Novel, rapid, low-cost screen-printed (bio)sensors for the direct analysis of boar taint compounds androstenone and skatole in porcine adipose tissue: Comparison with a high-resolution gas chromatographic method.

This is the first report on the fabrication, characterisation and application of an electrochemical (bio)sensor system for the simultaneous measurement of skatole and androstenone. A biosensor for androstenone was fabricated using a Meldola's Blue modified SPCE (MB-SPCE) by depositing NADH and the enzyme 3α-hydroxysteroid dehydrogenase onto the MB-SPCE surface; samples of adipose tissue were analysed using the biosensors in conjunction with chronoamperometry. Cyclic voltammetry was used to investigate the electrochemical behaviour of skatole at a screen-printed carbon electrode (SPCE vs. Ag/AgCl). An oxidation peak was observed around +0.55 V (vs. Ag/AgCl) and differential pulse voltammetry was applied for quantification of skatole in adipose tissue (in-situ). Quantitative analysis was achieved using calibration plots obtained from fortified meat samples. The concentrations obtained by the electrochemical and gas chromatographic (GC) methods demonstrated a good positive correlation. The (bio)sensor system completed both measurements within 60 s, as compared to several hours for GC, and at a considerably reduced cost and complexity. Consequently, the novel (bio)sensor system should have applications for analysis of carcasses on the abattoir processing line.

Temperature-dependent Henry's Law constants of 4-alkyl branched-chain fatty acids and 3-methylindole in an oil-air matrix and analysis of volatiles in lamb fat using selected ion flow tube mass spectrometry.

RATIONALE: 4-Alkyl branched-chain fatty acids and 3-methylindole are characteristic flavor compounds associated with sheep meat. Determining their partitioning behavior between the gas and condensed phase and ultimately developing a correlation between the compound's headspace concentration and sensory descriptive grouping are important for high-throughput characterization and grading classification. METHODS: The headspace concentrations of 3-methylindole, 4-methyloctanoic acid, 4-ethyl-octanoic acid, and 4-methylnonanoic acid above corn-oil-based standard solutions and lamb fat samples were measured using selected ion flow tube-mass spectrometry (SIFT-MS). The standard solutions were equilibrated at 80, 100, 110 and 125°C while the fat samples were equilibrated at 125°C. Statistical evaluation, linear and polynomial regression analyses were performed to establish the compound-specific and temperature-dependent Henry's Law constants, enthalpy (ΔH) and entropy (ΔS) of phase changes. RESULTS: The Henry's Law constants (kHcp ) were calculated from the regression analysis with a high degree of confidence (p < 0.05) and linearity (r2  > 0.99). The kHcp increased with increase in equilibrium temperature. The empirical calculation of ΔH and ΔS at different temperatures confirmed the temperature-dependence of the Henry's Law constants. The headspace concentrations of the lamb-flavor compounds were determined above actual lamb fat samples and the corresponding condensed-phase concentrations were successfully derived. CONCLUSIONS: The temperature-dependent Henry's Law constants, ΔH, and ΔS of phase changes for 3-methylindole, 4-methyloctanoic acid, 4-ethyloctanoic acid, and 4-methylnonanoic acid in an air-oil matrix were empirically derived. The effectiveness of SIFT-MS for the direct, real-time, and rapid determination of key flavor compounds in lamb fat samples was established.

Estimates of genetic parameters for content of boar taint compounds in adipose tissue of intact males at 160 and 220 days of age.

