Micellar effect on the scavenging of singlet molecular oxygen by hydroxybenzenes

The antioxidative effectiveness of three hydroxyaromatic derivatives (OHAD), namely phenol, resorcinol and phloroglucinol, as reflected by their scavenging ability of the photochemically generated species singlet molecular oxygen [O(2)((1)Delta(g))], was studied in aqueous solution and micellar media. Kinetic results, obtained through time-resolved phosphorescence detection of O(2)((1)Delta(g)) emission and polarographic methods, at pH 7 and 12 in homogeneous and CTAB micellar media, were compared. Calculated photooxidation quantum efficiencies (phi(r)), ranging from very low values ( approximately 0.01) to relatively high ones (approximately 0.3), depend on the preferential solubilization sites of the hydroxyaromatic derivatives either in the micellar or homogeneous media. OHAD in water pH 7 and in CTAB 0.02 M pH 12 exhibit their highest antioxidative efficiency as O(2)((1)Delta(g)) scavengers. In these conditions, they present the best degree of self protection against O(2)((1)Delta(g))-mediated photooxidation with relatively high values for the overall quenching rate constant and, simultaneously, the lowest phi(r).     

Singlet molecular oxygen [O<Subscript>2</Subscript>(<Superscript>1</Superscript>Δ<Subscript>g</Subscript>)]-mediated photodegradation of tyrosine derivatives in the presence of cationic and neutral micellar systems

The kinetics of rose bengal-sensitized photooxidation of tyrosine and several tyrosine-derivatives (tyr-D) named tyrosine methyl ester, tyrosine ethyl ester and tyrosine benzyl ester was studied in buffered pH 11 water, and buffered pH 11 micellar aqueous solutions of 0.01 M cetyltrimethylammonium chloride (CTAC) and 0.01 M-octylphenoxypolyethoxyethanol [triton X100 (TX100)]. Through time-resolved phosphorescence detection of singlet molecular oxygen (O(2)((1)Delta(g))) and polarographic determination of oxygen consumption, the respective bimolecular rate constants for reactive (k(r)) and overall (k(t)) quenching of the oxidative species by tyr-D were evaluated. Both rate constants behave in different fashion depending on the particular reaction medium. k(r)/k(t) values, increase in the sense CTAC相似文献   

The balance between charge transfer and non-charge transfer pathways in the sensitization of singlet oxygen by pi pi* triplet states.

A charge transfer (CT) channel and a non-CT deactivation channel, both leading to formation of O(2)((1)Sigma (g)(+)), O(2)((1) Delta(g)) and O(2)((3)Sigma(g)(-)), compete in the quenching of triplet states by O(2). Recent studies by our group demonstrated that these channels are described by rather simple and general quantitative relations. In the present paper we use the detailed kinetic data on the quenching by O(2) of pi pi* triplet sensitizers of three homologous aromatic series in CCl(4) to derive a parameter, which describes the balance between CT and non-CT deactivation. This quantity, p(CT), is the relative contribution of CT mediated deactivation and is easily calculated for a sensitizer of known triplet energy from its quenching rate constant. The parameter p(CT) quantitatively describes the balance between both deactivation channels without requiring any knowledge of oxidation potentials. It is shown how the variation of p(CT) influences the efficiencies and the rate constants of O(2)((1)Sigma(g)(+)), O(2)((1)Delta(g)) and O(2)((3)Sigma(g)(-)) formation in the quenching process.     

