枯草芽孢杆菌6-磷酸葡萄糖脱氢酶的分离纯化和部分性质
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摘要
枯草芽孢杆菌通过超声破壁、(NH_4)_2SO_4分段盐析、DEAE-Sepharose FF离子交换层析、Blue Sepharose CL-6B亲和层析、Sephadex G-200凝胶过滤等纯化步骤，分离出6-磷酸葡萄糖脱氢酶(G6PD)。经PAGE和SDS-PAGE检测为单一蛋白区带，比活力为1.7375IU／mg，纯化倍数为72.7,收率为13.6％。凝胶过滤法测定该酶表观分子量约为220kD，SDS-PAGE测定亚基分子量约为50.5kD，可见该酶是由四个相同亚基构成的四聚体。等电聚焦电泳测得等电点为6.3，用动力学方法测得该酶的最适反应pH为8.5，最适反应温度为37℃，以6-磷酸葡萄糖(G6P)为底物的Km值为0.177mmol／L。
考察了部分金属离子及EDTA对该酶活性和紫外、荧光光谱的影响：Mg~(2+)起激活作用；Zn~(2+)低浓度时表现激活作用，高浓度时表现抑制作用；Ag~+、Fe~(2+)起抑制作用；而EDTA和Mn~(2+)基本无影响。部分金属离子使G6PD的构象发生改变，发色团所处的微环境随之发生变化，使紫外吸收峰变宽变平。Ag~+和Fe~(2+)引起色氨酸(Trp)残基的荧光淬灭；Mg~(2+)和EDTA均使G6PD的荧光强度增强，说明它们使Trp残基重新包裹在分子内部而处于疏水的微环境中；Zn~(2+)和Mn~(2+)均使G6PD的荧光强度变小，说明它们使酶分子构象变得疏松，原来处在分子内部的发色团暴露在极性环境中。
天然G6PD的远紫外圆二色谱显示在208nm和222nm两处的双负峰曲线，说明α-螺旋含量高，此时含有41.2％的α-螺旋，20.6％的β-折叠和38.2％的无规卷曲及其它构象单元。
用常见的荧光淬灭剂丙烯酰胺和KI研究G6PD分子中Trp残基的微环境，
发现它们对分子中T印残基的荧光均有淬灭作用，丙烯酸胺能淬灭100%的荧
光，Kl能淬灭78%的荧光，表明分子中小部分饰残基埋藏在分子内部，大
部分T印残基处于分子表面的极性环境中。
Glucose 6-phosphate dehydrogenase (G6PD) was isolated and purified from Bacillus subtilis BF by a procedure including cell sonicatioru salting out with (NH4) 2SO4, ion-exchange chromatographing with DEAE-Sepharose FF, affinity chromatographing with Blue Sepharose CL-6B and gel filtration with Sephadex G-200. The purified G6PD showed a single protein band on PAGE and SDS-PAGE determination respectively. The purified enzyme had a specific activity of 1. 7375IU/mg, a 72. 7-fold purification was obtained with a recovery of 13.6%. The apparent molecular weight of G6PD, determined by gel filtration, was 220KD; The subunit molecular weight was 50. 5KD as determined by SDS-PAGE, suggesting that the native enzyme was a tetramer consisting of four identical subunits. The result of isoelectrofocusing showed that the isoelectric point of G6PD was 6. 3. The optimal pH of G6PD was 8. 5 and the optimal temperature was 37℃.The Km value of G6P on G6PD was 0. 177mmol/L.
The effect of some metal ions and EDTA on enzyme activity, ultraviolet absorbance spectra and fluorescent spectra were all investigated: the G6PD activity was stimulated by Mg2+ while inhibited by Ag+ and Fe2+;the G6PD activity was activated by Zn2+ in low
concentration condition while inhibited by Zn2+in high; EDTA and Mn2+ had no influence on G6PD activity. Some metal ions made G6PD conformation changed and caused the micro-environment of chromophores varied, which made the ultraviolet absorbance peak of G6PD in 276nm wider and flatter. Ag+ and Fe2+ caused the fluorescence of Trp residues in G6PD quenched ;Mg2+ and EDTA made fluorescence intensity of G6PD increased, this indicates that they caused Trp residues wrapped and came to the inner core and located in the hydrophobic area; while Zn2+ or Mn2+ made fluorescence intensity of G6PD decreased, this indicates that they made the conformation of G6PD relaxed and chromophores exposed to polarity environments.