The aims of this study were to investigate variation in content of androstenone (AND), skatole (SKA), and indole (IND), quantified in adipose tissue of intact male pigs at 160 d of age (105 kg BW) and 220 d of age (155 kg BW), to estimate genetic parameters and to investigate the genetic relationships for AND, SKA, IND, and growth traits. A sample of adipose tissue was collected in vivo, using a biopsy device, from the neck of 500 intact males at the 2 ages and at slaughter from the ham of 100 of the investigated animals. Backfat depth was measured at 220 d of age, whereas BW was recorded at each sampling. Quantification of AND, SKA, and IND was performed by HPLC with fluorescence detection. Estimates of genetic parameters were obtained through Bayesian analyses after logarithmic transformations of original measures. Contents of boar taint compounds (BTC) measured at 220 d were higher than those at 160 d of age. Correlations between contents of BTC in backfat and ham fat ranged from 0.7 (IND) to 0.88 (SKA). Medium-high h were estimated for BTC at both ages, but estimates at 220 d (0.58, 0.60, and 0.69 for AND, SKA, and IND, respectively) were greater than those at 160 d. The genetic correlation between contents at 160 and 220 d of each BTC was positive, but the probability that such estimates were greater than 0.8 was very low, indicating that contents at 160 and 220 d were traits controlled by different genetic backgrounds. Different rankings were observed when breeding values for the content at 160 and 220 d of age were used to rank animals. As a consequence, performance testing programs for BTC should be based preferably on phenotypes measured at 220 d of age. Weak genetic correlations were observed between content of BT compounds and growth traits (BW, backfat depth, and daily gain from 160 to 220 d of age), indicating that selective breeding to reduce the risk of tainted pork is expected to exert trivial effects on growth performance and fat deposition. Results indicate that prevalence of BTC is high in mature and heavy pigs relative to young and light pigs. High heritability; positive genetic correlations between AND, SKA, and IND; and trivial effects on growth traits suggest that reduction of BTC through selective breeding is feasible and exploitable as an alternative to surgical castration also for pigs slaughtered at heavy BW.

Preliminary study of FMO1, FMO5, CYP21, ESR1, PLIN2 and SULT2A1 as candidate gene for compounds related to boar taint.

An association study between polymorphisms of six genes and boar taint related compounds androstenone, skatole and indole was performed in a boar population (n=370). Significant association (P<0.05) was detected for SNP of FMO5 (g.494A>G) with all boar taint compounds, SNP of CYP21 (g.3911T>C) with skatole and indole, and SNP of ESR1 (g.672C>T) with androstenone and indole. mRNA expression of CYP21 and ESR1 was higher in CAB (castrated boar) compared to non-castrated boars; whereas, the expression of FMO5 and ESR1 was higher in LBT (low boar taint) compared to HBT (high boar taint) in liver tissue. FMO5, CYP21 and ESR1 proteins were less detectable in HBT compared with LBT and CAB in liver tissues. These findings suggest that FMO5, CYP21 and ESR1 gene variants might have effects on the boar taint compounds.

Skatole metabolism in the pigs with reduced testicular oestrogen synthesis.

The objectives of the study were to investigate the involvement of oestrogens in the regulation of skatole levels in pigs. In total, 44 intact male pigs, siblings from 10 litters, were included in the study. Pigs were orally treated weekly with either 0.1 mg letrozole/kg body weight to reduce endogenous oestrogens or the canola oil vehicle. Fat and liver samples were collected at slaughter at 16, 20 and 40 weeks of age. Skatole and androstenone levels in fat and activities of hepatic cytochrome P4501A1, CYP1A2, CYP2A19 and CYP2E1 were analysed. Letrozole treatment did not significantly change either the levels of skatole or activities of skatole-metabolising enzymes, suggesting that oestrogens are not responsible for gender-related differences in skatole concentrations in porcine tissues.

Fluorescence of tryptophan in designed hairpin and Trp-cage miniproteins: measurements of fluorescence yields and calculations by quantum mechanical molecular dynamics simulations.

The quantum yield of tryptophan (Trp) fluorescence was measured in 30 designed miniproteins (17 ß-hairpins and 13 Trp-cage peptides), each containing a single Trp residue. Measurements were made in D(2)O and H(2)O to distinguish between fluorescence quenching mechanisms involving electron and proton transfer in the hairpin peptides, and at two temperatures to check for effects of partial unfolding of the Trp-cage peptides. The extent of folding of all the peptides also was measured by NMR. The fluorescence yields ranged from 0.01 in some of the Trp-cage peptides to 0.27 in some hairpins. Fluorescence quenching was found to occur by electron transfer from the excited indole ring of the Trp to a backbone amide group or the protonated side chain of a nearby histidine, glutamate, aspartate, tyrosine, or cysteine residue. Ionized tyrosine side chains quenched strongly by resonance energy transfer or electron transfer to the excited indole ring. Hybrid classical/quantum mechanical molecular dynamics simulations were performed by a method that optimized induced electric dipoles separately for the ground and excited states in multiple π-π* and charge-transfer (CT) excitations. Twenty 0.5 ns trajectories in the tryptophan's lowest excited singlet π-π* state were run for each peptide, beginning by projections from trajectories in the ground state. Fluorescence quenching was correlated with the availability of a CT or exciton state that was strongly coupled to the π-π* state and that matched or fell below the π-π* state in energy. The fluorescence yields predicted by summing the calculated rates of charge and energy transfer are in good accord with the measured yields.