Scavenging of photogenerated oxidative species by antimuscarinic drugs: atropine and derivatives

The quenching ability of photogenerated oxidative species by some antimuscarinic drugs generically named atropines (e.g. atropine [I] eucatropine [II], homatropine [III] and scopolamine [IV]) have been investigated employing stationary photolysis, polarographic detection of dissolved oxygen, stationary and time-resolved fluorescence spectroscopy, and laser flash photolysis. Using Rose Bengal as a dye sensitiser for singlet molecular oxygen, O(2)((1)Delta(g)), generation, compounds I-IV behave as moderate chemical plus physical quenchers of the oxidative species. Correlation between kinetic and electrochemical data indicates that the process is possibly driven by a charge-transfer interaction. The situation is somewhat more complicated employing the natural pigment riboflavin (Rf) as a sensitiser. Compounds I and II complex Rf ground state, diminishing the quenching ability towards singlet and triplet excited state of the pigment. On the other hand, compounds III and IV effectively quench Rf excited states, protecting the pigment against photodegradation. Under anaerobic conditions, semireduced Rf (Rf(.-)) is formed through quenching of excited triplet Rf. Nevertheless, although Rf(.-) is a well-known generator of the reactive species superoxide radical anion by reductive quenching in the presence of oxygen, the process of O(2)((1)Delta(g)) production prevails over superoxide radical generation, due to the relatively low rate constants for the quenching of triplet Rf by the atropines (in the order of 10(7) M(-1)s(-1) for compounds III and IV) in comparison to the rate constant for the quenching by ground state oxygen, approximately two orders of magnitude higher, yielding O(2)((1)Delta(g)). Compound I is the most promising O(2)((1)Delta(g)) physical scavenger, provided that it exhibits the higher value for the overall quenching rate constant and only 11% of the quenching process leads to its own chemical damage.     

The role of vitamin B6 as an antioxidant in the presence of vitamin B2-photogenerated reactive oxygen species. A kinetic and mechanistic study

We report on the photostability of a mixture of vitamins B6 and B2 (riboflavin, Rf) upon visible light irradiation and on the possible role of the vitamin B6 family (B6D) as deactivators of reactive oxygen species (ROS). The work is a systematic kinetic and mechanistic study under conditions in which only Rf absorbs photoirradiation. Pyridoxine, pyridoxal hydrochloride, pyridoxal phosphate and pyridoxamine dihydrochloride were studied as representative members of the vitamin B6 family. The visible light irradiation of dissolved Rf and B6D in pH 7.4 aqueous medium under aerobic conditions induces photoprocesses that mainly produce B6D degradation. The overall oxidative mechanism involves the participation of ROS. Photogenerated (3)Rf* is quenched either by oxygen, giving rise to O(2)((1)Δ(g)) by electronic energy transfer to dissolved ground state oxygen, or by B6D yielding, through an electron transfer process, the neutral radical RfH˙, and O(2)˙(-) in an subsequent step. B6D act as quenchers of O(2)((1)Δ(g)) and O(2)˙(-), the former in a totally reactive event that also inhibits Rf photoconsumption. The common chromophoric moiety of B6D represented by 3-hydroxypyridine, constitutes an excellent model that mimics the kinetic behavior of the vitamin as an antioxidant towards Rf-generated ROS. The protein lysozyme, taken as an O(2)((1)Δ(g))-mediated oxidizable biological target, is photoprotected by B6D from Rf-sensitized photodegradation through the quenching of electronically excited triplet state of the pigment, in a process that competes with O(2)((1)Δ(g)) generation.     

Vitamin B2-sensitised photooxidation of the ophthalmic drugs Timolol and Pindolol: kinetics and mechanism

The kinetics and mechanistic aspects of the riboflavin-photosensitised oxidation of the topically administrable ophthalmic drugs Timolol (Tim) and Pindolol (Pin) were investigated in water-MeOH (9:1, v/v) solution employing light of wavelength > 400 nm. riboflavin, belonging to the vitamin B(2) complex, is a known human endogenous photosensitiser. The irradiation of riboflavin in the presence of ophthalmic drugs triggers a complex picture of competitive reactions which produces the photodegradation of both the drugs and the pigment itself. The mechanism was elucidated employing stationary photolysis, polarographic detection of dissolved oxygen, stationary and time-resolved fluorescence spectroscopy, and laser flash photolysis. Ophthalmic drugs quench riboflavin-excited singlet and triplet states. From the quenching of excited triplet riboflavin, the semireduced form of the pigment is generated, through an electron transfer process from the drug, with the subsequent production of superoxide anion radical (O(2)(*-)) by reaction with dissolved molecular oxygen. Through the interaction of dissolved oxygen with excited triplet riboflavin, the species singlet oxygen (O(2)((1)Delta(g))) is also generated to a lesser extent. Both O(2)(*-) and O(2)((1)Delta(g)) induce photodegradation of ophthalmic drugs, Tim being approximately 3-fold more easily photooxidisable than Pin, as estimated by oxygen consumption experiments.     

