Visible Difference Spectra Research Articles

Fourier transform visible and infrared difference spectroscopy for the study of P700 in photosystem I from Fischerella thermalis PCC 7521 cells grown under white light and far-red light: Evidence that the A–1 cofactor is chlorophyll f

(P700+ – P700) Fourier transform visible and infrared difference spectra (DS) have been obtained using photosystem I (PSI) complexes isolated from cells of Fischerella thermalis PCC 7521 grown under white light (WL) or far-red light (FRL). PSI from cells grown under FRL (FRL-PSI) contain ~8 chlorophyll f (Chl f) molecules (Shen et al., Photosynth. Res. Jan. 2019). Both the visible and infrared DS indicate that neither the PA or PB pigments of P700 are Chl f molecules, but do support the conclusion that at least one of the A–1 cofactors is a Chl f molecule.The FTIR DS indicate that the hydrogen bond to the 131-keto CO group of the PA pigment of P700 is weakened in FRL-PSI, as might be expected given that the proteins that bind the P700 pigments are substantially different in FRL-PSI (Gan et al., Science 345, 1312–1317, 2014).The FTIR DS obtained using FRL-PSI display a band at 1664 cm−1 that is assigned (based on density functional theory calculations) to the 21-formyl CO group of Chl f, that upshifts 5 cm−1 upon P700+ formation. This is much less than expected for a cation-induced upshift, indicating that the Chl f molecule is not one of the pigments of P700.In WL-PSI the A–1 cofactor is a Chl a molecule with 131-keto and 133-methylester CO mode vibrations at 1696 and 1750 cm−1, respectively. In FRL-PSI the A–1 cofactor is a Chl f molecule with 131-keto and 133-methylester CO mode vibrations at 1702 and 1754 cm−1, respectively.

Resonance Raman Characterization of the Ammonia-Generated Oxo Intermediate of Cytochrome c Oxidase from Paracoccus denitrificans

A novel oxo state of cytochrome c oxidase from Paracoccus denitrificans generated by successive addition of excess H2O2 and ammonia was investigated using resonance Raman (RR) spectroscopy. Addition of ammonia to the H2O2-generated artificial F state resulted in an upshift of the oxoferryl stretching vibration from 790 to 796 cm(-1), indicating that ammonia influences ligation of the heme-bound oxygen in the binuclear center. Concomitantly performed RR measurements in the high-frequency region between 1300 and 1700 cm(-1) showed a high-spin to low-spin transition of heme a3 upon generation of the F state that was not altered by addition of ammonia. Removal of H2O2 by addition of catalase resulted in the disappearance of the oxoferryl stretching vibration and major back transformation of heme a3 into the high-spin state. The ratio of high-spin to low-spin states was identical for intermediates created with and without ammonia, leading to the conclusion that ammonia does not interact directly with heme a3. Only for the ammonia-created state was a band at 612 nm observed in the UV-visible difference spectrum that was shifted to 608 nm after addition of catalase. Our results support the hypothesis by von der Hocht et al. [von der Hocht, I., et al. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 3964-3969] that addition of ammonia creates a novel oxo intermediate state called PN where ammonia binds to CuB once the oxo intermediate F state has been formed.

Infrared and visible absolute and difference spectra of bacteriorhodopsin photocycle intermediates.

We have used new kinetic fitting procedures to obtain infrared (IR) absolute spectra for intermediates of the main bacteriorhodopsin (bR) photocycle(s). The linear-algebra-based procedures of Hendler et al. (J. Phys. Chem. B, 105, 3319-3228 (2001)) for obtaining clean absolute visible spectra of bR photocycle intermediates were adapted for use with IR data. This led to isolation, for the first time, of corresponding clean absolute IR spectra, including the separation of the M intermediate into its M(F) and M(S) components from parallel photocycles. This in turn permitted the computation of clean IR difference spectra between pairs of successive intermediates, allowing for the most rigorous analysis to date of changes occurring at each step of the photocycle. The statistical accuracy of the spectral calculation methods allows us to identify, with great confidence, new spectral features. One of these is a very strong differential IR band at 1650 cm(-1) for the L intermediate at room temperature that is not present in analogous L spectra measured at cryogenic temperatures. This band, in one of the noisiest spectral regions, has not been identified in any previous time-resolved IR papers, although retrospectively it is apparent as one of the strongest L absorbance changes in their raw data, considered collectively. Additionally, our results are most consistent with Arg82 as the primary proton-release group (PRG), rather than a protonated water cluster or H-bonded grouping of carboxylic residues. Notably, the Arg82 deprotonation occurs exclusively in the M(F) pathway of the parallel cycles model of the photocycle.

