天花粉凝集素的荧光光谱研究

天花粉凝集素在天然状态下荧光发射峰位于３３２ｎｍ处,以丙烯酰胺,ＫＩ及ＣｓＣ１等淬灭剂研究ＴＫＬ分子中Ｔｒｐ残基的微环境,发现只有丙烯酰胺能淬灭ＴＫＬ分子Ｔｒｐ的荧光,同此推断大部分的Ｔｒｐ残基位于ＴＫＬ分子内部,其荧光不易为Ｉ＾－或Ｃｓ＾＋接近而淬灭。疏水探针ＴＮＳ能够检测到ＴＫＬ中疏水微区的存在,并且这一疏水微区亦不同于ＴＫＬ的半乳糖结合位点,ＴＫＬ中不存在金属离子的结合部位。     

油麻藤凝集素的荧光光谱研究

用化学修饰、内源荧光和荧光淬灭等方法研究了油麻藤凝集素（ＭＳＬ）的溶液构象变化和微环境的构象特征。研究发现ＭＳＬ分子中总共有９个色氨酸（Ｔｒｐ）残基,它们的荧光能被丙烯酰胺淬灭,但不易为ＫＩ接近而淬灭,ＭＳＬ经Ｎ－溴代琥珀酰亚胺（ＮＢＳ）修饰后,其内源性荧光发射谱发生相应变化,结果表明ＭＳＬ分子中部分Ｔｒｐ残基埋藏于分子内部,而位于分子表面的Ｔｒｐ残基可能处于分子的疏水袋中。     

油麻藤凝集素的荧光光谱研究

用化学修饰,内源荧光和荧光淬灭等方法研究了油麻藤集素（ＭＳＬ）的溶液的象变化和微环境的构象特征,研究发现ＭＳＬ分子中总共有９个色氨酸（Ｔｒｐ）残基,它们的荧光能被丙烯酰胺淬灭,但不易为ＫＩ接近而淬灭,ＭＳＬ经Ｎ－溴化琥珀酰亚胺（ＮＢＳ）修饰后,其内源性荧光发射谱发生相应变化,结果表明ＭＳＬ分子中部分Ｔｒｐ残基埋藏于分子内部,而位于分子表面的Ｔｒｐ残基可能处于分子的疏水袋中。     

野花生豆凝集素的化学修饰与荧光光谱研究

野花生豆凝集素（ＣＭＬ）经ＳｅｐｈａｄｅｘＧ－２００测得分子量为１０３．ＯｋＤ．用对二甲基氨基苯甲醛（ＤＡＢ）为显色剂,测得每个ＣＭＬ分子含有５．９个色氨酸残基．在ｐＨ５．１,含８ｍｏｌ／Ｌ脲的醋酸缓冲液中,Ｎ－溴代丁二酰亚胺（ＮＢＳ）可修饰ＣＭＬ分子中的５．６个色氨酸（Ｔｒｐ）残基,同时使ＣＭＬ的凝血活性完全丧失．用焦碳酸二乙酯（ＤＥＰＣ）和Ｎ－乙酰顺丁烯酰胺（ＮＥＭ）分别修饰ＣＭＬ的组氨酸残基和半胱氨酸巯基后,ＣＭＬ的活性均无变化．ＣＭＬ在天然状态下荧光发射峰位于３３６ｎｍ处,用ＣＭＬ的专一性抑制糖Ｎ－乙酰半乳糖胺研究色氨酸的微环境,发现Ｎ－乙酰半乳糖胺可以淬灭ＣＭＬ中８８％的色氨酸残基萤光,Ｓｔｅｒｎ－Ｖｏｌｍｅｒ常数Ｋ＝１．７３Ｌ／ｍｏｌ．同时发现Ｎ－乙酰半乳糖胺能够保护ＣＭＬ,避免ＮＢＳ对ＣＭＬ的修饰作用,表明色氨酸可能是ＣＭＬ维待活性所必需,并直接参与和专一性抑制糖的结合,其微环境较为疏水．     

