm


(2)蛋白质在低于等电点时会带上正电荷,在高于等电点时带负电荷.物理吸附的毛细管涂层

目前使用的聚合物:

  中性的聚合物-已经广泛用于DNA和其它一些生物大分子的分析,但是由于蛋白质的吸附作用,使得对蛋白质的分析仍有一定难度。

  亲水性的聚合物-如甲基纤维素,聚糖等不能很好的吸附在管壁上,稳定性差。

  带疏水基团的聚合物-聚乙烯基吡咯烷酮,聚N,N-二甲基丙烯酰胺可以形成稳定的涂层,但是由于疏水基团与蛋白质之间的相互作用使得分离效率下降。

Madabhushi R S. Electrophoresis,1998,19:224~230.

Verzola B, Gelfi C, Righetti P G. J Chromatogr A,2000,874:293~303.


羟乙基纤维素的改性蛋白质在低于等电点时会带上正电荷,在高于等电点时带负电荷.

(1) Cat-HEC

Formation of the cat-HEC

Schematic diagram of adsorption of cat-HEC onto silica surface.

Preparation and properties of Cat-HEC


Electroosmotic flow as a function of pH. Comparison between 蛋白质在低于等电点时会带上正电荷,在高于等电点时带负电荷.

a bare fused-silica capillary, HEC and cat-HEC coated capillary

Electropherograms of a mixture of standard proteins.

Separations were carried out using cat-HEC-2 coated capillary

1 = Lysozyme; 2 = Cytochrome C; 3 = Ribonuclease A.

Electropherograms of a mixture of standard proteins at pH 4.6.


Electropherograms of a mixture of standard proteins in different pH. Separations were carried out using cat-HEC-2 coated capillary 1 = Lysozyme; 2 = Cytochrome C; 3 = Ribonuclease A.

Migrationtime reproducibility (n = 3) and peak efficiency of proteins separated in polymer-coated capillaries at pH 4.6

Electrophoresis,2008, 29, 1460-1466.


( different pH. Separations were carried out using cat-HEC-2 coated capillary 1 = Lysozyme; 2 = Cytochrome C; 3 = Ribonuclease A.2) HEC-g-P4VP

Electroosmotic flow as a function of pH. Comparison between

a bare fused-silica capillary, HEC and HEC-g-P4VP coated capillary

Formation of the HEC-g-P4VP

Electropherograms of a mixture of standard proteins. Separations were carried out using cat-HEC-g-P4VP coated capillary. 1 = Lysozyme; 2 = Cytochrome C; 3 = Ribonuclease A.


O different pH. Separations were carried out using cat-HEC-2 coated capillary 1 = Lysozyme; 2 = Cytochrome C; 3 = Ribonuclease A.

O

O

B

r

O

B

r

+

O

O

N

N

N

2.结构规整的聚合物

PEO-b-P4VP

Formation of the PEO-b-P4VP

1H NMR and GPC data of PEO-b-P4VP copolymers

a. Mn of PEO-b-P4VP estimated by 1H NMR; b. Mn of PEO-b-P4VP determined by GPC.


(A) Typical different pH. Separations were carried out using cat-HEC-2 coated capillary 1 = Lysozyme; 2 = Cytochrome C; 3 = Ribonuclease A.TEM images of (a) PEO113-b-P4VP45, (b) PEO113-b-P4VP90,

(c) PEO113-b-P4VP113, and (d) PEO113-b-P4VP294;

(B) Schematic illustration of copolymers of different morphologies capillary coating procedure

(a) PEO113-b-P4VP45, (b) PEO113-b-P4VP90, (c) PEO113-b-P4VP113, and (d) PEO113-b-P4VP294

Effect of molecular weight of P4VP block on the separation of basic proteins at pH 4.6.

1, lysozyme; 2, cytochrome c; 3, ribonuclease.


Analysis of saliva samples by PEO different pH. Separations were carried out using cat-HEC-2 coated capillary 1 = Lysozyme; 2 = Cytochrome C; 3 = Ribonuclease A.113-b-P4VP294 coated capillary.

Saliva samples were diluted to 2-fold using deionized water. Samples (A)

without and (B) with spiking of 0.2mg / mL lysozyme were injected.

Separation conditions: 40mM phosphate-citrate buffer, pH 5.1; 500V/cm;

40cm capillary (30 cm to the detector); temperature 25℃.

Effect of buffer pH on the separation of basic proteins.

separations were taken in PEO113-b-P4VP294 coated capillary


Electrophoresis different pH. Separations were carried out using cat-HEC-2 coated capillary 1 = Lysozyme; 2 = Cytochrome C; 3 = Ribonuclease A.2008,

29, 2812-2819

Electropherograms of plasma sample in bare capillary (A) and a PEO113-b-P4VP294 coated capillary (B).

Separation buffer: 19mM NaOH-Na2B4O7 at pH 9.7. HSA, human serum albumin; α1-AT, α1-antitrypsin; α2-M, α2-macroglobulin; β-lp, β-lipoprotein; Tf, transferrin; IgA, immunoglobulin A; IgG, immunoglobulin G.


3. different pH. Separations were carried out using cat-HEC-2 coated capillary 1 = Lysozyme; 2 = Cytochrome C; 3 = Ribonuclease A.多功能分离介质的合成及应用研究

研究背景

  能进行DNA、蛋白质、氨基酸、同分异构体等的分离

(1) HEC-g-PDMA

Electrophoresis,

2008, 29, 4351–4354.

Separation of Φ×174/HaeIII digest by CE with

HEC-g-PDMA at (a) 1.0% w/v; (b) 1.5% w/v; (c) 2.0% w/v

Effect of pH values on separations of basic proteins using

HEC-g-PDMA coated capillary.1, lysozyme; 2, cytochrome c; 3, ribonuclease A.


(2) quasi-interpenetrating network of LPA andPDMA different pH. Separations were carried out using cat-HEC-2 coated capillary 1 = Lysozyme; 2 = Cytochrome C; 3 = Ribonuclease A.

Properties of quasi-IPN containing LPA with different molecular masses

EOF as a function of pH.

Electropherograms of a mixture of standard proteins.


Separation of basic proteins using quasi-IPN-3 coated capillary

at different pH values. 1=cytochrome c; 2=Lysozyme; 3= Ribonuclease A.

J. Sep. Sci., 2009, 32, 671-680.

Separation of Φ×174/HaeIII digest by CE with quasi-IPN-3 at

(a) 2% w/v; (b) 3% w/v; (c) 4% w/v; (d) 4% w/v. Conditions: Separation electric

field strength of (a), (b), and (c) is –6 kV, separation electric field strength of (d) is –8 kV.



Thank you !目前仍未找到一种普适性的涂层对所有种类的蛋白质分离都是有效的,因为不同种类的蛋白质等电点不同,这就需要用于管壁涂覆的聚合物涂层有较宽的


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