Vaughan D E, Declerck P J, Reilly T M, Park K, Collen D, Fasman G D
Cardiology Division, West Roxbury VAMC, Boston, MA.
Biochim Biophys Acta. 1993 Oct 6;1202(2):221-9. doi: 10.1016/0167-4838(93)90008-f.
The conformational characteristics of active, latent, and denatured recombinant plasminogen activator inhibitor-1 (rPAI-1) were compared using UV spectroscopy, spectrofluorimetry and circular dichroism (CD) techniques. The UV absorbance wavelength maxima in all preparations approximated 280 nm, while the extinction coefficients of active and latent rPAI-1 differed by up to 60%. When incubated at 37 degrees C, the A280 of latent rPAI-1 was quite stable while the A280 of active rPAI-1 spontaneously increased, eventually approximating that of latent rPAI-1. Alkali difference spectroscopy yielded markedly divergent titration patterns for active and latent rPAI-1, suggesting that the tyrosine residues present in active and latent rPAI-1 differ in terms of solvent exposure. At an excitation wavelength of 280 nm, active rPAI-1 exhibited the greatest relative fluorescence quantum yield. The relative fluorescence of latent and denatured rPAI-1 were less than that of active PAI-1, and the emission maxima of both species were slightly red-shifted in comparison to that of active rPAI-1, suggesting that at least one of the four tryptophan residues present in rPAI-1 is less exposed to the aqueous environment in the active form of the molecule. In contrast, the derived secondary structures based on CD of active and latent rPAI-1 were nearly identical, with both moieties exhibiting approx. 40% alpha-helix and 15% beta-sheet. Taken together, these spectroscopic data provide evidence supporting the hypothesis that active and latent PAI-1 differ in terms of their tertiary conformation and aromatic residue exposure, while their secondary structures appear generally comparable. Furthermore, denaturant-induced reactivation of latent rPAI-1 produces a partially active rPAI-1 with spectroscopic properties similar to that of latent rPAI-1, suggesting that denatured rPAI-1 more closely resembles the latent rPAI-1 conformation after refolding. The spontaneous spectroscopic changes observed in rPAI-1 may reflect conformational transitions that are critical to the regulation of endogenous PAI-1 activity.
运用紫外光谱法、荧光光谱法和圆二色（CD）技术比较了活性、潜伏性和变性重组纤溶酶原激活物抑制剂-1（rPAI-1）的构象特征。所有制剂的紫外吸收波长最大值接近280nm，而活性和潜伏性rPAI-1的消光系数相差高达60%。在37℃孵育时，潜伏性rPAI-1的A280相当稳定，而活性rPAI-1的A280自发增加，最终接近潜伏性rPAI-1的A280。碱差光谱法得出活性和潜伏性rPAI-1的滴定模式明显不同，表明活性和潜伏性rPAI-1中存在的酪氨酸残基在溶剂暴露方面存在差异。在280nm的激发波长下，活性rPAI-1表现出最大的相对荧光量子产率。潜伏性和变性rPAI-1的相对荧光低于活性PAI-1，并且与活性rPAI-1相比，这两种物质的发射最大值都略有红移，表明rPAI-1中存在的四个色氨酸残基中至少有一个在分子的活性形式中较少暴露于水环境。相反，基于活性和潜伏性rPAI-1的CD推导的二级结构几乎相同，两者都表现出约4%的α-螺旋和15%的β-折叠。综上所述，这些光谱数据提供了证据支持以下假设：活性和潜伏性PAI-1在三级构象和芳香族残基暴露方面存在差异，而它们的二级结构总体上似乎具有可比性。此外，变性剂诱导的潜伏性rPAI-1再激活产生一种部分活性的rPAI-1，其光谱性质与潜伏性rPAI-1相似，表明变性rPAI-1在重折叠后更类似于潜伏性rPAI-1的构象。在rPAI-1中观察到的自发光谱变化可能反映了对内源性PAI-1活性调节至关重要的构象转变。
