Casals C, Miguel E, Perez-Gil J
Department of Biochemistry and Molecular Biology, Faculty of Chemistry, Complutense University of Madrid, Spain.
Biochem J. 1993 Dec 15;296 ( Pt 3)(Pt 3):585-93. doi: 10.1042/bj2960585.
The fluorescence characteristics of surfactant protein A (SP-A) from porcine and human bronchoalveolar lavage were determined in the presence and absence of lipids. After excitation at either 275 or 295 nm, the fluorescence emission spectrum of both proteins was characterized by two maxima at about 326 and 337 nm, indicating heterogeneity in the emission of the two tryptophan residues of SP-A, and also revealing a partially buried character for these fluorophores. Interaction of both human and porcine SP-A with various phospholipid vesicles resulted in an increase in the fluorescence emission of tryptophan without any shift in the emission wavelength maxima. This change in intrinsic fluorescence was found to be more pronounced in the presence of dipalmitoyl phosphatidylcholine (DPPC) than with dipalmitoyl phosphatidylglycerol (DPPG), DPPC/DPPG (7:3, w/w) and 1-palmitoyl-sn-glycerol-3-phosphocholine (LPC). Intrinsic fluorescence of SP-A was almost completely unaffected in the presence of egg phosphatidylcholine (egg-PC). In addition, we demonstrated a shielding of the tryptophan fluorescence from quenching by acrylamide on interaction of porcine SP-A with DPPC, DPPG or LPC. This shielding was most pronounced in the presence of DPPC. In the case of human SP-A, shielding was only observed on interaction with DPPC. From the intrinsic fluorescence measurements as well as from the quenching experiments, we concluded that the interaction of some phospholipid vesicles with SP-A produces a conformational change on the protein molecule and that the interaction of SP-A with DPPC is stronger than with other phospholipids. This interaction appeared to be independent of Ca2+ ions. Physiological ionic strength was found to be required for the interaction of SP-A with negatively charged vesicles of either DPPG or DPPC/DPPG (7:3, w/w). Intrinsic fluorescence of SP-A was sensitive to the physical state of the DPPC vesicles. The increase in intrinsic fluorescence of SP-A in the presence of DPPC vesicles was much stronger when the vesicles were in the gel state than when they were in the liquid-crystalline state. The effect produced by SP-A on the lipid vesicles was also dependent on temperature. The aggregation of DPPC, DPPC/DPPG (7:3, w/w) or dimyristoyl phosphatidylglycerol (DMPG) was many times higher below the phase-transition temperature of the corresponding phospholipids. These results strongly indicate that the interaction of SP-A with phospholipid vesicles requires the lipids to be in the gel phase.
在有脂质和无脂质存在的情况下，测定了猪和人支气管肺泡灌洗中的表面活性蛋白A（SP-A）的荧光特性。在275或295nm激发后，两种蛋白的荧光发射光谱均在约326和337nm处有两个最大值，这表明SP-A的两个色氨酸残基发射存在异质性，也揭示了这些荧光团具有部分埋藏的特性。人和猪的SP-A与各种磷脂囊泡相互作用，导致色氨酸荧光发射增加，而发射波长最大值无任何偏移。发现这种内在荧光的变化在二棕榈酰磷脂酰胆碱（DPPC）存在时比在二棕榈酰磷脂酰甘油（DPPG）、DPPC/DPPG（7:3，w/w）和1-棕榈酰-sn-甘油-3-磷酸胆碱（LPC）存在时更明显。在卵磷脂（egg-PC）存在时，SP-A的内在荧光几乎完全不受影响。此外，我们证明了猪SP-A与DPPC、DPPG或LPC相互作用时，色氨酸荧光可免受丙烯酰胺淬灭的屏蔽作用。这种屏蔽作用在DPPC存在时最为明显。对于人SP-A，仅在与DPPC相互作用时观察到屏蔽作用。从内在荧光测量以及淬灭实验中，我们得出结论，一些磷脂囊泡与SP-A的相互作用会使蛋白质分子发生构象变化，且SP-A与DPPC的相互作用强于与其他磷脂的相互作用。这种相互作用似乎与Ca2+离子无关。发现生理离子强度是SP-A与带负电荷的DPPG或DPPC/DPPG（7:3，w/w）囊泡相互作用所必需的。SP-A的内在荧光对DPPC囊泡的物理状态敏感。当囊泡处于凝胶态时，DPPC囊泡存在下SP-A内在荧光的增加比处于液晶态时要强得多。SP-A对脂质囊泡产生的影响也取决于温度。在相应磷脂的相变温度以下，DPPC、DPPC/DPPG（7:3，w/w）或二肉豆蔻酰磷脂酰甘油（DMPG）的聚集程度要高很多倍。这些结果有力地表明，SP-A与磷脂囊泡的相互作用要求脂质处于凝胶相。