Directed evolution reveals requisite sequence elements in the functional expression of P450 2F1 in Escherichia coli.

Cytochrome P450 2F1 (P450 2F1) is expressed exclusively in the human respiratory tract and is implicated in 3-methylindole (3MI)-induced pneumotoxicity via dehydrogenation of 3MI to a reactive electrophilic intermediate, 3-methyleneindolenine (3-MEI). Studies of P450 2F1 to date have been limited by the failure to express this enzyme in Escherichia coli. By contrast, P450 2F3, a caprine homologue that shares 84% sequence identity with P450 2F1 (86 amino acid differences), has been expressed in E. coli at yields greater than 250 nmol/L culture. We hypothesized that a limited number of sequence differences between P450s 2F1 and 2F3 could limit P450 2F1 expression in E. coli and that problematic P450 2F1 sequence elements could be identified by directed evolution. A library of P450 2F1/2F3 mutants was created by DNA family shuffling and screened for expression in E. coli. Three generations of DNA shuffling revealed a mutant (named JH_2F_F3_1_007) with 96.5% nucleotide sequence identity to P450 2F1 and which expressed 119 ± 40 pmol (n = 3, mean ± SD) hemoprotein in 1 mL microaerobic cultures. Across all three generations, two regions were observed where P450 2F3-derived sequence was consistently substituted for P450 2F1 sequence in expressing mutants, encoding nine amino acid differences between P450s 2F1 and 2F3: nucleotides 191-278 (amino acids 65-92) and 794-924 (amino acids 265-305). Chimeras constructed to specifically test the importance of these two regions confirmed that P450 2F3 sequence is essential in both regions for expression in E. coli but that other non-P450 2F1 sequence elements outside of these regions also improved the expression of mutant JH_2F_F3_1_007. Mutant JH_2F_F3_1_007 catalyzed the dehydrogenation of 3MI to 3-MEI as indicated by the observation of glutathione adducts after incubation in the presence of glutathione. The JH_2F_F3_1_007 protein differs from P450 2F1 at only 20 amino acids and should facilitate further studies of the structure-activity relationships of P450s of the 2F subfamily.

Investigation of the sunscreen octocrylene's interaction with amino acid analogs in the presence of UV radiation.

Octocrylene is an organic UV filter, commonly used in sunscreens and cosmetics, which can give rise to both contact and photocontact allergy. Our aim was to investigate octocrylene's interaction with amino acid analogs in the presence of UV radiation to better understand the reason for octocrylene's photoallergenic capacity. The amino acid analogs were photolysed in presence and absence of octocrylene for 1 h in cyclohexane. The rate of degradation was considerably slower for all amino acid analogs when octocrylene was present in the mixture. Benzylamine, the lysine analog, did react with octocrylene during the photolysis and the corresponding amide was formed in an acylation reaction. By varying the benzylamine concentration and keeping the octocrylene concentration fixed the reaction rate was shown to be independent of the amine concentration. The same type of acylation reaction took place when octocrylene alone was photolysed in ethanol in which the ethyl ester was formed from octocrylene and ethanol. Our results suggest that octocrylene's ability to cause photocontact allergy could be due to its photoinduced reactivity toward primary amines and alcohols.

Potent mutagenicity of 3-methylindole requires pulmonary cytochrome P450-mediated bioactivation: a comparison to the prototype cigarette smoke mutagens B(a)P and NNK.