In native condition and in the far circular dichroic (CD) region, G6PD exhibited two characteristic negative band centered at 208nm and 222nm respectively, thus it is estimated to contain about 41.2% a -helix, 20.6% β-pleated sheet and 38.2% random coil and turn.
I used acrylamide (Acr) and KI to probe the micro-environments of Trp residues in the G6PD molecule to show that the Trp residues fluorescence of G6PD were quenched by the two quenching reagents, 100% and 78% was quenched by Acr and KI respectively. I deduced that some Trp residues of G6PD were located in the inner core and the others located in the polarity surface.
考察了部分金属离子及EDTA对该酶活性和紫外、荧光光谱的影响：Mg~(2+)起激活作用；Zn~(2+)低浓度时表现激活作用，高浓度时表现抑制作用；Ag~+、Fe~(2+)起抑制作用；而EDTA和Mn~(2+)基本无影响。部分金属离子使G6PD的构象发生改变，发色团所处的微环境随之发生变化，使紫外吸收峰变宽变平。Ag~+和Fe~(2+)引起色氨酸(Trp)残基的荧光淬灭；Mg~(2+)和EDTA均使G6PD的荧光强度增强，说明它们使Trp残基重新包裹在分子内部而处于疏水的微环境中；Zn~(2+)和Mn~(2+)均使G6PD的荧光强度变小，说明它们使酶分子构象变得疏松，原来处在分子内部的发色团暴露在极性环境中。
天然G6PD的远紫外圆二色谱显示在208nm和222nm两处的双负峰曲线，说明α-螺旋含量高，此时含有41.2％的α-螺旋，20.6％的β-折叠和38.2％的无规卷曲及其它构象单元。
用常见的荧光淬灭剂丙烯酰胺和KI研究G6PD分子中Trp残基的微环境，
发现它们对分子中T印残基的荧光均有淬灭作用，丙烯酸胺能淬灭100%的荧
光，Kl能淬灭78%的荧光，表明分子中小部分饰残基埋藏在分子内部，大
部分T印残基处于分子表面的极性环境中。
Glucose 6-phosphate dehydrogenase (G6PD) was isolated and purified from Bacillus subtilis BF by a procedure including cell sonicatioru salting out with (NH4) 2SO4, ion-exchange chromatographing with DEAE-Sepharose FF, affinity chromatographing with Blue Sepharose CL-6B and gel filtration with Sephadex G-200. The purified G6PD showed a single protein band on PAGE and SDS-PAGE determination respectively. The purified enzyme had a specific activity of 1. 7375IU/mg, a 72. 7-fold purification was obtained with a recovery of 13.6%. The apparent molecular weight of G6PD, determined by gel filtration, was 220KD; The subunit molecular weight was 50. 5KD as determined by SDS-PAGE, suggesting that the native enzyme was a tetramer consisting of four identical subunits. The result of isoelectrofocusing showed that the isoelectric point of G6PD was 6. 3. The optimal pH of G6PD was 8. 5 and the optimal temperature was 37℃.The Km value of G6P on G6PD was 0. 177mmol/L.
The effect of some metal ions and EDTA on enzyme activity, ultraviolet absorbance spectra and fluorescent spectra were all investigated: the G6PD activity was stimulated by Mg2+ while inhibited by Ag+ and Fe2+;the G6PD activity was activated by Zn2+ in low
concentration condition while inhibited by Zn2+in high; EDTA and Mn2+ had no influence on G6PD activity. Some metal ions made G6PD conformation changed and caused the micro-environment of chromophores varied, which made the ultraviolet absorbance peak of G6PD in 276nm wider and flatter. Ag+ and Fe2+ caused the fluorescence of Trp residues in G6PD quenched ;Mg2+ and EDTA made fluorescence intensity of G6PD increased, this indicates that they caused Trp residues wrapped and came to the inner core and located in the hydrophobic area; while Zn2+ or Mn2+ made fluorescence intensity of G6PD decreased, this indicates that they made the conformation of G6PD relaxed and chromophores exposed to polarity environments.