红花菜豆（矮生红花变种）凝集素分子的色氨酸残基修饰与其活性的关系

用各种化学试剂修饰红花菜豆凝集素分子,测定与其活性相关的氨基酸残基,经ＮＢＳ修饰表明ＰＣＬ具有８个Ｔｒｐ残基,其中４个暴露于分子表面,此４个Ｔｒｐ残基被修饰后,ＰＬＣ的凝血活性完全丧失,比较ＰＣＬ修饰前后的ＣＤ光谱表明修饰不改变其二级结构。修饰Ｔｙｒ,Ａｒｇ,Ｈｉｓ残基和游离氨基及羧基不影响ＰＣＬ的血凝活性,巯基也不是血凝活性所必需,但是ＰＣＬ分子中二硫键被还的,或被ＣＮＢｒ分解为两个片断则使蛋白     

红花菜豆(矮生红花变种)凝集素分子的色氨酸残基修饰与其活性的关系

用各种化学试剂修饰红花菜豆（Ｐｈａｓｅｏｌｕｓｃｏｃｃｉｎｅｕｓｖａｒｒｕｂｒｏｎａｎｕｓ,Ｂｅｒｒｙ）凝集素（简称ＰＣＬ）分子,测定与其活性相关的氨基酸残基．经ＮＢＳ修饰表明ＰＣＬ具有８个Ｔｒｐ残基,其中４个暴露于分子表面,此４个Ｔｒｐ残基被修饰后,ＰＣＬ的凝血活性完全丧失．比较ＰＣＬ修饰前后的ＣＤ光谱表明修饰不改变其二级结构。修饰Ｔｙｒ,Ａｒｇ,Ｈｉｓ残基和游离氨基及羧基不影响ＰＣＬ的血凝活性．巯基也不是血凝活性所必需,但是ＰＣＬ分子中的二硫键被还原,或被ＣＮＢｒ分解为两个片断则使蛋白质丧失血凝活性,提示分子的完整结构对ＰＣＬ的血凝活力是重要的     

红花菜豆凝集素的荧光光谱学研究

利用荧光光谱方法研究了红花菜豆凝集素（Ｐｈａｓｅｏｌｕｓｃｏｃｃｉｎｅｕｓｖａｒ．ｒｕｂｒｏｎａｎｕｓｌｅｃｔｉｎ,简称ＰＣＬ）,结果表明ＰＣＬ分子各亚基中的两个色氨酸（Ｔｒｐ）残基分别位于ＰＣＬ分子表面和分子内。标记了ＤＮＳ的ＰＣＬ荧光偏振研究指出,致使ＰＣＬ在１０ｍｍｏｌ／ＬＳＤＳ条件下失活的主要原因可能是亚基解离。荧光偏振研究还表明,甲状腺球蛋白、甘露聚糖、海参多糖硫酸酯可与ＰＣＬ结合。荧光探针ｂｉｓ－ＡＮＳ与ＰＣＬ的结合可引起明显的荧光增强和发射谱蓝移,表明ＰＣＬ分子中存有疏水区域。结合了的ｂｉｓ－ＡＮＳ还可和ＰＣＬ中的Ｔｒｐ发生能量传递。     

皖南尖吻蝮蛇蛇毒中纤溶组分的荧光特性

用荧光光谱方法研究了皖南尖吻蝮蛇蛇毒中纤溶组分。研究了Ａｃｒ对ＦＰ内源发光的影响,结果表明,每个ＦＰ分子含有多个Ｔｒｐ残基,这些Ｔｒｐ残基约有８３％可被Ａｃｒ所淬灭,而且这些Ｔｒｐ残基位于ＦＰ分子的较疏水环境中。     

钙离子对江浙蝮蛇蛇毒中性磷脂酶A2溶液构象的研究

用荧光光谱方法研究了钙离子对江浙蝮蛇毒中性磷脂酶Ａ２（简称ＮＰＬＡ２）构象的影响。结果表明,Ｃａ２＋能使酶中唯一的色氨酸残基的荧光增强：只有在Ｃａ２＋存在时,底物卵磷脂才明显改变酶分子中Ｔｒｐ周围的环境,使其光谱的兰移达７ｎｍ,荧光增强约一倍：酶中唯一的Ｈｉｓ残基被修饰以后,则没有上述两种现象发生;结合在ＮＰＬＡ２上的ｂｉｓＡＮＳ的荧光强度,随Ｃａ２＋浓度的增加而增强,提示Ｃａ２＋对ｂｉｓ－ＡＮＳ结合区域的构象有明显影响。     