On the antioxidant properties of therapeutic drugs: quenching of singlet molecular oxygen by aminosalicylic acids

The ability of the widely employed therapeutic drugs 4-aminosalicylic acid and 5-aminosalicylic acid to act as singlet molecular oxygen (O(2)((1)delta(g))) scavengers was investigated at pH 7 and pH 12. The isomer 3-aminosalicylic acid was also included in the study for comparative purposes. All three compounds quench photochemically generated O(2)((1)delta(g)) with rate constants in the range of 10(7)-10(8) x M(-1)s(-1), depending on the experimental conditions. No chemical reaction (oxidation of the aminosalicylic acids) was detected at the neutral pH, whereas at pH 12 both chemical and physical interactions with O(2)((1)delta(g)) operated. The physical process implies the de-activation of the oxidant species without destruction of the aminosalicylic acid. The quotients between the overall and reactive rate constants for O(2)((1)delta(g)) quenching at pH 12 (k(r)/k(t) ratios), which account for the actual effectiveness of photodegradation, were relatively low (0.22, 0.04, and 0.06 for 3-, 4- and 5-aminosalicylic acids, respectively). This indicates that the drugs, particularly the 4- and 5-amino derivatives, de-activate the excited oxygen species, at both pH values studied, mainly in a physical fashion, preventing its photodegradation and providing an antioxidative protection for possible photo-oxidizable biological targets in the surroundings.     

Synthesis and photodynamic activity of zinc(II) phthalocyanine derivatives bearing methoxy and trifluoromethylbenzyloxy substituents in homogeneous and biological media

Two zinc(II) phthalocyanines bearing either four methoxy (ZnPc 3) or trifluoromethylbenzyloxy (ZnPc 4) substituents have been synthesized by a two-step procedure starting from 4-nitrophthalonitrile. Absorption and fluorescence spectroscopic studies were analyzed in different media. These compounds are essentially non-aggregated in the organic solvent. Fluorescence quantum yields (phi(F)) of 0.26 for ZnPc 3 and 0.25 for ZnPc 4 were calculated in tetrahydrofuran (THF). The photodynamic activity of these compounds was compared in both THF containing photooxidizable substrates and in vitro on Hep-2 human larynx-carcinoma cell line. The production of singlet molecular oxygen, O(2)((1)Delta(g)), was determined using 9,10-dimethylanthracene yielding values of approximately 0.56 for both sensitizers. Under these conditions, the addition of beta-carotene (Car) suppresses the O(2)((1)Delta(g))-mediated photooxidation. In biological medium, no dark cytotoxicity was found for cells incubated with 0.1 microM of phthalocyanines 3 and 4 for 24 h. However, under similar conditions 0.5 microM of ZnPc 4 was toxic (70% cell survival). The uptake into Hep-2 cells was evaluated using 0.1muM of sensitizer, reaching values of approximately 0.05 nmol/10(6) cells after 3h of incubation at 37 degrees C. The cell survival after irradiation of the cultures with visible light was dependent upon both light exposure level and intracellular sensitizer concentration. A higher photocytotoxic effect was found for ZnPc 3 with respect to 4 (32%/70% cell survival after 15 min of irradiation). Also, these studies were performed treating the cells with 0.5 microM of ZnPc 3. In this case, an increase in the uptake (approximately 0.28 nmol/10(6) cells) was observed, which is accompanied by a higher photocytotoxic activity (20% cell survival). These results show that even though both sensitizer present similar photophysical properties in homogeneous medium, the photodynamic behavior in cellular media can significantly be changed.     