3-Methylindole (3MI) is a preferential pneumotoxicant found in cigarette smoke. A number of lung-expressed human cytochrome P450 enzymes, including 1A1, 2F1, and 2A13, catalyze the metabolism of 3MI to reactive intermediates that fragment DNA, measured with the Comet assay to assess DNA damage, in a cytochrome P450-dependent manner in primary normal human lung cells in culture, but the mutagenesis of 3MI has been controversial. In the present study, the mutagenic potential of 3MI was compared to the prototypical cigarette smoke carcinogens benzo(a)pyrene (B(a)P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). 3MI, B(a)P, and NNK were incubated with the Salmonella typhimurium strain TA98, which is known to detect the most common subtype of cigarette smoke-induced mutagenicity, frameshift mutations in DNA, and with Salmonella typhimurium strain TA100, which detects base pair substitution mutants, with five sources of P450-mediated bioactivation: rat liver S9, human lung microsomes, recombinant CYP2A13, purified CYP2F3, and recombinant CYP1A1. Only B(a)P was mutagenic in TA100, and it was bioactivated by human lung microsomes and rat liver S9 sources of P450s. However, with the TA98 strain, CYP1A1, CYP2A13, CYP2F3, and human lung microsomes bioactivated 3MI to highly mutagenic intermediates, whereas neither human nor rat liver S9 subcellular fractions formed mutagenic intermediates from 3MI. Quantitative Western blot analysis verified that all three respiratory enzymes were present in human lung microsomes in widely varying amounts. These results indicate that metabolism of 3MI by human lung-expressed cytochrome P450 enzymes but not hepatic P450s elicits equivalent or higher mutagenicity than the prototype cigarette smoke mutagens B(a)P and NNK and indicates that 3MI is a likely human pulmonary carcinogen.

Insulin-like growth factor binding protein-2: contributions of the C-terminal domain to insulin-like growth factor-1 binding.

Signaling by the insulin-like growth factor (IGF)-1 receptor (IGF-1R) has been implicated in the promotion and aggressiveness of breast, prostate, colorectal, and lung cancers. The IGF binding proteins (IGFBPs) represent a class of natural IGF antagonists that bind to and sequester IGF-1/2 from the IGF-1R, making them attractive candidates as therapeutics for cancer prevention and control. Recombinant human IGFBP-2 significantly attenuated IGF-1-stimulated MCF-7 cell proliferation with coaddition of 20 or 100 nM IGFBP-2 (50 or 80% inhibition, respectively). We previously identified IGF-1 contact sites both upstream and downstream of the CWCV motif (residues 247-250) in human IGFBP-2 (J Biol Chem 276:2880-2889, 2001). To further test their contributions to IGFBP-2 function, the single tryptophan in human IGFBP-2, Trp-248, was selectively cleaved with 2-(2'nitrophenylsulfenyl)-3-methyl-3 bromoindolenine (BNPS-skatole) and the BNPS-skatole products IGFBP-2(1-248) and IGFBP-2(249-289) as well as IGFBP-2(1-190) were expressed as glutathione S-transferase-fusion proteins and purified. Based on competition binding analysis, deletion of residues 249 to 289 caused an approximately 20-fold decrease in IGF-1 binding affinity (IGFBP-2 EC50 = 0.35 nM and IGFBP-2(1-248) = 7 nM). Removal of the remainder of the C-terminal domain had no further effect on affinity (IGFBP-2(1-190) EC50 = 9.2 nM). In kinetic assays, IGFBP-2(1-248) and IGFBP-2(1-190) exhibited more rapid association and dissociation rates than full-length IGFBP-2. These results confirm that regions upstream and downstream of the CWCV motif participate in IGF-1 binding. They further support the development of full-length IGFBP-2 as a cancer therapeutic.

Use of a chemical trigger for electron transfer to characterize a precursor to cluster X in assembly of the iron-radical cofactor of Escherichia coli ribonucleotide reductase.