野花生豆凝集素的化学修饰与荧光光谱研究

野花生豆凝集素（ＣＭＬ）经Ｓｅｐｈａｄｅｘ Ｇ－２００测得分子量为１０３．Ｏ ｋＤ。用对二甲基氨基苯甲醛（ＤＡＢ）为显色剂,测得每个ＣＭＬ分子含有５．９个色氨酸残基。在ｐＨ５．１,含８ｍｏｌ／Ｌ脲的醋酸缓冲中,Ｎ－溴代丁二酰亚胺（ＮＢＳ）可修饰ＣＭＬ分子中的５．６个色氨酸（Ｔｒｐ）残基,同时使ＣＭＬ的凝血活性完全丧失。用焦碳酸二乙酯（ＤＥＰＣ）和Ｎ－乙酰顺丁烯酰胺（ＮＥＭ）分别修饰ＣＭＬ的组氨酸残     

Venom exonuclease. IV. Intrinsic fluorescence of the exonuclease from Crotalus adamanteus venom

The intrinsic fluorescence of the exonuclease isolated from Crotalus adamanteus venom, was studied. The position of its maximum at 335 nm and half-width of the emission band 55 nm (lambda exc. 295 nm) suggested the existence of at least two types of tryptophan residues in the enzyme molecule. Differential analysis of the fluorescence spectra obtained by excitation at 280 and 295 nm revealed about 12.5% contribution of the tyrosine fluorescence in the overall emission excited at 280 nm. The environment of the tryptophan residues in the exonuclease was studied by quenching of their fluorescence with various ionic (NO3-, NO2-, I-, Br- and Cs+) and non-ionic agents (acrylamide, chloroform-methanol). On this basis, fractions of inner (non-polar) and surface tryptophan residues located in charged and neutral regions of the enzyme molecule were evaluated. More than half of the residues (60%) was found in the inner part of the exonuclease while most of its surface tryptophans--in a neutral region(s).     

N-溴代琥珀酰亚胺修饰菌紫质中色氨酸的光谱学研究

采用紫外-可见吸收光谱和荧光光谱方法研究了菌紫质（Bacteriorhodopsin,bR）中的8个色氨酸（Tryptophan,Trp）残基在被N-溴代琥珀酰亚胺（N-bromosuccinimide,NBS）修饰过程中的残基数目及对应的光谱变化。研究结果显示：随着NBS／bR摩尔比例增加逐渐被修饰的Trp残基有4个左右,如果NBS过量,则Trp残基的修饰个数最终可迭6～7个;伴随化学修饰出现Trp残基特征荧光峰值下降及峰位蓝移。研究结果揭示了bR中Trp残基可能的三种结构分布,对于进一步弄清bR中Trp-视黄醛（Retinal）偶联能量传递、单独Trp残基的荧光寿命和Tm残基在膜蛋白结构和功能中的作用具有积极而重要的意义。     

Organization and dynamics of tryptophan residues in tetrameric and monomeric soybean agglutinin: Studies by steady-state and time-resolved fluorescence,phosphorescence and chemical modification