Important parameters affecting efficiency of protein refolding by reversed micelles

Refolding of denatured RNase A as a model of inclusion bodies was performed by reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) in isooctane. In the novel refolding process, a solid-liquid extraction was utilized as an alternative to the ordinary protein extraction by reversed micelles based on a liquid-liquid extraction. First, the effects of operational parameters such as concentration of AOT, W(o) (= [H(2)O]/[AOT]), and pH were examined on the solubilization of solid denatured proteins into a reversed micellar solution. The solubilization was facilitated by a high AOT concentration, a high W(o) value, and a high pH in water pools. These conditions are favorable for the dispersion of the solid protein aggregates in an organic solvent. Second, the renaturation of the denatured RNase A solubilized into the reversed micellar solution was conducted by addition of glutathione as a redox reagent. A complete renaturation of RNase A was accomplished by adjusting the composition of the redox reagent even at a high protein concentration in which protein aggregation would usually occur in aqueous media. In addition, the renaturation rates were improved by optimizing water content (W(o)) and the pH of water pools in reversed micelles. Finally, the recovery of renatured RNase A from the reversed micellar solution was performed by adding a polar organic solvent such as acetone into the reversed micellar solution. This precipitation method was effective for recovering proteins from reversed micellar media without any significant reduction in enzymatic activity.     

Identification of the main oxidation products of 8-methoxy-2'-deoxyguanosine by singlet molecular oxygen

It is now well established that oxidation of 2'-deoxyguanosine (dGuo) in DNA by singlet molecular oxygen [O2 (1Delta(g))] produces 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), whereas the main degradation products of free dGuo in aqueous solution have been identified as the two diastereomers of spiroiminodihydantoin nucleoside. Interestingly, O2 (1Delta(g))-mediated oxidation of free 8-oxodGuo gives rise to a pattern of degradation products that is different from that observed when the nucleoside is inserted into DNA. The reasons for these differences and the mechanisms involved in the oxidation reactions are not yet completely understood for either dGuo or 8-oxodGuo, either free or within DNA. In the present work, we report a study of the reaction of O2 (1Delta(g)) toward a modified nucleoside, 8-methoxy-2'-deoxyguanosine (8-MeOdGuo), either free or incorporated into an oligonucleotide. The reason for the choice of 8-MeOdGuo as a chemical model to study in more detail the oxidation pathways of 8-oxodGuo or, more precisely, of the tautomeric 8-hydroxy-2'-deoxyguanosine was dictated by the fact that only the 7,8-enolic tautomer is present in the molecule. The thermolysis of an endoperoxide of a naphthalene derivative as a clean chemical source of 18O-labeled O2 (1Delta(g)) was used to oxidize 8-MeOdGuo. The main O2 (1Delta(g)) oxidation products that were separated and analyzed by HPLC coupled to tandem mass spectrometry were identified as the 2'-deoxyribonucleoside derivatives of 2,2,4-triamino-5-(2H)oxazolone, 2,5-diamino-4H-imidazol-4-one together with the methyl-substituted derivatives of spiroiminodihydantoin, oxidized iminoallantoin and urea. On the other hand, O2 (1Delta(g)) oxidation of 8-MeOdGuo-containing oligonucleotide generated imidazolone as the predominant degradation product. These results provided new mechanistic insights into the reactions of O2 (1Delta(g)) with purine nucleosides.     

Photodynamic activity of cationic and non-charged Zn(II) tetrapyridinoporphyrazine derivatives: biological consequences in human erythrocytes and Escherichia coli.

