采用大分子单体稳定印迹牛血清白蛋白

doi:10.7503/cjcu20160437

摘要/Abstract

摘要：

以牛血清白蛋白为模板蛋白质, 聚(丙烯酸羟乙酯-乙烯基咪唑-1-(烯丙基乙酯)-3-乙烯基咪唑氯)[P(HEA-co-VIM-co-[AVIM]Cl)]为大分子功能单体和交联剂, 通过氧化还原引发聚合法制备了蛋白质印迹水凝胶. 圆二色光谱和同步荧光光谱结果表明, P(HEA-co-VIM-co-[AVIM]Cl)能够较好地维持牛血清白蛋白结构稳定性, 而相同质量的HEA, VIM和氯化1-(烯丙基乙酯)-3-乙烯基咪唑([AVIM]Cl)的混合物对牛血清白蛋白的结构破坏严重. 选择性吸附和竞争吸附的结果表明, 用P(HEA-co-VIM-co-[AVIM]Cl)制备的印迹水凝胶比用HEA, VIM和[AVIM]Cl制备的印迹水凝胶具有更强的选择性和识别能力. 在制备过程中维持模板蛋白质结构的稳定性有利于得到具有高识别能力和选择性的印迹聚合物. 采用大分子单体印迹蛋白质的方法, 可以有效地克服蛋白质在印迹过程中的结构容易变性的缺点, 对蛋白质印迹技术的发展具有重要意义.

关键词: 分子印迹技术, 蛋白质印迹, 蛋白质结构稳定, 圆二色光谱, 同步荧光光谱

Abstract:

Protein imprinted hydrogels were prepared via redox initiated polymerization by utilizing bovine serum albumin(BSA) as a template and poly(hydroxyethylacrylate-vinylimidazole-[1-(allylacetate)-3-vinyl-imidazolium]chloride) [P(HEA-co-VIM-co-[AVIM]Cl)] as the macromolecularly functional monomer and crosslinker. The analytical results of circular dichroism and synchronous fluorescence spectrum demonstrated that P(HEA-co-VIM-co-[AVIM]Cl) could effectively maintain the structural stability of BSA, while the equivalent HEA, VIM and [AVIM]Cl denatured the template protein. The selective and competitive adsorption experiments showed that the imprinted hydrogels made by P(HEA-co-VIM-co-[AVIM]Cl) obtained better selectivity and recognition ability compared with those made by HEA, VIM and [AVIM]Cl. Therefore, the above results suggested the significant advantage of maintaining the structural and conformational stability of template protein during the preparation of imprinted polymers. The strategy of using macromolecule monomer to imprint protein could effectively overcome the difficulty of mutability of protein, therefore would promote the development and application of protein imprinting technology.

Key words: Molecularly imprinting technology, Protein imprinting, Stability of protein, Circular dichroism, Synchronous fluorescence spectrum

中图分类号:

TrendMD:

钱立伟, 李季, 宋文琦, 胡小玲, 管萍. 采用大分子单体稳定印迹牛血清白蛋白. 高等学校化学学报, 2016, 37(11): 2092.

QIAN Liwei, LI Ji, SONG Wenqi, HU Xiaoling, GUAN Ping. Utilizing Macromolecular Chain as Functional Monomer and Crosslinker to Imprint BSA with Preserving the Structural Integrity of Template^†. Chem. J. Chinese Universities, 2016, 37(11): 2092.

图/表 14

Scheme 1 Influence of macromolecular chain and micromolecular monomer on the conformation of template protein[6,7]

Fig.1 1H NMR spectrum of [AVIM]Cl in CDCl3

Fig.2 1H NMR spectra of P(HEA-co-VIM)(a) and P(HEA-co-VIM-co-[AVIM]Cl)(b)

Fig.3 XPS spectra of P(HEA-co-VIM)(a) and P(HEA-co-VIM-co-[AVIM]Cl)(b)

Fig.4 GPC analysis of P(HEA-co-VIM)(A) and P(HEA-co-VIM-co-[AVIM]Cl)(B)

Fig.5 Effects of micromolecular monomers(A) and P(HEA-co-VIM-co-[AVIM]Cl)(B)

Scheme 2 Influence of different concentrations of macromolecular chain on the conformation of protein

Fig.6 Synchronous fluorescence spectra of BSA influenced by micromolecular monomers(A, C) or macromolecular chains(B, D) (A), (B) Δλ =15 nm; (C), (D) Δλ =60 nm. M and C are represented micromolecular monomers and macromolecular chain, respectively.

Fig.7 SEM images of MIH-C-BSA(A), MIH-M-BSA(B), NIH-C-BSA(C) and NIH-M-BSA(D)

Fig.8 Adsorption dynamic curves of MIH-C-BSA(a), NIH-C-BSA(b), MIH-M-BSA(c) and NIH-M-BSA(d) for BSA, respectively

Fig.9 Adsorption isotherm of MIH-C-BSA(a), NIH-C-BSA(b), MIH-M-BSA(c) and NIH-M-BSA(d) for BSA, respectively

Table 1 Theoretical maximum capacity(Qm) and Langmuir adsorption equilibrium constant(Km) from the Langmuir model

Sample	K_m/(mL·mg^-1)	Q_m/(mg·g^-1)	R²
MIH-C-BSA	2.79	91.7	0.9929
NIH-C-BSA	0.96	52.6	0.9926
MIH-M-BSA	1.65	78.7	0.9969
NIH-M-BSA	0.88	64.1	0.9829

Table 2 Selective adsorption experiments for MIH and NIH

Protein	Q(MIH-C-BSA)/ (mg·mg^-1)	Q(NIH-C-BSA)/ (mg·mg^-1)	IF^a	β^a	Q(MIH-M-BSA)/ (mg·mg^-1)	Q(NIH-M-BSA)/ (mg·mg^-1)	IF^b	β^b
BSA	67.6	25.5	2.65	49.2	30.1	1.63
OVA	19.8	28.9	0.68	3.86	25.7	28.2	0.91	1.79
Hb	16.3	27.8	0.58	4.52	27.9	26.2	1.06	1.53
Lyz	6.7	13.1	0.51	5.18	9.2	13.4	0.68	2.38

Fig.10 SDS-PAGE analysis of competitive adsorption of MIH-C-BSA and MIH-M-BSALane 1: protein molecular weight marker; lane 2: the mixture solution of BSA, OVA and Lyz with each concentration of 1.0 mg/mL; lane 3: the protein eluted from MIH-C-BSA; lane 4: the protein eluted from NIH-C-BSA; lane 5: the protein eluted from MIH-M-BSA; lane 6: the protein eluted from NIH-M-BSA.

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