Complex photophysics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex.
Complex photophysics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex.
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猪胰磷脂酶 A2 的单个色氨酸、其酶原和酶/胶束复合物的复杂光物理学。
DOI：
10.1021/bi00346a033
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Hudson,BS
中科院分区：
文献类型：
--
作者：
Ludescher,RD;Volwerk,JJ;deHaas,GH;Hudson,BS
材料与方法样品制备.从猪胰腺中分离出磷脂酶A2的酶原，并通过有限的蛋白水解将其转化为完全活性的酶，如Niewenhuizen等人所述。（1974年）。C16 PN如货车Dam-Mieras et al.（1975年）。所有蛋白质和脂质溶液均在0.1 M乙酸钠缓冲液（pH 6.0）中用玻璃蒸馏水制备。将蛋白质作为冻干粉末储存在-20 ℃下，并在使用前以接近10 µ的浓度溶解在缓冲液中。以约500的去污剂/蛋白质比制备PLA 2和C16 PN的复合物，最终C16 PN浓度为5 mM。通过使用PLA 2为13.0和proPLA 2为12.3的E2 ib，由280 nm处的光密度确定蛋白质浓度（货车Dam-Mieras等人，1975年）。使用购自Sigma Chemical Co. Fluorescence Spectra的色氨酸和N-乙酰基色氨酸酰胺。荧光发射光谱在23 ℃下用与微计算机连接的SLM-8000分光荧光计（SLM Industries，Urbana，IL）收集。激发波长为295 nm（狭缝4 nm），发射波长为300 - 475 nm（狭缝2 nm），增量为1 nm，积分时间为2 s。为了消除激发单色器的明显偏振偏置，有必要在激发光束中放置一个石英消偏器。收集光谱作为发射与灯参考的比率，并通过使用SLM提供的校正曲线针对发射单色器和光电倍增管的波长依赖性偏置进行校正。该方法的有效性通过与色氨酸和NATA的已发表光谱参数的一致性得到证实。校正光谱的最大发射波长由发射强度与波长的一阶导数曲线确定。通过图解分析确定校正光谱的半宽。相对量子产率由校正的发射光谱的积分与校正的发射光谱的积分的比率确定。
Materials and MethodsSample Preparation. The zymogen of phospholipase A2 was isolated from hog pancreas and convertedinto fully active enzyme by limited proteolysis as described in Niewenhuizen et al.(1974). C16PN was synthesized as described in Van Dam-Mieras et al.(1975). All protein and lipid solutions were prepared in 0.1 M sodium acetate buffer, pH 6.0, with glass distilled water. Protein was stored as a lyophilized powder at-20 C and dissolved in buffer prior to use at concentrations near 10 µ. The complex of PLA2 andC16PN was prepared at a detergent/protein ratio of about500 with a final C16PN concentration of 5 mM. The concentration of protein was determined from the optical density at 280 nm by using£ 2ib of 13.0 for PLA2 and 12.3 for proPLA2 (Van Dam-Mieras et al., 1975). Tryptophan and TV-acetyltryptophanamide were used as purchased from Sigma Chemical Co. Fluorescence Spectra. Fluorescence emission spectra were collected at 23 C with an SLM-8000 spectrofluorometer (SLM Industries, Urbana, IL) interfaced to a microcomputer. Excitation was at 295 nm(slit 4 nm), and emission was scanned from 300 to 475 nm(slit 2 nm) in increments of 1 nm with a 2-s integration time. It was necessary to place a quartz depolarizer in the excitation beam to eliminate the pronounced polarization bias of the excitation monochromator. Spectra were collected as a ratio of emission to lamp reference and were corrected for the wavelength-dependent bias of the emission monochromator and photomultiplier tube by using a correction curve supplied by SLM. The validity of this procedure was confirmed by agreement with published spectral parameters for tryptophan and NATA. The wavelength of maximum emission of the corrected spectra was determined from plots of the first derivative of the emission intensity vs. wavelength. The half-width of the corrected spectra was determined by graphical analysis. Relative quantum yields were determined from the ratio of the integral of the corrected emission spectra to the integral of the corrected emission