The photodynamic activity of a cationic Zn(II) tetramethyltetrapyridinoporphyrazinium salt (ZnPc ) was compared with that of a non-charged Zn(II) tetrapyridinoporphyrazine (ZnPc 1), both in vitro using human red blood (HRB) cells and a typical Gram-negative bacterium Escherichia coli. Absorption and fluorescence spectroscopic studies were analyzed in different media. Fluorescence quantum yields (phi(F)) of 0.35 for ZnPc 1 and 0.30 for ZnPc 2 were calculated in N,N-dimethylformamide (DMF). The singlet molecular oxygen, O(2)((1)Delta(g)), production was evaluated using 9,10-dimethylanthracene (DMA) in DMF yielding values of Phi(Delta)= 0.56 for ZnPc 1 and 0.50 for ZnPc 2. In biological medium, the photodynamic effect was first evaluated in HRB cells. Both phthalocyanines produce similar photohemolysis of HRB cells, reaching values >90% of lysis after 5 min of irradiation with visible light. The photodynamic effect is accompanied by an increase in the membrane fluidity of HRB cells. However, these studies on E. coli cells showed that the cationic ZnPc 2 produces a higher photoinactivation of Gram-negative bacteria than ZnPc 1. Also, these results were established by stopped of growth curves for E. coli. Therefore the studies show that cationic ZnPc 2 is an efficient phototherapeutic agent with potential applications in tumor cell and Gram-negative bacteria inactivation by photodynamic therapy.     

Improvement in extraction and catalytic activity of Mucor javanicus lipase by modification of AOT reverse micelle

Reverse micelles are formed in apolar solvents by spontaneous aggregation of surfactants. Surfactant sodium bis (2-ethylhexyl) sulfosuccinate (AOT) is most often used for the reverse micellar extraction of enzymes. However, the inactivation of enzyme due to strong interaction with AOT molecules is a severe problem. To overcome this problem, the AOT/water/isooctane reverse micellar system was modified by adding short chain polyethylene glycol 400 (PEG 400). The modified AOT reverse micellar system was used to extract Mucor javanicus lipase from the aqueous phase to the reverse micellar phase. The extraction efficiency (E) increased with the increase in PEG 400 addition and the maximum E in PEG 400 modified system was twofold higher than that in the PEG 400-free system. Upon addition of PEG 400, the water activity (a(w)) of aqueous phase decreased, whereas a(w) of reverse micellar phase increased. The circular dichroism spectroscopy analysis revealed that PEG 400 changes the secondary and tertiary structure of lipase. The maximum specific activity of lipase extracted in PEG 400-modified reverse micellar system was threefold higher than that in the PEG-free system.     

Changes in the spin state and reactivity of cytochrome C induced by photochemically generated singlet oxygen and free radicals

This work compares the effect of photogenerated singlet oxygen (O(2)((1)Delta(g))) (type II mechanism) and free radicals (type I mechanism) on cytochrome c structure and reactivity. Both reactive species were obtained by photoexcitation of methylene blue (MB(+)) in the monomer and dimer forms, respectively. The monomer form is predominant at low dye concentrations (up to 8 microm) or in the presence of an excess of SDS micelles, while dimers are predominant at 0.7 mm SDS. Over a pH range in which cytochrome c is in the native form, O(2) ((1)Delta(g)) and free radicals induced a Soret band blue shift (from 409 to 405 nm), predominantly. EPR measurements revealed that the blue shift of the Soret band was compatible with conversion of the heme iron from its native low spin state to a high spin state with axial symmetry (g approximately 6.0). Soret band bleaching, due to direct attack on the heme group, was only detected under conditions that favored free radical production (MB(+) dimer in SDS micelles) or in the presence of a less structured form of the protein (above pH 9.3). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of the heme group and the polypeptide chain of cytochrome c with Soret band at 405 nm (cytc405) revealed no alterations in the mass of the cytc405 heme group but oxidative modifications on methionine (Met(65) and Met(80)) and tyrosine (Tyr(74)) residues. Damage of cytc405 tyrosine residue impaired its reduction by diphenylacetaldehyde, but not by beta-mercaptoethanol, which was able to reduce cytc405, generating cytochrome c Fe(II) in the high spin state (spin 2).     

