Effects of salts on the interaction of 8-anilinonaphthalene 1-sulphonate and thermolysin

Neutral salts activate and stabilize thermolysin. In this study, to explore the mechanism, we analyzed the interaction of 8-anilinonaphthalene 1-sulphonate (ANS) and thermolysin by ANS fluorescence. At pH 7.5, the fluorescence of ANS increased and blue-shifted with increasing concentrations (0–2.0?μM) of thermolysin, indicating that the anilinonaphthalene group of ANS binds with thermolysin through hydrophobic interaction. ANS did not alter thermolysin activity. The dissociation constants (K_d) of the complex between ANS and thermolysin was 33?±?2?μM at 0?M NaCl at pH 7.5, decreased with increasing NaCl concentrations, and reached 9?±?3?μM at 4?M NaCl. The K_d values were not varied (31?34?μM) in a pH range of 5.5?8.5. This suggests that at high NaCl concentrations, Na⁺ and/or Cl^– ions bind with thermolysin and affect the binding of ANS with thermolysin. Our results also suggest that the activation and stabilization of thermolysin by NaCl are partially brought about by the binding of Na⁺ and/or Cl^– ions with thermolysin.     

Analysis of the mechanism of inhibition of human matrix metalloproteinase 7 (MMP-7) activity by green tea catechins

Green tea catechins inhibit human matrix metalloproteinase 7 (MMP-7) activity non-competitively, and the galloyl group is essential for potent inhibition (Oneda et al., J. Biochem., 133, 571-576 (2003)). In this study, we analyzed the mechanism of this inhibition. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2), the inhibitory effects of (-)-epigallocatechin-3-gallate (EGCG), (-)-gallocatechin-3-gallate (GCG), (-)-epicatechin-3-gallate (ECG), and (-)-catechin-3-gallate (CG) increased with increasing pH levels from 7.0 to 8.5. The inhibitory effects of EGCG and GCG were more potent than those of ECG and CG, and increased with increasing CaCl(2) concentrations from 10 to 50 mM. The fluorescence of EGCG and GCG decreased with increasing CaCl(2) concentrations and with the addition of MMP-7, while those of ECG and CG did not. Our results suggest that these differences result from that in the B ring, EGCG and GCG have phenol hydroxyl groups at the 3', 4', and 5' positions, while ECG and CG have them at the 3' and 4' positions.     

Expression in Escherichia coli, refolding, and purification of the recombinant mature form of human matrix metalloproteinase 7 (MMP-7)

In the latent pro-form of matrix metalloproteinase 7 (MMP-7), the cysteine residue in the pro-peptide binds the active-site zinc ion. Hence, recombinant active MMP-7 was prepared from pro-MMP-7 by modification of this cysteine residue with a mercuric reagent. In this study, mature MMP-7 was expressed in Escherichia coli as inclusion bodies, solubilized, and refolded with 1 M L-arginine. The purified product was indistinguishable from the one prepared from pro-MMP-7 as assessed by hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2).     

1-Anilino-8-Naphthalene Sulfonate (ANS) Is Not a Desirable Probe for Determining the Molten Globule State of Chymopapain

The molten globule (MG) state of proteins is widely detected through binding with 1-anilino-8-naphthalene sulphonate (ANS), a fluorescent dye. This strategy is based upon the assumption that when in molten globule state, the exposed hydrophobic clusters of protein are readily bound by the nonpolar anilino-naphthalene moiety of ANS molecules which then produce brilliant fluorescence. In this work, we explored the acid-induced unfolding pathway of chymopapain, a cysteine proteases from Carica papaya, by monitoring the conformational changes over a pH range 1.0–7.4 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). The spectroscopic measurements showed that although maximum ANS fluorescence intensity was observed at pH 1.0, however protein exhibited ∼80% loss of secondary structure which does not comply with the characteristics of a typical MG-state. In contrast at pH 1.5, chymopapain retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii and nearly 30-fold increase in ANS fluorescence with respect to the native state, indicating that MG-state exists at pH 1.5 and not at pH 1.0. ITC measurements revealed that ANS molecules bound to chymopapain via hydrophobic interaction were more at pH 1.5 than at pH 1.0. However, a large number of ANS molecules were also involved in electrostatic interaction with protein at pH 1.0 which, together with hydrophobically interacted molecules, may be responsible for maximum ANS fluorescence. We conclude that maximum ANS-fluorescence alone may not be the criteria for determining the MG of chymopapain. Hence a comprehensive structural analysis of the intermediate is essentially required.     