In native condition and in the far circular dichroic (CD) region, G6PD exhibited two characteristic negative band centered at 208nm and 222nm respectively, thus it is estimated to contain about 41.2% a -helix, 20.6% β-pleated sheet and 38.2% random coil and turn.
I used acrylamide (Acr) and KI to probe the micro-environments of Trp residues in the G6PD molecule to show that the Trp residues fluorescence of G6PD were quenched by the two quenching reagents, 100% and 78% was quenched by Acr and KI respectively. I deduced that some Trp residues of G6PD were located in the inner core and the others located in the polarity surface.
引文
[1] Stryer L. Biochemistry (third edition).New York:W.H. Freeman and Company, 1988, 427-428.
[2] Beutler E. Bkood. 1959, 14: 103.
[3] Okuno H, Nagata K, Nakajima H.J. Appl. Biochem. 1985, 7(3): 192-201.
[4] Donohue T M, Mahowald T A, Adams D J, et al. Biochim. Biophys. Acta. 1981, 658: 356-368.
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[6] Lindblom H. J. Chromatogr. 1983, 266: 265-271.
[7] Levy H R, Cook C. Arch. Biochem. Biophys. 1991, 291(1): 161-167.
[8] Anderson B M, Anderson C D. Arch. Biochem. Biophys. 1995, 321(1): 94-100.
[9] 穆虹，李明启，吴光耀，等．植物学报．1992，34(1)：37-42．
[10] Lowry O H. J. Biol. Chem. 1951, 193:265-275.
[11] 何忠效，张树政．电泳(第二版)．北京：科学出版社，1999，36-39．
[12] 奥斯伯 F，金斯顿 R E，布伦特 R，等．精编分子生物学实验指南．北京：科学出版社，1998，334-338．
[13] Nakatsuji T, Miwa S. Anal. Biochem. 1981, 112:52-54.
[14] Farmer E E, Easterby J S. Anal. Biochem. 1984, 141(1): 79-82.
[15] 汪家政，范明．蛋白质技术手册．北京：科学出版社，2000，231-238．
[16] Scott W A, Tatum E L. J. Biol. Chem. 1971, 246:6347.
[17] 张龙翔，张庭芳，李令媛．生化实验方法和技术(第二版)．北京：高等教育出版社，1997，116-124．
[18] 哈密斯 B D 等著，刘毓秀等译．蛋白质的凝胶电泳实践方法．北京：科学出版社，1994．
[19] 张洪渊，刘克武，石安静，等．水生生物学报．1996，20(1)：57-62．
[20] Chen Y H, Yang J T, Chan K H. Biochemistry. 1974, 13(6): 3350-3359.
[21] 张洪渊，刘克武．四川大学学报(自然科学版)．1996，33(1)：100-105．
[22] Shyamali S S. Biochemistry. 1979, 18: 1026.
[23] Niehaus W G, Mallet T C. Arch. Biochem. Biophys. 1994, 313(2): 304-309.
[24] Niehaus W G, Dilts R P. Arch. Biochem. Biophys. 1984, 228(1): 113-119.
[25] 赵南明，周海梦．生物物理学．北京：高等教育出版社，2000，287-298．
[26] Yip Y K, Waks M, Beychok S. J. Biol. Chem. 1972, 247: 7237-7244.
[27] Litman G W. Biochem. Biophys. Acta. 1971, 236: 647-654.
[28] Cathou R E, Kulczyki A J. Biochemistry. 1968, 7: 3958-3964.
[29] Bailey G S, Lee J, Tu A T. J. Biol. Chem. 1979, 254: 8922-8926.
[30] Lakowicz J R. principles of Fluorescence Spectroscopy. New York: Plenum Press, 1983, 257.