Synthesis and biological evaluation of methoxyphenyl porphyrin derivatives as potential photodynamic agents

A new meso-2,4,6-trimethoxyphenyl porphyrin covalently linked to a 2',6'-dinitro-4'-trifluoromethylphenyl group by an amine bond 5 and its metal complex with Cd(II) 6 was prepared. The photodynamic activities of 5 and 6 were evaluated in vitro on Hep-2 cells. A considerable increase in the photocytotoxic effect was found for 6, which has higher singlet molecular oxygen, O(2)((1)Delta(g)), production.     

Internal conductance to CO(2) diffusion and C(18)OO discrimination in C(3) leaves

(18)O discrimination in CO(2) stems from the oxygen exchange between (18)O-enriched water and CO(2) in the chloroplast, a process catalyzed by carbonic anhydrase (CA). A proportion of this (18)O-labeled CO(2) escapes back to the atmosphere, resulting in an effective discrimination against C(18)OO during photosynthesis (Delta(18)O). By constraining the delta(18)O of chloroplast water (delta(e)) by analysis of transpired water and the extent of CO(2)-H(2)O isotopic equilibrium (theta(eq)) by measurements of CA activity (theta(eq) = 0.75-1.0 for tobacco, soybean, and oak), we could apply measured Delta(18)O in a leaf cuvette attached to a mass spectrometer to derive the CO(2) concentration at the physical limit of CA activity, i.e. the chloroplast surface (c(cs)). From the CO(2) drawdown sequence between stomatal cavities from gas exchange (c(i)), from Delta(18)O (c(cs)), and at Rubisco sites from Delta(13)C (c(c)), the internal CO(2) conductance (g(i)) was partitioned into cell wall (g(w)) and chloroplast (g(ch)) components. The results indicated that g(ch) is variable (0.42-1.13 mol m(-2) s(-1)) and proportional to CA activity. We suggest that the influence of CA activity on the CO(2) assimilation rate should be important mainly in plants with low internal conductances.     

Comparison of Quasielastic Light Scattering and Laser Diffractometry as Nondestructive Probes into the Structure of Bacillus sphaericus Spores Produced at Different Temperatures

Volume 62, no. 5, p. 1702, column 2, equation 3: the equation should read as follows. g(sup1)((tau)) = [g(sup2)((tau)) - 1](sup1/2) = exp[-K(sup2)(D(inf1) cos(sup2)(alpha) + D(inf2) sin(sup2)(alpha))(tau)] (3) [This corrects the article on p. 1699 in vol. 62.].     

Immobilized enzymes in reverse micelles: studies with gel-entrapped trypsin and alpha-chymotrypsin in AOT reverse micelles

Trypsin and alpha-chymotrypsin were immobilized by gelentrapment in polyacrylamide cross-linked with N,N(1)-methylenebisacrylamide. The immobilized enzymes are catalytically efficient in suspensions of reverse micelles formed in isooctane by bis(2-ethylhexyl) sodium sulfosuccinate (AOT) and water. Both entrapped enzymes are stable in reverse micellar suspension at room temperature and pH 8.2 for 3 days and lose 30-40% activity after 1 week. The enzymes obey Michaelis-Menten kinetics in the investigated concentration range with K(m) values higher than those in solution. Activity of the enzymes is independent of the water content of the micellar solution. No shift in pH optimum was observed for immobilized trypsin activity toward Nalpha-benzoyl-L-arginine ethyl ester. The utility of the procedure, which combines the advantage of enzyme immobilization and enzymology in reverse micelles, is illustrated by an example of peptide synthesis. In particular, peptide synthesis (e. g., Z--Ala--Phe--Leu--NH(2)) using water-insoluble substrate has been performed with gelentrapped alpha-chymotrypsin in reverse micellar suspension with the advantage of efficient enzyme recycling.     