A key step in generation of the catalytically essential tyrosyl radical (Y122(*)) in protein R2 of Escherichia coli ribonucleotide reductase is electron transfer (ET) from the near-surface residue, tryptophan 48 (W48), to a (Fe(2)O(2))(4+) complex formed by addition of O(2) to the carboxylate-bridged diiron(II) cluster. Because this step is rapid, the (Fe(2)O(2))(4+) complex does not accumulate and, therefore, has not been characterized. The product of the ET step is a "diradical" intermediate state containing the well-characterized Fe(IV)Fe(III) cluster, X, and a W48 cation radical (W48(+)(*)). The latter may be reduced from solution to complete the two-step transfer of an electron to the buried diiron site. In this study, a (Fe(2)O(2))(4+) state that is probably the precursor to the X-W48(+)(*) diradical state in the reaction of the wild-type protein (R2-wt) has been characterized by exploitation of the observation that in R2 variants with W48 replaced with alanine (A), the otherwise disabled ET step can be mediated by indole compounds. Mixing of the Fe(II) complex of R2-W48A/Y122F with O(2) results in accumulation of an intermediate state that rapidly converts to X upon mixing with 3-methylindole (3-MI). The state comprises at least two species, of which each exhibits an apparent Mössbauer quadrupole doublet with parameters characteristic of high-spin Fe(III) ions. The isomer shifts of these complexes and absence of magnetic hyperfine coupling in their Mössbauer spectra suggest that both are antiferromagnetically coupled diiron(III) clusters. The fact that both rapidly convert to X upon treatment with a molecule (3-MI) shown in the preceding paper to mediate ET in W48A R2 variants indicates that they are more oxidized than X by one electron, which suggests that they have a bound peroxide equivalent. Their failure to exhibit either the long-wavelength absorption (at 650-750 nm) or Mössbauer doublet with high isomer shift (>0.6 mm/s) that are characteristic of the putatively mu-1,2-peroxo-bridged diiron(III) intermediates that have been detected in the reactions of methane monooxygenase (P or H(peroxo)) and variants of R2 with the D84E ligand substitution suggests that they have geometries and electronic structures different from those of the previously characterized complexes. Supporting this deduction, the peroxodiiron(III) complex that accumulates in R2-W48A/D84E is much less reactive toward 3-MI-mediated reduction than the (Fe(2)O(2))(4+) state in R2-W48A/Y122F. It is postulated that the new (Fe(2)O(2))(4+) state is either an early adduct in an orthogonal pathway for oxygen activation or, more likely, the successor to a (mu-1,2-peroxo)diiron(III) complex that is extremely fleeting in R2 proteins with the wild-type ligand set but longer lived in D84E-containing variants.

A direct access to 3-(2-Oxoalkyl)indoles via aluminum chloride induced C-C bond formation.

3-Methylindole is acylated regioselectively at the methyl group when treated with a variety of acyl chlorides in 1,2-dichloroethane in the presence of AlCl(3), affording a mild and direct method for the synthesis of 3-(2-oxoalkyl)indoles. The product formation in this one-pot reaction largely depends on the conditions of the reaction employed. The methodology does not require protection-deprotection steps and is amenable for the scale-up synthesis of these indole derivatives.

Photoaffinity labeling identifies the substrate-binding site of mammalian squalene epoxidase.

Squalene epoxidase (SE) catalyzes the conversion of squalene to (3S)-2,3-oxidosqualene. Photolabeling and site-directed mutagenesis were performed on recombinant rat SE (rrSE) in order to identify the location of the substrate-binding site and the roles of key residues in catalysis. Truncated 50-kDa rrSE was purified and photoaffinity labeled by competitive SE inhibitor (Ki=18.4 microM), [(3)H]TNSA-Dza. An 8-kDa CNBr/BNPS-skatole peptide was purified and the first 24 amino acids were sequenced by Edman degradation. The sequence PASFLPPSSVNKRGVLLLGDAYNL corresponded to residues 388-411 of the full-length rat SE. Three nucleophilic residues (Lys-399, Arg-400, and Asp-407) were labeled by [(3)H]TNSA-Dza. Triple mutants were prepared in which bulky groups were used to replace the labeled charged residues. Purified mutant enzymes showed lower enzymatic activity and reduced photoaffinity labeling by [(3)H]TNSA-Dza. This constitutes the first evidence as to the identity of the substrate-binding site of SE.

Phase II in vitro metabolism of 3-methylindole metabolites in porcine liver.

1. The Phase II in vitro metabolism of 3-methylindole (3MI) metabolites was investigated in pigs to determine the possible relationship between 3MI Phase II metabolism and 3MI accumulation in fat. Sulphation and glucuronidation of five of the seven major metabolites found to be produced by porcine microsomes was investigated using porcine cytosol and microsomes, respectively. The possible formation of glutathione conjugates was also investigated using microsomally activated 3MI intermediate(s). 2. No sulphation or glucuronidation was observed for metabolites 3-hydroxy-3-methyloxindole, 3-methyloxindole, indole-3-carbinol or 2-aminoacetophenone; however, 5-hydroxy-3-methylindole (5-OH-3MI) was conjugated with both sulphate and glucuronic acid. 3. The enzyme responsible for sulphation of 5-OH-3MI was identified as the thermostable form of phenol-sulphotransferase (TS-PST) based on its susceptibility to TS-PST inhibitors and the correlation between sulphation of 5-OH-3MI and sulphation of the prototype substrate p-nitrophenol (r = 0.94, p < 0.001). 4. A 3MI-glutathione adduct was identified in microsomal incubations containing 3MI and glutathione. 5. Sulphation of 5-OH-3MI was high in pigs with low levels of 3MI in fat. No relationship was observed between 3MI levels in fat and either glutathione transferase or glucuronidation activities in liver.