Thermodynamic analysis of ionizable groups involved in the catalytic mechanism of human matrix metalloproteinase 7 (MMP-7)

Human matrix metalloproteinase 7 (MMP-7) exhibits a broad bell-shaped pH-dependence with the acidic and alkaline pK(e) (pK(e1) and pK(e2)) values of about 4 and 10. In this study, we estimated the ionizable groups involved in its catalytic mechanism by thermodynamic analysis. pK(a) of side chains of L-Asp, L-Glu, L-His, L-Cys, L-Tyr, L-Lys, and L-Arg at 25-45°C were determined by the pH titration of amino-acid solutions, from which their enthalpy changes, ?H°, of deprotonation were calculated. pK(e1) and pK(e2) of MMP-7 at 15-45°C were determined in the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2), from which ?H(o) for pK(e1) and pK(e2) was calculated. The ?H(o) for pK(e1) (-20.6±6.1kJmol(-1)) was similar to that for L-Glu (-23.6±5.8kJmol(-1)), and the ?H(o) for pK(e2) (89.9±4.0kJmol(-1)) was similar to those for L-Arg (87.6±5.5kJmol(-1)) and L-Lys (70.4±4.4kJmol(-1)). The mutation of the active-site residue Glu198 into Ala completely abolished the activity, suggesting that Glu198 is the ionizable group for pK(e1). On the other hand, no arginine or lysine residues are found in the active site of MMP-7. We proposed a possibility that a protein-bound water is the ionizable group for pK(e2).     

Effect of protons and cations on chloroplast membranes as visualized by the bound ANS fluorescence

Structural changes in the chloroplast membranes caused by acidification and heat-treatment are studied by observing the changes in the fluorescence of ANS bound to thylakoid membranes. On addition of acids to buffered suspension of isolated pea chloroplasts, the fluorescence intensity of bound ANS shows a sigmoidal rise on reaching a pH value of about 4.5. A part of the fluorescence enhancement of bound ANS brought about by protons is not reversible on back titration with alkali. The reversible part of acid induced rise in ANS fluorescence possibly reflects structural changes expected to be associated with photophosphorylation. Divalent cations enhance the fluorescence of ANS bound to chloroplasts between a pH range 4.5–7.0 but diminish it if the pH is below 4.5.Addition of acid to heat-treated chloroplasts shows similar sigmoidal rise in ANS fluorescence intensity on lowering the pH to about 4.5. On addition of acid upto a pH of 3.1, the ANS fluorescence is greater than that of untreated chloroplasts, however, at pH below 3.1, the fluorescence of bound ANS is lower than the control chloroplasts. This observation indicates that heat-treatment caused some alteration of the microstructure of thylakoid membranes of chloroplasts besides the usual loss in the O₂ evolving capacity.This is further confirmed from the studies of Hill-activity and ANS binding to chloroplasts incubated at various temperatures in the absence and presence of aliphatic alcohol. Hill-activity (DCPIP reduction) of chloroplasts incubated at temperatures between 25 C and 55 C first increases reaching a maximum at 45 C and then declines rather sharply, when the chloroplasts are heated beyond 45 C (Tmax). The presence of 200 mM n-butyl alcohol or 40 mM n-amyl alcohol during the warming treatment lowers the temperature by 8 C at which the decline in the Hill-activity is observed. An enhancement in the fluorescence intensity and a blue shift of the emission spectrum of bound ANS are noted if the chloroplasts are heated beyond the Tmax either in absence or presence of alcohol. The changes in the fluorescence of ANS bound to heat-treated chloroplasts plausibly reflect the nature of the structural changes in chloroplasts during the heating upto 55 C.Abbreviations ANS 1-anilino-8-naphthalene sulphonate - DCPIP 2,6-dichlorophenol indophenol     

