Quantitative fluorescence analysis of the adsorption of lysozyme to phospholipid vesicles

Experiments directed to measure the interaction of lysozyme with liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) have been conducted by monitoring both protein and lipid fluorescence and fluorescence anisotropy of the protein. The binding of lysozyme to the unilamellar vesicles was quantified using a novel method of analysis in which the fractional contribution at moderate binding conditions is determined from either total fluorescence decay or anisotropy decay curves of tryptophan at limiting binding conditions. In the energy transfer experiments PC and PS lipids labelled with two pyrene acyl chains served as energy acceptors of the excited tryptophan residues in lysozyme. The binding was strongly dependent on the molar fraction of negatively charged PS in neutral PC membranes and on the ionic strength. Changes in the tryptophan fluorescence decay characteristics were found to be connected with long correlation times, indicating conformational rearrangements induced by binding of the protein to these lipid membranes. The dynamics of membrane bound protein appeared to be dependent on the physical state of the membrane. Independent of protein fluorescence studies, formation of a protein-membrane complex can also be observed from the lipid properties of the system. The interaction of lysozyme with di-pyrenyl-labelled phosphatidylserine in anionic PS/PC membranes resulted in a substantial decrease of the intramolecular excimer formation, while the excimer formation of dipyrenyl-labelled phosphatidylcholine in neutral PC membranes barely changed in the presence of lysozyme.Abbreviations dipyr₄ sn-1,2-(pyrenylbutyl) - dipyr₁₀ sn-1,2-(pyrenyldecanoyl). - DMPC dimyristoyl-phosphatidylcholine - DOPC dioleoyl-phosphatidylcholine - DPPC dipalmitoyl-phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - PC phosphatidylcholine - PS phosphatidylserine Correspondence to: A. J. W. G. Visser     

Molecular investigation of the interaction between ionic liquid type gemini surfactant and lysozyme: A spectroscopic and computational approach

Herein, we are reporting the interaction of ionic liquid type gemini surfactant, 1,4‐bis(3‐dodecylimidazolium‐1‐yl) butane bromide ([C₁₂?4‐C₁₂im]Br₂) with lysozyme by using Steady state fluorescence, UV‐visible, Time resolved fluorescence, Fourier transform‐infrared (FT‐IR) spectroscopy techniques in combination with molecular modeling and docking method. The steady state fluorescence spectra suggested that the fluorescence of lysozyme was quenched by [C₁₂?4‐C₁₂im]Br₂ through static quenching mechanism as confirmed by time resolved fluorescence spectroscopy. The binding constant for lysozyme‐[C₁₂?4‐C₁₂im]Br₂ interaction have been measured by UV‐visible spectroscopy and found to be 2.541 × 10⁵M^?1. The FT‐IR results show conformational changes in the secondary structure of lysozyme by the addition of [C₁₂?4‐C₁₂im]Br₂. Moreover, the molecular docking study suggested that hydrogen bonding and hydrophobic interactions play a key role in the protein‐surfactant binding. Additionally, the molecular dynamic simulation results revealed that the lysozyme‐[C₁₂?4‐C₁₂im]Br₂ complex reaches an equilibrium state at around 3 ns. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 406–415, 2015.     

Light scattering,CD, and ligand binding studies of ferrihemoglobin–polyelectrolyte complexes

Quasi‐elastic light scattering (QELS), electrophoretic light scattering (ELS), CD spectroscopy, and azide binding titrations were used to study the complexation at pH 6.8 between ferrihemoglobin and three polyelectrolytes that varied in charge density and sign. Both QELS and ELS show that the structure of the soluble complex formed between ferrihemoglobin and poly(diallyldimethylammonium chloride) [PDADMAC] varies with protein concentration. At fixed 1.0 mg/mL polyelectrolyte concentration, protein addition increases complex size and decreases complex mobility in a tightly correlated manner. At 1.0 mg/mL or greater protein concentration, a stable complex is formed between one polyelectrolyte chain and many protein molecules (i.e., an intra‐polymer complex) with apparent diameter approximately 2.5 times that of the protein‐free polyelectrolyte. Under conditions of excess polyelectrolyte, each of the three ferrihemoglobin–polyelectrolyte solutions exhibits a single diffusion mode in QELS, which indicates that all protein molecules are complexed. CD spectra suggest little or no structural disruption of ferrihemoglobin upon complexation. Azide binding to the ferrihemoglobin–poly(2‐acrylamide‐2‐methylpropanesulfonate) [PAMPS] complex is substantially altered relative to the polyelectrolyte‐free protein, but minimal change is induced by complexation with an AMPS‐based copolymer of reduced linear charge density. The change in azide binding induced by PDADMAC is intermediate between that of PAMPS and its copolymer. © 1999 John Wiley & Sons, Inc. Biopoly 50: 153–161, 1999     