Solubilizing water involved in protein extraction using reversed micelles

The extraction of protein using reversed micelles was investigated in relation to the amount of solubilizing water in the reversed micellar organic phase. The minimal concentration of amphiphilic molecule di-2-ethylhexyl sodium sulfosuccinate (C(20)H(37)O(7)Na) (AOT) required for 100% cytochrome c extraction was recognized. This critical AOT concentration increased with protein concentration in the aqueous phase. On this minimal AOT condition, the molar ratio of solubilizing water to extracted protein was found to be a constant of 3500 under C(KCI) = 1.0 x 10(2) mol . m(-3) in this system. This ratio means the hydrophillic surroundings required for extracting one protein molecule into the micellar organic phase under the suitable pH and salt concentration for the forward extraction. In this regard, AOT molecules seemed to take the part of water solubilizing agent in the reversed micellar extraction. This role of AOT is important to extract protein under the suitable pH and salt concentration. The amount of solubilizing water in the protein-containing system was larger than in the protein-free system. This difference shows that the water molecules accompany the extracted protein into the reversed micellar organic phase at constant ratio 2200 under C(KCI) = 1.0 x 10(2) mol . m(-3), i.e., accompanying water molecules per one extracted protein. The minimal AOT concentration increased with ionic strength. On this minimal AOT condition, the molar ratio of solubilizing water to extracted protein also increased with ionic strength, so that in higher ionic strength, more solubilizing water was required. Then more AOT was required to provide the hydrophillic surroundings for protein. The pH affected the minimal AOT concentration required for 100% protein extraction.     

Residues 88-109 of factor IXa are important for assembly of the factor X activating complex.

Activated platelets and phospholipid vesicles promote assembly of the intrinsic factor X (FX) activating complex by presenting high-affinity binding sites for blood coagulation FIXa, FVIIIa, and FX. Previous reports suggest that the second epidermal growth factor (EGF)-like domain of FIXa mediates assembly of the FX activating complex (Ahmad, S. S., Rawala, R., Cheung, W. F., Stafford, D. W., and Walsh, P. N. (1995) Biochem. J. 310, 427-431; Wong, M. Y., Gurr, J. A., and Walsh, P. N. (1999) Biochemistry 38, 8948-8960). To identify important residues, we prepared several chimeric FIXa proteins using homologous sequences from FVII: FIXa(FVIIEGF2) (FIX Delta 88-124,inverted Delta FVII91-127), FIXa(loop1) (FIX Delta 88-99,inverted Delta FVII91-102), FIXa(loop2) (FIX Delta 95-109,inverted Delta FVII98-112), FIXa(loop3) (FIX Delta 111-124,inverted Delta FVII114-127), and point mutants (FIXaR94D and FIXa(loop1)G94R). In the presence and absence of FVIIIa, a 2- to 10-fold reduced V(max) of FX activation (nm FXa min(-1)) was observed for FIXa(FVIIEGF2), FIXa(loop1), FIXa(loop2), and FIXa(loop1)G94R, whereas FIXa(loop3) and FIXaR94D were normal. For all of the FIXa proteins, K(m)((app)) values were normal as were EC(50) values for interactions with FVIIIa. However, K(d)((app)) (in nm) for the FX activating complex assembled on phospholipid vesicles was increased for FIXa(FVIIEGF2) (43.3 +/- 2.70), FIXa(loop1)(10.9 +/- 2.8), FIXa(loop2) (70.5 +/- 1.60), and FIXa(loop1)G94R (17.1 +/- 2.90) relative to FIXa(N) (3.9 +/- 0.11), FIXa(WT) (4.6 +/- 0.17), FIXa(loop3) (4.5 +/- 0.20), and FIXaR94D (2.2 +/- 0.09) suggesting that reduced V(max) is a result of impaired complex assembly. These data indicate that residues 88-109 (but not Arg(94)) are important for normal assembly of the FX activating complex on phospholipid vesicles.     

Solubilization of ribosomes in reverse micelles.

Pig liver ribosomes have been solubilized in reverse micelles constituted by bis (2-ethyl hexyl) sodium sulfosuccinate (AOT) in isooctane and 3.6% water, v:v. The micellar ribosomal solutions are transparent, show no significant scattering and permit direct spectroscopic observation of the ribosomes to be made. Ultraviolet absorption and circular dichroic spectra have been recorded and indicate that the ribosomes maintain in the micellar environment their structural integrity. Some possible applications of these micellar systems are discussed.