The Puzzle of Ligand Binding to Corynebacterium ammoniagenes FAD Synthetase

In bacteria, riboflavin phosphorylation and subsequent conversion of FMN into FAD are carried out by FAD synthetase, a single bifunctional enzyme. Both reactions require ATP and Mg(2+). The N-terminal domain of FAD synthetase appears to be responsible for the adenylyltransferase activity, whereas the C-terminal domain would be in charge of the kinase activity. Binding to Corynebacterium ammoniagenes FAD synthetase of its products and substrates, as well as of several analogues, is analyzed. Binding parameters for adenine nucleotides to each one of the two adenine nucleotide sites are reported. In addition, it is demonstrated for the first time that the enzyme presents two independent flavin sites, each one related with one of the enzymatic activities. The binding parameters of flavins to these sites are also provided. The presence of Mg(2+) and of both adenine nucleotides and flavins cooperatively modulates the interaction parameters for the other ligands. Our data also suggest that during its double catalytic cycle FAD synthetase must suffer conformational changes induced by adenine nucleotide-Mg(2+) or flavin binding. They might include not only rearrangement of the different protein loops but also alternative conformations between domains.

Virtual cooperativity in myoglobin oxygen saturation curve in skeletal muscle in vivo

BackgroundMyoglobin (Mb) is the simplest monomeric hemoprotein and its physicochemical properties including reversible oxygen (O2)binding in aqueous solution are well known. Unexpectedly, however, its physiological role in intact muscle has not yet been established in spite of the fact that the role of the more complex tetrameric hemoprotein, hemoglobin (Hb), in red cells is well established. Here, I report my new findings on an overlooked property of skeletal Mb.MethodsI directly observed the oxygenation of Mb in perfused rat skeletal muscle under various states of tissue respiration. A computer-controlled rapid scanning spectrophotometer was used to measure the oxygenation of Mb in the transmission mode. The light beam was focused on the thigh (quadriceps) through a 5-mm-diameter light guide. The transmitted light was conducted to the spectrophotometer through another 5-mm-diameter light guide. Visible difference spectra in the range of 500–650 nm were recorded when O2 uptake in the hindlimb muscle reached a constant value after every stepwise change in the O2 concentration of the buffer.ResultsThe O2 dissociation curve (ODC) of Mb, when the effluent buffer O2 pressure was used as the abscissa, was of a sigmoid shape under normal and increased respiratory conditions whereas it was of rectangular hyperbolic shape under a suppressed respiratory condition. The dissociation curve was shifted toward the right and became more sigmoid with an increase in tissue respiration activity. These observations indicate that an increase in O2 demand in tissues makes the O2 saturation of Mb more sensitive to O2 pressure change in the capillaries and enhances the Mb-mediated O2 transfer from Hb to cytochrome oxidase (Cyt. aa3), especially under heavy O2 demands.ConclusionThe virtual cooperativity and O2 demand-dependent shifts of the ODC may provide a basis for explaining why Mb has been preserved as monomer during molecular evolution.

Cardiolipin Is Essential for Organization of Complexes III and IV into a Supercomplex in Intact Yeast Mitochondria

Digitonin extracts of mitochondria from cardiolipin-containing (wild type) and cardiolipin-lacking (crd1Delta mutant) Saccharomyces cerevisiae subjected to colorless native polyacrylamide gel electrophoresis in the presence of 0.003% digitonin displayed a supercomplex composed of homodimers of complexes III and IV in the former case but only the individual homodimers in the latter case. To avoid treatment with any detergent or dye, we compared organization of the respiratory chain in intact mitochondria from wild type and cardiolipin-lacking cells by using a functional analysis developed previously for the study of the organization of the respiratory chain of S. cerevisiae (Boumans, H., Grivell, L. A., and Berden, J. A. (1998) J. Biol. Chem. 273, 4872-4877). Dependence of the kinetics of NADH oxidation via complexes III, IV, and cytochrome c on the concentration of the complex III-specific inhibitor antimycin A was studied. A linear relationship between respiratory activity and saturation of complex III with antimycin A was obtained for wild type mitochondria consistent with single functional unit kinetics of the respiratory chain. Under the same conditions, cardiolipin-lacking mitochondria displayed a hyperbolic relationship indicating cytochrome c pool behavior. No release of cytochrome c from cardiolipin-lacking mitochondria or mitoplasts under our standard experimental conditions was detected. Identical cytochrome c pool behavior was observed for both wild type and cardiolipin-lacking mitochondria in the presence of a chaotropic agent, which disrupts the interaction between respiratory complexes. The results demonstrate that cardiolipin is essential for association of complexes III and IV into a supercomplex in intact yeast mitochondria.