The binding of 1,8 ANS congeners to I-FABP and comparison of some hypotheses about ANS' spectral sensitivity to environment

The ANS congeners 1-anilinonaphthalene and 1-amino,8-sulfonato naphthalene were investigated as analogs of 1,8 anilinonaphthalene sulfonate. Like 1,8 ANS, they also bind to I-FABP, and the fully-bound spectra reveal interesting similarities and differences with respect to ANS binding. The nature of these differences suggests that certain hypotheses in the literature about ANS photophysics ought to be revised. The conceptual decomposition of energetic effects in the thermodynamics of ANS binding proposed earlier [W. Kirk, E. Kurian, F. Prendergast, Characterization of the sources of protein-ligand affinity: 1-sulfonato-8-(1')anilinonaphthalene binding to intestinal fatty acid binding protein. Biophys. J. (1996) 70 69-83.] is extended further in this report.     

8-anilino-1-naphthalene sulfonic acid (ANS) induces folding of acid unfolded cytochrome c to molten globule state as a result of electrostatic interactions.

Hydrophobic interaction of 8-anilino-1-naphthalene sulfonic acid (ANS) with proteins is one of the widely used methods for characterizing/detecting partially folded states of proteins. We have carried out a systematic investigation on the effect of ANS, a charged hydrophobic fluorescent dye, on structural properties of acid-unfolded horse heart cytochrome c at pH 2.0 by a combination of optical methods and electrospray ionization mass spectroscopy (ESI MS). ANS was found to induce, a secondary structure similar to native protein and quenching of fluorescence of tryptophan residue, in the acid-unfolded protein. However, the tertiary structure was found to be disrupted thus indicating that ANS stabilizes a molten globule state in acid-unfolded protein. To understand the mechanism of ANS-induced folding of acid-unfolded cytochrome c, comparative ESI MS, soret absorption, and tryptophan fluorescence studies using nile red, a neutral hydrophobic dye, and ANS were carried out. These studies suggested that, at low pH, electrostatic interactions between negatively charged ANS molecules and positively charged amino acid residues present in acid-unfolded cytochrome c are probably responsible for ANS-induced folding of acid-unfolded protein to partially folded compact state or molten globule state. This is the first experimental demonstration of ANS induced folding of unfolded protein and puts to question the usefulness of ANS for characterization/determination of partially folded intermediates of proteins observed under low pH conditions.     

胰蛋白酶与ANS的相互作用

利用荧光光谱法研究了在不同ｐＨ、压力及不同浓度的脲作用时荧光探针１,８－ＡＮＳ（１－ａｎｉｌｉｏｎｎａｐｈｔｈａｌｅｎｅ－８－ｓｕｌｆｏｎｉｃａｃｉｄ）与胰蛋白酶的相互作用．发现在低ｐＨ时ＡＮＳ可以结合到胰蛋白酶上,其中以ｐＨ２．０、３．０时结合最强．进一步的研究发现脲变性对胰蛋白酶结合ＡＮＳ的能力有很大的影响：１．５ｍｏｌ／Ｌ的脲即可使得胰蛋白酶结合ＡＮＳ的能力大大降低,但有趣的是即使高达４ｍｏｌ／Ｌ的脲对胰蛋白酶色氨酸残基荧光也无明显影响．另外,在ｐＨ猝变、脲变性、及逐渐改变压力时,胰蛋白酶色氨酸残基荧光和结合到胰蛋白酶分子上的ＡＮＳ的荧光的变化大不相同．上述结果暗示胰蛋白酶的色氨酸残基所在的区域和其结合ＡＮＳ的区域是两个不相同的区域．     

Dynamics of ANS binding to tuna apomyoglobin measured with fluorescence correlation spectroscopy.