We have investigated the organization and dynamics of tryptophan residues in tetrameric, monomeric and unfolded states of soybean agglutinin (SBA) by selective chemical modification, steady-state and time-resolved fluorescence, and phosphorescence. Oxidation with N-bromosuccinimide (NBS) modifies two tryptophans (Trp 60 and Trp 132) in tetramer, four (Trp 8, Trp 203 and previous two) in monomer, and all six (Trp 8, Trp 60, Trp 132, Trp 154, Trp 203 and Trp 226) in unfolded state. Utilizing wavelength-selective fluorescence approach, we have observed a red-edge excitation shift (REES) of 10 and 5 nm for tetramer and monomer, respectively. A more pronounced REES (21 nm) is observed after NBS oxidation. These results are supported by fluorescence anisotropy experiments. Acrylamide quenching shows the Stern–Volmer constant (K_SV) for tetramer, monomer and unfolded SBA being 2.2, 5.0 and 14.6 M⁻¹, respectively. Time-resolved fluorescence studies exhibit biexponential decay with the mean lifetime increasing along tetramer (1.0 ns) to monomer (1.9 ns) to unfolded (3.6 ns). Phosphorescence studies at 77 K give more structured spectra, with two (0,0) bands at 408.6 (weak) and 413.2 nm for tetramer. However, a single (0,0) band appears at 411.8 and 407.2 nm for monomer and unfolded SBA, respectively. The exposure of hydrophobic surface in SBA monomer has been examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, which shows ∼20-fold increase in ANS fluorescence compared to that for tetramer. The mean lifetime of ANS also shows a large increase (12.0 ns) upon binding to monomer. These results may provide important insight into the role of tryptophans in the folding and association of SBA, and oligomeric proteins in general.     

Fluorescence and chemical modification of tryptophan as probes of structure of GTP:AMP phosphotransferase from beef heart mitochondria

Selective modification of the two Trp residues of GTP:AMP phosphotransferase from beef heart mitochondria (Mr 26 000; MgGTP + AMP in equilibrium MgGDP + ADP) has been attained by treatment of the enzyme with N-bromosuccinimide at pH 4.0. Almost complete loss of activity is observed when one Trp is oxidized. Fluorescence emission spectra (lambda exc 295 nm) were recorded over the pH range 1.9-12.2. Quenching constants, K, with acrylamide were 4.9, 3.4, 3.1, 2.4, 9.2 and 9.4 M-1 at respective pH values of 11.1, 7.5, 5.5, 4.0, 1.9 and 7.5 with 6 M guanidine/HCl. Over the pH range 8.0-5.5 the fluorescence peak has a constant height with maximum at 333-334 nm, which can be segregated by acrylamide quenching into a peak with maximum at 338 nm and another with maximum at 330 nm. Dropping the pH from 5.5 to 4.0 results in the fluorescence at 338 nm decreasing to 335 nm (indicative of less exposure of the Trp) while that at 330 nm remains constant. Thus the limitation of reactivity to N-bromosuccinimide to pH 4.0 or lower cannot be accounted for by increased exposure of the Trp residues but rather must be explained by a change in the microenvironment of each Trp. As shown by K values above, at pH 2.0 Trp residues are exposed to the solvent, as in the case of treatment with 6 M guanidine hydrochloride. In raising the pH from 8.0 to 12.0 a number of changes occur: (a) the lambda max of emission shifts from 333-334 nm to 343 nm; (b) residue(s) become(s) more available to acrylamide quenching; (c) fluorescence decreases and enzymatic activity increases, both with a midpoint at about 10.6; (d) absorption difference spectra show a maximum at 295 nm typical of Tyr ionization. These data are consistent with conformational change as the pH becomes more alkaline making the Trp residue(s) more exposed to the solvent and/or to non-radiative energy transfer to tyrosinate.     

Fluorescence quenching studies of Trp repressor using single-tryptophan mutants

Time-resolved and steady-state fluorescence have been used to resolve the heterogeneous emission of single-tryptophan-containing mutants of Trp repressors W19F and W99F into components. Using iodide as the quencher, the fluorescence-quenching-resolved spectra (FQRS) have been obtained The FQRS method shows that the fluorescence emission of Trp99 can be resolved into two component spectra characterized by maxima of fluorescence emission at 338 and 328 nm. The redder component is exposed to the solvent and participates in about 21% of the total fluorescence emission of TrpR W19F. The second component is inacessible to iodide, but is quenched by acrylamide. The tryptophan residue 19 present in TrpR W99F can be resolved into two component spectra using the FQRS method and iodide as a quencher. Both components of Trp19 exhibit similar maxima of emission at 322–324 nm and both are quenchable by iodide. The component more quenchable by iodide participates in about 38% of the total TrpR W99F emission. The fluorescence lifetime measurements as a function of iodide concentration support the existence of two classes of Trp99 and Trp19 in the Trp repressor. Our results suggest that the Trp aporepressor can exist in the ground state in two distinct conformational states which differ in the microenvironment of the Trp residues.Abbreviations TrpR tryptophan aporepressor fromE. coli - TrpR W19F TrpR mutant with phenylalanine substituted for tryptophan at position 19 - TrpR W99F TrpR mutant with phenylalanine substituted for tryptophan at position 99 - FQRS fluorescence-quenching-resolved spectra - FPLC fast protein liquid chromatography     