Detection and characterization of DNA adducts of 3-methylindole.

The pneumotoxin 3-methylindole is metabolized to the reactive intermediate 3-methyleneindolenine which has been shown to form adducts with glutathione and proteins. Reported here is the synthesis, detection, and characterization of nucleoside adducts of 3-methylindole. Adducted nucleoside standards were synthesized by the reaction of indole-3-carbinol with each of the four nucleosides under slightly acidic conditions, which catalyze the dehydration of indole-3-carbinol to 3-methyleneindolenine. Following solid phase extraction, the individual adducts were infused via an electrospray source into an ion trap mass spectrometer for molecular weight determination and characterization of the fragmentation patterns. The molecular ions and fragmentation of the dGuo, dAdo, and dCyd adducts were consistent with nucleophilic addition of the exocyclic primary amine of the nucleosides to the methylene carbon of 3-methyleneindolenine. The apparent chemical preference of this addition lead primarily to dAdo and dGuo adducts, with substantially less of the dCyd adduct formed. No adduct with dThd was detected. The adducts were purified by HPLC and subsequent NMR analysis of the dGuo and dCyd adducts confirmed the proposed structures. Mass spectral fragmentation of the three adducts produced primarily two ions which were the result of the loss of either the 3-methylindole moiety or the sugar. On a triple quadrupole electrospray mass spectrometer, the neutral loss of the sugar, [M + H - 116](+), was utilized for selected reaction monitoring of the calf thymus DNA adducts, formed by incubations of 3-methylindole with various microsomes (rat liver, goat lung, and human liver). All three adducts were detected from each of the microsomal incubations, following extraction and cleavage of the DNA to the nucleoside level. The dGuo adduct was the primary adduct formed, with smaller amounts of the dAdo and dCyd adducts. Rat hepatocytes incubated with 3-methylindole produced the same three adducts, in approximately the same proportions, while no adducts were detected in untreated hepatocytes. Microsomal incubations in the presence of ([3-(2)H(3)]-methyl)indole confirmed the formation and identification of the adducts as well as the fragmentation patterns. These results demonstrate that bioactivated 3-methylindole forms specific adducts with exogenous or intact cellular DNA, and indicates that 3-methylindole may be a potential mutagenic and/or carcinogenic chemical.

Chemical cleavage of bovine beta-lactoglobulin by BNPS-skatole for preparative purposes: comparative study of hydrolytic procedures and peptide characterization.

A comparative study of various procedures for tryptophanyl peptide bond cleavage by BNPS-skatole [2-(2-nitrophenyl)-3-methyl-3-bromoindolenine] was carried out on native and on reduced and alkylated bovine beta-lactoglobulin (BLG). The reaction yield and the composition of the derived products were studied in acetic acid, trifluoroacetic acid (TFA), and ethanol/TFA. For BNPS-skatole removal, extraction by water or ethyl ether was compared with dialysis and gel filtration. The three expected peptides (1-19, 20-61, 62-162) and incomplete cleaved fragments (1-61, 20-162) were separated and characterized by electrophoresis, reverse-phase high-performance liquid chromatography, and mass spectrometry. The highest hydrolysis yield (67.4%) occurred with native BLG cleaved in 88% acetic acid at 47 degrees C for 60 min. Subsequent water extraction and gel filtration led to total recovery of the material, but reagent elimination was only quantitative after gel filtration. Cleavage specificity was ensured by mass spectrometry and the amino acid composition of peptides 1-19 and 62-162. The chemical side reactions identified are discussed.

Toward understanding tryptophan fluorescence in proteins.

A general approach to dissecting the complex photophysics of tryptophan is presented and used to elucidate the effects of amino acid functional groups on tryptophan fluorescence. We have definitively identified the amino acid side chains that quench tryptophan fluorescence and delineated the respective quenching mechanisms in a simple model system. Steady-state and time-resolved fluorescence techniques, photochemical H-D exchange experiments, and transient absorption techniques were used to measure individual contributions to the total nonradiative rate for deactivation of the excited state, including intersystem crossing, solvent quenching, and excited-state proton and electron transfer rates. Eight amino acid side chains representing six functional groups quench 3-methylindole fluorescence with a 100-fold range in quenching rate constant. Lysine and tyrosine side chains quench by excited-state proton transfer; glutamine, asparagine, glutamic and aspartic acid, cysteine, and histidine side chains quench by excited-state electron transfer. These studies provide a framework for deriving detailed structural and dynamical information from tryptophan fluorescence intensity and lifetime data in peptides and proteins.