The dynamics of the binding reaction of ANS to native and partly folded (molten globule) tuna and horse apomyoglobins has been investigated by fluorescence correlation spectroscopy and frequency domain fluorometry. The reaction rate has been measured as a function of apomyoglobin and ANS concentrations, pH, and temperature. Examination of the autocorrelation functions shows that the reaction rate is fast enough to be observed in tuna apomyoglobin, whereas the reaction rate in horse apomyoglobin is on the same time scale as diffusion through the volume or longer. Specifically, for tuna apomyoglobin at pH 7 and room temperature the on rate is 2200 microM(-1) s(-1) and the off rate is 5900 s(-1), in comparison with k(on) = 640 microM(-1) s(-1) and k(off) = 560 s(-1) for horse myoglobin as measured previously. The independence of the reaction rate from the ANS concentration indicates that the reaction rate is dominated by the off rate. The temperature dependence of the on-rate shows that this rate is diffusion limited. The temperature dependence of the off rates analyzed by Arrhenius and Ferry models indicates that the off rate depends on the dynamics of the protein. The differences between horse and tuna apomyoglobins in the ANS binding rate can be explained in terms of the three-dimensional apoprotein structures obtained by energy minimization after heme removal starting from crystallographic coordinates. The comparison of the calculated apomyoglobin surfaces shows a 15% smaller cavity for tuna apomyoglobin. Furthermore, a negative charge (D44) is present in the heme cavity of tuna apomyoglobin that could decrease the strength of ANS binding. At pH 5 the fluorescence lifetime distribution of ANS-apomyoglobin is bimodal, suggesting the presence of an additional binding site in the protein. The binding rates determined by FCS under these conditions show that the protein is either in the open configuration or is more flexible, making it much easier to bind. At pH 3, the protein is in a partially denatured state with multiple potential binding sites for ANS molecule, and the interpretation of the autocorrelation function is not possible by simple models. This conclusion is consistent with the broad distribution of ANS fluorescence lifetimes observed in frequency domain measurements.     

Effect of a laminin amphiphatic sequence on DPPC ordered bilayers.

A peptide sequence, stearoyl-GESIKVAVS(NH2), related to a laminin fragment, has been synthesized. Formation of aggregates was controlled by titrating a sodium anilinonaphthalene sulphonate (ANS) solution with peptide and recording fluorescence intensity increases. The results show that this system experiences a sudden increase in fluorescence at peptide concentrations around 2.5 x 10(-4) mol/L. The interaction of this hydrophobic peptide with DPPC vesicles has been studied using fluorescence techniques. Its influence on the microviscosity of bilayers was determined by studying polarization/temperature dependence for ANS and diphenyl hexatriene (DPH) fluorescent probes. With both markers the presence of peptide promotes a clear increase in anisotropy values. This indicates a rigidifying effect. Leakage studies carried out with liposomes loaded with carboxyfluorescein (CF) indicate a stabilizing effect of the peptide on bilayers, in agreement with results obtained with fluorescent probes.     

The binding of 1,8 ANS congeners to I-FABP and comparison of some hypotheses about ANS' spectral sensitivity to environment

The ANS congeners 1-anilinonaphthalene and 1-amino,8-sulfonato naphthalene were investigated as analogs of 1,8 anilinonaphthalene sulfonate. Like 1,8 ANS, they also bind to I-FABP, and the fully-bound spectra reveal interesting similarities and differences with respect to ANS binding. The nature of these differences suggests that certain hypotheses in the literature about ANS photophysics ought to be revised. The conceptual decomposition of energetic effects in the thermodynamics of ANS binding proposed earlier [1] [W. Kirk, E. Kurian, F. Prendergast, Characterization of the sources of protein–ligand affinity: 1-sulfonato-8-(1′)anilinonaphthalene binding to intestinal fatty acid binding protein. Biophys. J. (1996) 70 69–83.] is extended further in this report.     

A partially folded intermediate conformation is induced in pectate lyase C by the addition of 8-anilino-1-naphthalenesulfonate (ANS)

Addition of 8-anilino-1-naphthalenesulfonate (ANS) to acid-denatured pectate lyase C (pelC) leads to a large increase in the fluorescence quantum yield near 480 nm. The conventional interpretation of such an observation is that the ANS is binding to a partially folded intermediate such as a molten globule. Far-ultraviolet circular dichroism demonstrates that the enhanced fluorescence results from the induction of a partially folded protein species that adopts a large fraction of native-like secondary structure on binding ANS. Thus, ANS does not act as a probe to detect a partially folded species, but induces such a species. Near-ultraviolet circular dichroism suggests that ANS is bound to the protein in a specific conformation. The mechanism of ANS binding and structure induction was probed. The interaction of acid-unfolded pelC with several ANS analogs was investigated. The results strongly indicate that the combined effects of hydrophobic and electrostatic interactions account for the relatively high binding affinity of ANS for acid-unfolded pelC. These results demonstrate the need for caution in interpreting enhancement of ANS fluorescence as evidence for the presence of molten globule or other partially folded protein intermediates.     