An unusual fluorescence spectrum of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor, a dimeric protein proteinase inhibitor isolated in crystalline form by Murae et al. in 1972, contains three tyrosine and one tryptophan residues per monomer unit and has unusual fluorescence properties. When excited at 280 nm, it shows a characteristic fluorescence spectrum having a peak at 307 nm and a shoulder near 340 nm, a feature which has been recognized only for a very few cases in proteins containing both tryosine and tryptophan residues. When excited at 295 nm, at which tryrosine scarcely absorbs, the inhibitor shows an emission spectrum with a peak at 340 nm characteristic of a tryptophan residue. The emission with a peak at 307 nm is considered to arise from the tryrosine residues. The tryptophan quantum yield of Streptomyces subtilisin inhibitor excited at 295 nm is very small, indicating that the tryptophan florescence is strongly quenched in the native state of the inhibitor. Below pH 4 the peak of the fluorescence spectrum of the inhibitor excited at 280 nm shifts toward 340-350 nm with a concomitant increase in the quantum yield. The structural change induced by low pH seems to release the tryptophan fluorescence from the quenching.     

Penetration of lipid chains into transmembrane surfaces of membrane proteins: studies with MscL

The transmembrane surface of a multi-helix membrane protein will be rough with cavities of various sizes between the transmembrane alpha-helices. Efficient solvation of the surface by the lipid molecules that surround the protein in a membrane requires that the lipid fatty acyl chains be able to enter the cavities. This possibility has been investigated using fluorescence quenching methods. Trp residues have been introduced into lipid-facing sites in the first transmembrane alpha-helix (M1) of the mechanosensitive channel of large-conductance MscL; lipid-facing residues at the N-terminal end of M1 are buried below the transmembrane surface of the protein. Fluorescence emission maxima for lipid-facing Trp residues in M1 vary with position in the bilayer comparably to those for Trp residues in the second transmembrane alpha-helix (M2) despite the fact that lipid-facing residues in M2 are on the surface of the protein. Fluorescence emission spectra for most Trp residues on the periplasmic sides of M1 and M2 fit well to a model proposing a trough-like variation of dielectric constant across the membrane, but the relationship between location and fluorescence emission maximum on the cytoplasmic side of the membrane is more complex. The fluorescence of Trp residues in M1 is quenched efficiently by phospholipids with bromine-containing fatty acyl chains, showing that the lipid chains must be able to enter the Trp-containing cavities on the surface of MscL, resulting in efficient solvation of the surface.     

Anomalous fluorescence of yeast 3-phosphoglucerate kinase.

The 3-phosphoglycerate kinase (EC 2.7.2.3) of yeast which contains two tryptophyl and eight tyrosyl residues per molecule, displayed an unusualy fluorescence emission spectrum with a maximum at 308 nm when excited at 280 nm. The emission peak shifted to 329 nm when excited at 295 nm. We could confirm that it was due to the efficient quenching of tryptophyl fluorescence as well as to the incomplete energy transfer from tyrosyl to tryptophyl residues. The average fluorescence quantum yield of this protein was 0.076 (excitation at 280 nm) and that of tryptophyl residues was 0.046 (excitation at 295 nm). As the pH of the solution was lowered, the fluorescence intensity of phosphoglycerate kinase at 329 nm dramatically increased between pH 5 and 4, while the position of the peak remained unchanged. When denatured in 4 M guanidine hydrochloride, the protein showed two emission peaks, one at 343 nm and the other at 303 nm.