Diffusion of bovine serum albumin in a neutral polymer solution

The diffusion coefficient D of bovine serum albumin through various solutions (pH 7.0, 0.5M NaCl) of polythylene oxide (M_w ～ 1 × 10⁵, 3 × 10⁵) was studied with quasielastic light scattering. In solutions of the 1 × 10⁵ polymer solution at polymer concentrations above 0.5 g/L, D is considerably greater than would have been expected from the viscosity of water:polymer mixtures, the deviations being larger at low protein concentration that at high protein concentration. With either polymer, D falls with increasing protein concentration.     

The interaction of lysozyme with caffeine,theophylline and theobromine in solution

The interactions of lysozyme with caffeine (Caf), theophylline (Tph) and theobromine (Tbr) were investigated using UV–Vis absorption, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques. The results revealed that Caf (Tph or Tbr) caused the fluorescence quenching of lysozyme by the formation of Caf (Tph or Tbr)–lysozyme complex. The binding constants (K _A) and thermodynamic parameters (ΔG°, ΔH°, ΔS°) at two different temperatures, the binding locality, and the binding power were obtained. The results showed that the process of binding Caf (Tph or Tbr) to lysozyme was a spontaneous molecular interaction procedure and the hydrophobic and electrostatic interactions play a major role in stabilizing the complex; The distance r between donor (lysozyme) and acceptor (Caf, Tph or Tbr) was obtained according to fluorescence resonance energy transfer. The effect of Caf (Tph or Tbr) on the conformation of lysozyme was analyzed using synchronous fluorescence and three-dimensional fluorescence spectra techniques. The results showed that the binding of Caf (Tph or Tbr) to lysozyme induced some micro-environmental and conformational changes in lysozyme and disturbed the environment of the polypeptide of lysozyme.     

Static and dynamic quenching of protein fluorescence. II. lysozyme.

The fluorescence parameters—lifetime, relative quantum yield, wavelength of maximum fluorescence intensity, half-width, and polarization—of 0.01% lysozyme were measured at 15°C in aqueous solution, in glycerol–water mixtures (0–90% v/v glycerol), in aqueous urea (0–8M) solutions, and in aqueous guanidine hydrochloride (0–6.4M) solutions. The changes in the static and dynamic quenching of lysozyme fluorescence, monitored by the quantum yield and lifetime measurements, were correlated with the other fluorescence parameters and compared with our earlier results with bovine serum albumin. The results were interpreted in terms of induced conformational changes. The various perturbants altered the fluorescence parameters of lysozyme and bovine serum albumin very differently. The differences were shown to be entirely consistent with our earlier conclusion that bovine serum albumin fluorophores are nonsurface residues and with the conclusion of others that lysozyme fluorophores are surface residues. Unlike their effects on bovine serum albumin, urea and guanidine hydrochloride affect lysozyme structure quite differently, both in nature and degree. We have suggested that the affect of urea on lysozyme fluorescence is an indirect result of reduction in the size of the cleft brought about by the structure-breaking action of urea on water in the cleft. 4M Urea is sufficient for this reaction. Large decreases in the polarization of the fluorescence of lysozyme in the 0.8–1.6M and 3.2–4.8M guanidine hydrochloride ranges demonstrated two guanidine hydrochloride-induced conformation changes. A red shift of the fluorescence maximum to 354 nm indicated that the second transition completely exposes all fluorescing tryptophan residues of lysozyme to mobile solvent water. However, even 6.4M guanidine hydrochloride did not completely unravel the lysozyme molecule at 15°C, as evidenced by its failure to cause any of the tyrosine residues to become fluorescent.     