Binding of amelogenin to MMP-9 and their co-expression in developing mouse teeth

Amelogenin is the most abundant matrix protein in enamel. Proper amelogenin processing by proteinases is necessary for its biological functions during amelogenesis. Matrix metalloproteinase 9 (MMP-9) is responsible for the turnover of matrix components. The relationship between MMP-9 and amelogenin during tooth development remains unknown. We tested the hypothesis that MMP-9 binds to amelogenin and they are co-expressed in ameloblasts during amelogenesis. We evaluated the distribution of both proteins in the mouse teeth using immunohistochemistry and confocal microscopy. At postnatal day 2, the spatial distribution of amelogenin and MMP-9 was co-localized in preameloblasts, secretory ameloblasts, enamel matrix and odontoblasts. At the late stages of mouse tooth development, expression patterns of amelogenin and MMP-9 were similar to that seen in postnatal day 2. Their co-expression was further confirmed by RT-PCR, Western blot and enzymatic zymography analyses in enamel organ epithelial and odontoblast-like cells. Immunoprecipitation assay revealed that MMP-9 binds to amelogenin. The MMP-9 cleavage sites in amelogenin proteins across species were found using bio-informative software program. Analyses of these data suggest that MMP-9 may be involved in controlling amelogenin processing and enamel formation.     

Prediction of interaction mode between a typical ACE inhibitor and MMP-9 active site

To characterize the inhibitory specificity of angiotensin converting enzyme (ACE) inhibitors for matrix metalloproteinase 9 (MMP-9) activity, molecular modeling of these complex was performed referring the recent X-ray structure analyses using lisinopril as an ACE inhibitor. Two interaction modes differing in the orientation of the inhibitor on the active site were identified. Lisinopril was effectively stabilized by specific hydrogen bonds and hydrophobic interactions in the active site of MMP-9, and its hydrophobic group appeared to interact preferentially with the S1 site compared with the S1' site. These findings showed that ACE inhibitors could become important seeds for cardiovascular protection and the development of MMP inhibitors.     

Conformational Changes of Myoglobin Upon Interaction with Negatively-Charged Phospholipid Vesicles

Abstract

The interaction between myoglobin and negatively-charged liposomes composed of phosphatidylcholine/phosphatidylglycerol (1:1) was studied at low ionic strength under acidic conditions. Changes in the absorbance and the fluorescence spectra of myoglobin were recorded upon addition of liposomes to partially unfolded (pH 3.5) and native (pH 4.5 and pH 6.5) myoglobin. Association of myoglobin with liposomes was a relatively fast process at pH 3.5 and pH 4.5. Although at pH 3.5 myoglobin was unfolded partially before the addition of the liposomes while at pH 4.5 before the addition of liposomes myoglobin retained its native form, similar interaction patterns of myoglobin with liposomes were observed. The fluorescence and absorption spectra in the Soret region of myoglobin clearly indicated that at these pH values myoglobin was associated with the liposomes in a (partially) unfolded state. At pH 6.5 the kinetics of myoglobin association with liposomes was much slower than at pH 3.5 and 4.5. The spectroscopic measurements also indicated that the interaction of myoglobin with liposomes at pH 6.5 followed a different pattern and resulted in different protein structures in comparison with pH 3.5/4.5.     