[31] 孙建忠等．生物化学和生物物理学报．1994，6：649．
[32] Ganzhorn A J, et al. Biochem. Biophys. Acta. 1993, 1161:303.
[33] 郭尧君．荧光实验技术及其在分子生物学中的运用．北京：科学出版社，1983，123．
[34] 许根俊．酶的作用原理．北京：科学出版社，1984，108．
[35] 杨斌盛，杨频．生物化学和生物物理进展．1992，19(2)：110-114．
[2] Beutler E. Bkood. 1959, 14: 103.
[3] Okuno H, Nagata K, Nakajima H.J. Appl. Biochem. 1985, 7(3): 192-201.
[4] Donohue T M, Mahowald T A, Adams D J, et al. Biochim. Biophys. Acta. 1981, 658: 356-368.
[5] Kadir C T. Prep. Biochem. Biotech. 2002, 32(2): 173-187.
[6] Lindblom H. J. Chromatogr. 1983, 266: 265-271.
[7] Levy H R, Cook C. Arch. Biochem. Biophys. 1991, 291(1): 161-167.
[8] Anderson B M, Anderson C D. Arch. Biochem. Biophys. 1995, 321(1): 94-100.
[9] 穆虹，李明启，吴光耀，等．植物学报．1992，34(1)：37-42．
[10] Lowry O H. J. Biol. Chem. 1951, 193:265-275.
[11] 何忠效，张树政．电泳(第二版)．北京：科学出版社，1999，36-39．
[12] 奥斯伯 F，金斯顿 R E，布伦特 R，等．精编分子生物学实验指南．北京：科学出版社，1998，334-338．
[13] Nakatsuji T, Miwa S. Anal. Biochem. 1981, 112:52-54.
[14] Farmer E E, Easterby J S. Anal. Biochem. 1984, 141(1): 79-82.
[15] 汪家政，范明．蛋白质技术手册．北京：科学出版社，2000，231-238．
[16] Scott W A, Tatum E L. J. Biol. Chem. 1971, 246:6347.
[17] 张龙翔，张庭芳，李令媛．生化实验方法和技术(第二版)．北京：高等教育出版社，1997，116-124．
[18] 哈密斯 B D 等著，刘毓秀等译．蛋白质的凝胶电泳实践方法．北京：科学出版社，1994．
[19] 张洪渊，刘克武，石安静，等．水生生物学报．1996，20(1)：57-62．
[20] Chen Y H, Yang J T, Chan K H. Biochemistry. 1974, 13(6): 3350-3359.
[21] 张洪渊，刘克武．四川大学学报(自然科学版)．1996，33(1)：100-105．
[22] Shyamali S S. Biochemistry. 1979, 18: 1026.
[23] Niehaus W G, Mallet T C. Arch. Biochem. Biophys. 1994, 313(2): 304-309.
[24] Niehaus W G, Dilts R P. Arch. Biochem. Biophys. 1984, 228(1): 113-119.
[25] 赵南明，周海梦．生物物理学．北京：高等教育出版社，2000，287-298．
[26] Yip Y K, Waks M, Beychok S. J. Biol. Chem. 1972, 247: 7237-7244.
[27] Litman G W. Biochem. Biophys. Acta. 1971, 236: 647-654.
[28] Cathou R E, Kulczyki A J. Biochemistry. 1968, 7: 3958-3964.
[29] Bailey G S, Lee J, Tu A T. J. Biol. Chem. 1979, 254: 8922-8926.
[30] Lakowicz J R. principles of Fluorescence Spectroscopy. New York: Plenum Press, 1983, 257.
[31] 孙建忠等．生物化学和生物物理学报．1994，6：649．
[32] Ganzhorn A J, et al. Biochem. Biophys. Acta. 1993, 1161:303.
[33] 郭尧君．荧光实验技术及其在分子生物学中的运用．北京：科学出版社，1983，123．
[34] 许根俊．酶的作用原理．北京：科学出版社，1984，108．
[35] 杨斌盛，杨频．生物化学和生物物理进展．1992，19(2)：110-114．