Seneca Valley Virus 3Cpro Substrate Optimization Yields Efficient Substrates for Use in Peptide-Prodrug Therapy

The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3C^pro is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3C^pro, we measured cleavage kinetics of predicted SVV-001 3C^pro substrates. An efficient substrate, L/VP4 (k_cat/K_M = 1932 ± 183 M^-1s^-1), was further optimized by a P2’ N→P substitution yielding L/VP4.1 (k_cat/K_M = 17446 ± 2203 M^-1s^-1). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3C^pro in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3C^pro, with direct implications for anti-cancer therapeutic development.     

Photosystem I-dependent cyclic electron transport is important in controlling Photosystem II activity in leaves under conditions of water stress

Leaves of the C₃ plant Brassica oleracea were illuminated with red and/or far-red light of different photon flux densities, with or without additional short pulses of high intensity red light, in air or in an atmosphere containing reduced levels of CO₂ and/or oxygen. In the absence of CO₂, far-red light increased light scattering, an indicator of the transthylakoid proton gradient, more than red light, although the red and far-red beams were balanced so as to excite Photosystem II to a comparable extent. On red background light, far-red supported a transthylakoid electrical field as indicated by the electrochromic P₅₁₅ signal. Reducing the oxygen content of the gas phase increased far-red induced light scattering and caused a secondary decrease in the small light scattering signal induced by red light. CO₂ inhibited the light-induced scattering responses irrespective of the mode of excitation. Short pulses of high intensity red light given to a background to red and/or far-red light induced appreciable additional light scattering after the flashes only, when CO₂ levels were decreased to or below the CO₂ compensation point, and when far-red background light was present. While pulse-induced light scattering increased, non-photochemical fluorescence quenching increased and F₀ fluorescence decreased indicating increased radiationless dissipation of excitation energy even when the quinone acceptor Q_A in the reaction center of Photosystem II was largely oxidized. The observations indicate that in the presence of proper redox poising of the chloroplast electron transport chain cyclic electron transport supports a transthylakoid proton gradient which is capable of controlling Photosystem II activity. The data are discussed in relation to protection of the photosynthetic apparatus against photoinactivation.Abbreviations F, F_M, F'_M, F"_M, F₀, F'₀ chlorophyll fluorescence levels - _exc quantum efficiency of excitation energy capture by open Photosystem II - _{PS II} quantum efficiency of electron flow through Photosystem II - P₅₁₅ field indicating rapid absorbance change peaking at 522 nm - P₇₀₀ primary donor of Photosystem I - Q_A primary quinone acceptor in Photosystem II - Q_N non-photochemical fluorescence quenching - Q_q photochemical quenching of chlorophyll fluorescence     

Filipin and its interaction with cholesterol in aqueous media studied using static and dynamic light scattering

Aggregation of filipin in aqueous medium and filipin-induced changes in cholesterol micelles have been studied using intensity and dynamic light scattering. The dependencies of filipin aggregate dimensions on concentration, solvent, and temperature were studied, and revealed that the aggregates do not have a well-defined geometry, i.e., a critical micelle concentration cannot be detected and stable structures are not formed. The aggregates are of size R_g ≈ 110 nm and R_h ≈ 63 nm, referring to the radius of gyration and hydrodynamic radius, respectively. In the concentration range studied (1 μM < C < 30 μM), a low molecular weight species (monomer/dimer) is always present together with the aggregates. In ethanol/ water mixtures, large (R_g ≈ 500 nm), narrow distribution aggregates are formed in the water volume fraction range 0.45 < Φ_H2_O < 0.65. Aggregation also occurs on changing the temperature; In the range 7–37°C, smaller aggregates (10–30 nm) form and the process is only partially reversible. No pronounced effect of filipin on the structure of the cholesterol micelles was observed (a small increase in R_g and R_h is noted). These results rule out any “specificity” for the filipin interactions with cholesterol, which has been considered a key event in the filipin biochemical mode of action. A reevaluatiori Of this question is suggested and some alternatives are advanced. © 1994 John Wiley & Sons, Inc.     