Detection of MMP-2 and MMP-9 activity in vivo with a triple-helical peptide optical probe

We report a novel activatable NIR fluorescent probe for in vivo detection of cancer-related matrix metalloproteinase (MMP) activity. The probe is based on a triple-helical peptide substrate (THP) with high specificity for MMP-2 and MMP-9 relative to other members of the MMP family. MMP-2 and MMP-9 (also known as gelatinases) are specifically associated with cancer cell invasion and cancer-related angiogenesis. At the center of each 5 kDa peptide strand is a gelatinase sensitive sequence flanked by 2 Lys residues conjugated with NIR fluorescent dyes. Upon self-assembly of the triple-helical structure, the 3 peptide chains intertwine, bringing the fluorophores into close proximity and reducing fluorescence via quenching. Upon enzymatic cleavage of the triple-helical peptide, 6 labeled peptide chains are released, resulting in an amplified fluorescent signal. The fluorescence yield of the probe increases 3.8-fold upon activation. Kinetic analysis showed a rate of LS276-THP hydrolysis by MMP-2 (k(cat)/K(M) = 30,000 s(-1) M(-1)) similar to that of MMP-2 catalysis of an analogous fluorogenic THP. Administration of LS276-THP to mice bearing a human fibrosarcoma xenografted tumor resulted in a tumor fluorescence signal more than 5-fold greater than that of muscle. This signal enhancement was reduced by treatment with the MMP inhibitor Ilomostat, indicating that the observed tumor fluorescence was indeed enzyme mediated. These results are the first to demonstrate that triple-helical peptides are suitable for highly specific in vivo detection of tumor-related MMP-2 and MMP-9 activity.     

Characterization of stromelysin 1 (MMP-3), matrilysin (MMP-7), and membrane type 1 matrix metalloproteinase (MT1-MMP) derived fibrin(ogen) fragments D-dimer and D-like monomer: NH2-terminal sequences of late-stage digest fragments.

Matrix metalloproteinases (MMPs) participate in physiological remodeling of the extracellular matrix. Recently we determined that both fibrinogen (Fg) and cross-linked fibrin (XL-Fb) are substrates for selected MMPs. Specifically, XL-Fb clots were solubilized by MMP-3 (stromelysin 1) by cleavage at gamma Gly 404-Ala 405, resulting in a D-like monomer fragment. Similarly, MMP-7 (matrilysin) and MT1-MMP (membrane type 1 matrix metalloproteinase) solubilized XL-Fb clots. However, the molecular mass of fragment D-dimer, obtained after MMP-7 and MT1-MMP degradation of XL-Fb, is similar to that of fragment D-dimer from plasmin degradation ( approximately 186 kDa). In contrast, fragment D-like monomer, from MMP-3 degradation of both fibrinogen (Fg) and XL-Fb, is similar to fragment D from plasmin degradation of Fg ( approximately 94 kDa). Reduced chains from MMP-3, MMP-7, and MT1-MMP digests of Fg and XL-Fb were subjected to direct sequence analyses and D/D-dimer alpha-chain showed cleavage at both alpha Asp 97-Phe 98 and alpha Asn 102-Asn 103. Degradation of the beta-chain resulted in microheterogeneity of cleavage sites at beta Asp 123-Leu 124, beta Asn 137-Val 138, and beta Glu 141-Tyr 142, whereas all three enzymes cleaved the gamma-chain at gamma Thr 83-Leu 84. In both Fg and XL-Fb, several cleavage sites obtained by proteolysis with MMP-3, MMP-7, and MT1-MMP were found to be in very close proximity to those obtained by plasmin on these same substrates. That does not occur with other MMPs such as MMP-1, -2, and -9 and MT2-MMP. The degradation of XL-Fb by MMPs suggests both plasmin-dependent and independent mechanisms of fibrinolysis that might be relevant in inflammation, angiogenesis, arthritis, and atherosclerosis.     

Photophysics of ANS. III: Circular dichroism of ANS and anilinonaphthalene in I-FABP

We investigate the circular dichroism of the I-FABP system with the ligands ANS (1,8-anilinonaphthalene sulfonate) and AnN (anilinonaphthalene) as previously reported in our earlier publications in the series (referred to as I and II here) on ANS photophysics. We employ our semi-empirical calculated spectral functions (from II) to compute the actual CD spectra, without any additional assumptions or data except what we have previously presented with respect to binding geometry (in I). The common mechanisms fail to produce the observed spectra. However, we identify a novel mechanism of induced CD activity, which does succeed. This new mechanism also suggests how it is that near UV CD can often show extreme sensitivity to local 'order' effects.