Identification of skatolyl hydroperoxide and its role in the peroxidase-catalysed oxidation of indol-3-yl acetic acid.

Indol-3-yl acetic acid (IAA, auxin) is a plant hormone whose degradation is a key determinant of plant growth and development. The first evidence for skatolyl hydroperoxide formation during the plant peroxidase-catalysed degradation of IAA has been obtained by electrospray MS. Skatolyl hydroperoxide degrades predominantly non-enzymically to oxindol-3-yl carbinol but in part enzymically into indol-3-yl methanol via a peroxidase cycle in which IAA acts as an electron donor. Skatolyl hydroperoxide is degradable by catalase. Horseradish peroxidase isoenzyme C (HRP-C) and anionic tobacco peroxidase (TOP) exhibit differences in their mechanisms of reaction. The insensitivity of the HRP-C-catalysed reaction to catalase is ascribed to the formation of HRP-C Compound III at the initiation step and its subsequent role in radical propagation. This is in contrast with the TOP-catalysed process in which skatolyl hydroperoxide has a key role. Indol-3-yl aldehyde is produced not via the peroxidase cycle but by catalysis involving ferrous peroxidase. Because indol-3-yl aldehyde is one of the main IAA-derived products identified in planta, we conclude that ferrous peroxidases participate in IAA catalytic transformations in vivo. A general scheme for peroxidase-catalysed IAA oxidation is presented.

Interaction sites of the COOH-terminal region of the gamma subunit of cGMP phosphodiesterase with the GTP-bound alpha subunit of transducin.

In photoreceptor cells, visual transduction occurs through photoexcitation of rhodopsin, GTP activation of the alpha subunit of transducin, and interaction between GTP-bound transducin alpha subunit and the inhibitory gamma subunit of phosphodiesterase. The gamma subunit of phosphodiesterase, in turn, accelerates the hydrolysis of GTP on the alpha subunit of transducin. Within the COOH-terminal residues (46-87) of the phosphodiesterase gamma subunit, Trp-70 has been implicated in phosphodiesterase activation, transducin alpha subunit-phosphodiesterase gamma subunit interaction, and the GTP hydrolysis accelerating activity. We have derivatized the phosphodiesterase gamma subunit with a reversible photoactivatable reagent, [125I]N-[(3-iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) ]cysteine ([125I]ACTP), at cysteine (Cys-68). A light-dependent, cross-linked complex of guanosine 5'-(gamma-thio)triphosphate-bound transducin alpha subunit and ACTPderivatized phosphodiesterase gamma subunit formed after photolysis of a 1:1 stoichiometic complex of the two proteins. The specificity of complex formation between the transducin alpha subunit and the phosphodiesterase gamma subunit was demonstrated by specific protection by the C68A mutant of the phosphodiesterase gamma subunit. The cross-linked complex was treated with beta-mercaptoethanol to transfer the 125I photomoiety from the phosphodiesterase gamma subunit to the transducin alpha subunit. Combined techniques involving electrophoresis, chemical and enzymatic cleavage, and chemical and radiosequencing were used to identify photoinsertion sites on the alpha3 and alpha4/beta6 regions of the transducin alpha subunit. Three photo-labeled residues, His-244 (alpha3 helix), Met-308, and Arg-310 (alpha4/beta6 interface), were specifically identified as photoinsertion sites. Utilizing the crystal structure coordinates of the GTP-bound transducin alpha subunit and molecular modeling, we conclude that Cys-68 of the phosphodiesterase gamma subunit is located at a position between the exposed face of the alpha3 and alpha4 helices of the transducin alpha subunit. We propose that the phosphodiesterase gamma subunit interacts with GTP-bound transducin alpha subunit at multiple sites in which the cysteine 68 to tryptophan 70 sequence of the phosphodiesterase gamma subunit, which is critical for GTP hydrolysis accelerating activity, interacts in the alpha3/alpha4/beta6 region of GTP-bound transducin alpha subunit.