Identification of the adenine binding site in the ricin toxin A-chain by fluorescence,CD, and electron spin resonance spectroscopy

CD, electron spin resonance, and fluorescence spectroscopy have been utilized to study the adenine binding site of ricin and its toxic A-subunit. At acidic (4.5) and physiological (7.3) pH, adenine or a spin-labeled analogue of adenine, N⁶-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl) adenine, alters the near uv CD spectra of the ricin A-chain as well as intact ricin, whereas the far uv CD spectra of all proteins remain unchanged. Electron spin resonance data show that the adenine spin-labeled analogue interacts strongly with the A-chain both at pH 4.5 and 7.3, but no or very weak binding is observed for the intact ricin or the isolated B-chain. The adenine spin label gets highly immobilized (2A_II = 65.5G) by the A-chain. The apparent dissociation constant K_d for the toxic A-chain ligand complex is 1.55 × 10^?4 M and 5.6 × 10^?5 M at pH 7.3 and 4.5, respectively. Fluorescence intensity of ricin A-chain bound 1,8-anilinonaphthalenesulfonic acid (ANS) decreases by ～55% at pH 4.5 with the addition of the spin-labeled analogue of adenine, implying that both the ANS and adenine spin label (ADSL) bind to the hydrophobic domain of the A-chain. Fluorescence of the only intrinsic tryptophan probe of the A-chain is also efficiently quenched by ADSL, indicating that the tryptophan residue and the hydrophobic adenine binding site are closely located. All spectroscopic measurements indicate that adenine or its spin-labeled analogue has a single binding site adjacent to the TRP211 residue in the A-chain. Expansion of the A-chain globule and subsequent exposure of the hydrophobic binding site seem to be responsible for the increased binding of adenine at pH 4.5. © 1993 John Wiley & Sons, Inc.     

Binding of Paeonol to Human Serum Albumin: A Hybrid Spectroscopic Approach and Conformational Study

The purpose of this study was to elucidate the binding of paeonol to human serum albumin (HSA) through spectroscopic methods. The fluorescence quenching of HSA by paeonol was a result of the formation of the HSA–paeonol complex with low binding affinity (K = 4.45 × 10³ M^?1 at 298 K). Thermodynamic parameters (ΔG^○ = –2.08 × 10⁴ J·mol^?1, ΔS^○ = 77.9 J·mol^?1·K^?1, ΔH^○ = 2.41 × 10³ J·mol^?1, k_q = 9.67 × 10¹² M^?1·s^?1) revealed that paeonol mainly binds HSA through hydrophobic force following a static quenching mode. The binding distance was estimated to be 1.91 nm by fluorescence resonant energy transfer. The conformation of HSA was changed and aggregates were formed in the presence of paeonol, revealed by synchronous fluorescence, circular dichroism, Fourier transform infrared spectroscopy, three‐dimensional fluorescence spectroscopy, and resonance light scattering results.     

The Molecular Mechanism of Domain Swapping of the C-Terminal Domain of the SARS-Coronavirus Main Protease

In three-dimensional domain swapping, two protein monomers exchange a part of their structures to form an intertwined homodimer, whose subunits resemble the monomer. Several viral proteins domain swap to increase their structural complexity or functional avidity. The main protease (M^pro) of the severe acute respiratory syndrome (SARS) coronavirus proteolyzes viral polyproteins and has been a target for anti-SARS drug design. Domain swapping in the α-helical C-terminal domain of M^pro (M^proC) locks M^pro into a hyperactive octameric form that is hypothesized to promote the early stages of viral replication. However, in the absence of a complete molecular understanding of the mechanism of domain swapping, investigations into the biological relevance of this octameric M^pro have stalled. Isolated M^proC can exist as a monomer or a domain-swapped dimer. Here, we investigate the mechanism of domain swapping of M^proC using coarse-grained structure-based models and molecular dynamics simulations. Our simulations recapitulate several experimental features of M^proC folding. Further, we find that a contact between a tryptophan in the M^proC domain-swapping hinge and an arginine elsewhere forms early during folding, modulates the folding route, and promotes domain swapping to the native structure. An examination of the sequence and the structure of the tryptophan containing hinge loop shows that it has a propensity to form multiple secondary structures and contacts, indicating that it could be stabilized into either the monomer- or dimer-promoting conformations by mutations or ligand binding. Finally, because all residues in the tryptophan loop are identical in SARS-CoV and SARS-CoV-2, mutations that modulate domain swapping may provide insights into the role of octameric M^pro in the early-stage viral replication of both viruses.     

