水相合成荧光纳米晶与碱性蛋白酶的偶联及纯化

Labeling and Purification of Alcalase and Fluorescent Nanocrystals Synthesized in Water Phase

利用水相中合成的巯基丙酸包覆的Cd Te荧光纳米晶与碱性蛋白酶(Alcalase)进行偶联标记.荧光光谱分析表明,Cd Te纳米晶体具有较窄的粒度分布和较高的光稳定性,室温下30 d时荧光强度降低8.36%.透射电子显微镜结果显示,Cd Te纳米晶的直径约为3 nm;X射线衍射结果表明,Ce Te纳米晶具有闪锌矿结构.利用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺磺酸钠盐(NHSS)偶联方法将Cd Te纳米晶与Alcalase进行偶联,通过超滤离心、凝胶柱层析及凝胶电泳技术对偶联后的Cd Te-Alcalase进行分离和纯化.荧光光谱显示偶联产物具有很好的光稳定性和较高的酶活力(61.06 U/μg).在荧光显微镜下可观察到标记蛋白的荧光,为研究酶反应的示踪及酶的构效关系等提供了实验基础.

CdTe nanocrystals were synthesized in aqueous solution and capped with amercaptopropionic acid, which were used as a kind of fluorescent probe to label Alcalase. The fluorescence spectra showed that CdTe nanocrystals were accompanied by a narrow size distribution and high stability. The fluorescence intensity de- creased 8.36% after 30 d at room temperature. Transmission electron microscopy （TEM） imaging revealed that CdTe nanocrystal s diameter was about 3 nm, and X-ray diffraction （XRD） pattern showed it with zinc blende structure. After being conjugated with Alcalase using 1-ethyl-3-（ 3-dimethyl aminopropye） carbodii- mide hydroclloride （ EDC ） and N-hydroxysulfosucinimide sodium salt （ NHSS ） methods, the CdTe-Alcalase conjugates were separated and purified by uhra-filter-centrifugation, a Sephadex G-100 column and sodium de- decye sulfate（SDS）-polyacrylamide gel electrophoresis. Fluorescent spectral analysis showed that CdTe-Alca- lase conjugates also presented good optical stability compared with CdTe nanocrystals. The enzyme activity was also relatively high at 61.06 U/μg. The fluorescent emission of the CdTe-Alcalase conjugates was visualized with a fluorescence microscope, which would be suitable for tracing the enzyme reaction in the future.