Development of a fluorescently labeled thermostable DHFR for studying conformational changes associated with inhibitor binding

A fluorescently-labeled, conformationally-sensitive Bacillus stearothermophilus (Bs) dihydrofolate reductase (DHFR) (C73A/S131C_MDCC DHFR) was developed and used to investigate kinetics and protein conformational motions associated with methotrexate (MTX) binding. This construct bears a covalently-attached fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) attached at a distal cysteine, introduced by mutagenesis. The probe is sensitive to the local molecular environment, reporting on changes in the protein structure associated with ligand binding. Intrinsic tryptophan fluorescence of the unlabeled Bs DHFR construct (C73A/S131C DHFR) also showed changes upon MTX association. Stopped-flow analysis of all data can be understood by invoking the presence of two native state DHFR conformers that bind to MTX at different rates (20.2 and 0.067 μM⁻¹ s⁻¹), similar to previously published findings for Escherichia coli DHFR. Probe fluorescence of C73A/S131C_MDCC DHFR predominantly reports on MTX binding to one of the conformers while intrinsic tryptophan fluorescence of C73A/S131C DHFR reports on binding to the other conformer. This study demonstrates the use of an extrinsic fluorophore attached to a distal region to investigate ligand binding interactions that are not experimentally accessible via intrinsic tryptophan fluorescence alone. The thermostability of C73A/S131C_MDCC DHFR provides an important new tool with applications for investigating the temperature dependence of DHFR conformational changes associated with binding and catalysis.     

Interactions between staphylococcal protein A and immunoglobulin domains.

The affinity and stoichiometry of interaction between staphylococcal protein A and different domains of immunoglobulins have been studied. Light scattering and tryptophan fluorescence quenching titrations along with direct binding assays were performed. The lack of binding to protein A of pFc′ fragment (corresponding to C_H3 domain of IgG) or of Facb derivative of rabbit IgG (which is devoid of the C_H3) suggests that the locus of protein A binding is at the interface between the C_H2 and C_H3 domains. This assignment is also supported by results of the tryptophan fluorescence quenching and C$\text{1}$ binding experiments.     

以EV71 3Cpro为靶标的抗病毒药物筛选模型的建立及抗3Cpro化合物筛选

建立一种以EV71 3C蛋白酶为靶标的抗肠病毒药物筛选模型,并应用于小分子化合物库筛选具有抗EV71活性的化合物.从临床手足口病例标本中分离肠道病毒进行PCR鉴定及基因组测序.通过插入突变在黄色荧光YFP编码框合适位点处引入EV71 3C酶切位点,构建对3C蛋白酶敏感的报告质粒pc DNA3-m YFP,然后将其与表达3C的质粒共转293A细胞,在3C抑制剂Rupintrivir存在与否的情况下通过荧光显微镜和酶标仪检测Ex(500nm)/Em(535nm)荧光信号的变化,判断建模是否成功;利用建好的筛选模型在高通量药物筛选平台对小分子化合物库进行初筛和复筛;再利用空斑分析检测筛选出的活性化合物是否对临床分离的EV71毒株具有抑制作用.m YFP在293A细胞中表达良好,3C的表达使荧光信号下降80%,Rupintrivir的存在则几乎不影响荧光表达,说明以3C为靶位的筛选模型构建成功.经过高通量初筛和复筛从26 000多种小分子化合物中获得26种能够显著回复m YFP表达的活性化合物;空斑分析显示其中2种化合物具有较为明显的抑制EV71复制的活性.因此,我们所构建的3C-m YFP共表达系统是一种简便有效的、可用于高通量筛选抗EV71 3C~(pro)药物的筛选模型.     

Energy transfer from tryptophan residues of proteins to 8-anilinonaphthalene-1-sulfonate

The binding of the apolar fluorescent dye 8-anilinonaphthalene-1-sulfonate (ANS) to bovine serum albumin (BSA), phospholipase A₂ (PLA₂), ovalbumin, lysozyme, cobrotoxin and N-acetyltryptophanamide was used to assess the factors affecting the efficiency of energy transfer from Trp residues to the ANS molecule. We found that the efficiency of energy transfer from Trp residues to ANS was associated with the ability of proteins to enhance the ANS fluorescence. At the same molar concentration of protein, BSA enhanced ANS fluorescence most among these proteins; its Trp fluorescence was drastically quenched by the addition of ANS. Fluorescence enhancement of ANS in PLA₂-ANS complex increased upon addition of Ca²⁺ or change of the buffer to acidicpH, resulting in a higher efficiency of energy transfer from Trp residues to ANS. There was limited ANS fluorescence enhancement with ovalbumin, lysozyme, cobrotoxin, and N-acetyltryptophanamide and a less efficient quenching in Trp fluorescence. The capabilities of proteins for binding with ANS correlated with the decrease in their Trp fluorescence being quenching by ANS. However, the microenvironment surrounding Trp residues of proteins did not affect the energy transfer. Based on these results, the factors that affected the energy transfer from Trp residues to ANS are discussed.     

An Improvement in the Sensitivity of the Salkowski Reagent for Tryptamine, Tryptophan and Indoleacetic Acid

The reaction of indoles with the Salkowski reagent has been examined. It was found that the concentration of acid as well as the concentration and anionic component of the iron salt employed are critical factors in the choice of a reagent that will fail to react—or will react maximally with a given indole. Tryptamine can be reproducibly assayed with a reagent containing 0.01 M Fe(NO₃)₃ in 7.0 M HCIO₄. Two ml of this reagent are added to two ml of the sample. The absorbancy is read at 450 nm after 90 minutes under uniform light conditions. Versions of this reagent can also be used for the quantitative colorimetric determination of tryptophan or indoleacetic acid.     

Helix-coil transition of Poly(L-glutamic acid) in N-methylacetamide

The folding of randomly coiled poly(L -glutamic acid) to the helical state has been studied in N-methylacetamide by titration methods. Since this solvent would be expected to form amide-peptide group hydrogen bonds with the unfolded form of the polymer, to a first approximation no helix stabilization could come from intrapolymer hydrogen bonds. The titration data, collected from 30 to 70°C yield the following values per residue for the thermodynamic parameters governing the coil-helix reaction for the uncharged polymer: ΔG_30°C°, ?1. 9 ± 0.1 kcal; Δ H°, 0 ± 0.1 kcal; ΔS_30°C°, 6.3 ± 0.6 eu. In N-methyl acetamide, the helix is an order of magnitude more stable than in water, and this stabilization appears to be entirely the result of the entropy gained by solvent molecules which are released from the polymer upon folding.     

Activation and mechanism of Clostridium septicum alpha toxin

Clostridium septicum produces a single lethal factor, alpha toxin (AT), which is a cytolytic protein with a molecular mass of approximately 48kDa. The 48kDa toxin was found to be an inactive protoxin (AT^pro) which could be activated via a carboxy-terminal cleavage with trypsin. The cleavage site was located approximately 4kDa from the carboxy-terminus. Proteolytically activated AT^pro had a specific activity of approximately 1.5 × 10⁶ haemolytic units mg^-1. The trypsin-activated toxin (AT^act) was haemolytic, stimulated a prelytic release of potassium ions from erythrocytes which was followed by haemoglobin release, induced channel formation in planar membranes and aggregated into a complex of M_r >210000 on erythrocyte membranes. AT^pro did not exhibit these properties. AT^act formed pores with a diameter of at least 1.3-1.6 nm. We suggest that pore formation on target cell membranes is responsible for the cytolytic activity of alpha toxin.     

Chemical modification studies onCocos nucifera pollen allergens

Two highly active allergens C_n II (M _r 158,000) and C_nVII (M _r 2900) isolated fromCocos nucifera pollen extract were treated with various protein modifying reagents in order to ascertain the amino acid residues responsible for their allergenicity, In C_n II modification of carboxy group and tryptophan residue led to 30 and ≽ 75% loss in allergenicity and those of lysine and tyrosine reduced 62 and 38% activity, Lysine, tyrosine, tryptophan and carboxy group of C_nVII were also modified causing 81, 17, ≽ 70 and 26% loss of allergenicity respectively, Allergenicity of both was highly affected by pronase and moderately affected by heat, Periodate destroyed about 50% of their allergenicity and other chemical reagents except urea had no remarkable effect