Time-Resolved Step-Scan Fourier Transform Infrared and Visible Absorption Difference Spectroscopy for the Study of Photosystem I

Step-scan Fourier transform infrared and visible absorption difference spectroscopy, with nanosecond to millisecond time resolution, has been applied to the study of photosystem I particles from photosynthetic oxygen-evolving organisms. In particular, time-resolved infrared (1800–1200 cm−1) and visible (680–850 nm) difference spectra associated with flash induced oxidation of the primary electron donor in photosystem I particles from Synechocystis sp. 6803 and Acarychloris marina are presented. These spectra are compared directly to static, photoaccumulated Fourier transform infrared and visible difference spectra from both species. The detailed bonding interactions of P740, the primary donor in photosystem I particles from Acarychloris marina, are very similar to that found for P700, the primary donor in photosystem I particles from Synechocystis sp. 6803. P740 consists of at least two chlorophyll molecules, both of which are probably chlorophyll-d. Combined time-resolved step-scan Fourier transform infrared and visible absorption difference spectroscopy is shown to be a powerful tool for the study of single molecular bond dynamics in proteins as large as 0.4 mega-Daltons.

Intramolecular electron transfer and conformational changes in cytochrome c oxidase.

The photolysis intermediates of partially and fully reduced CO-bound cytochrome oxidase derivatives were investigated. A gated optical spectrometric multichannel analyzer was used to collect visible and near-infrared transient difference spectra on time scales from nanoseconds to milliseconds. The spectra were analyzed by a singular value decomposition method combined with a global exponential fitting procedure. Global analysis of the mixed-valence CO complex transient difference spectra shows that five intermediates are present with apparent lifetimes of 1.4 microseconds, 4.8 microseconds, 76.7 microseconds, 10.6 ms, and 21.6 ms. The data were fitted to a kinetic model involving a sequential pathway with accompanying equilibria. On the basis of this mechanism, the absorption spectra of the intermediates were determined. The first step, also present in the fully reduced enzyme, is attributed to a conformational change at cytochrome a3. The spectral changes associated with the second step are similar to those expected for 1:1 electron transfer from cytochrome a3 to cytochrome a, except for a higher absorbance between 480 and 550 nm. A comparison of the experimental spectral change associated with this step, (a2+ minus a3+) minus (a3(2+) minus a3(3+), and the calculated spectral change, (a2+ CuA+ minus a3+ CuA2+) minus a3(2+) CuB+ minus a3(3+) CuB2+), allowed extraction of the absorbance spectrum of CuA2+ in the 480-550 nm region. The spectral change associated with the third step is consistent with the oxidation of cytochrome a. A decrease in the 830 nm band on the same time scale indicates that the electron acceptor is CuA. The data also suggest that the redox state of CuB significantly affects the absorption spectrum of oxidized cytochrome a3 in the visible region. The two processes on a millisecond time scale are attributed to CO recombination and intramolecular electron transfer.

Inactivation of yeast hexokinase by Cibacron Blue 3G-A: spectral, kinetic and structural investigations.

Yeast hexokinase, a homodimer (100 kDa), is an important enzyme in the glycolytic pathway. Although Cibacron Blue 3G-A (Reactive Blue 2) has been previously shown to inactivate yeast hexokinase, no comprehensive study exists concerning the nature of interaction(s) between hexokinase and the blue dye. A comparison of the computer-generated three-dimensional (3D) representations showed considerable overlap of the purine ring of ATP, a nucleotide substrate of hexokinase, with the hydrophobic anthraquinone moiety of the blue dye. The visible spectrum of the blue dye showed a characteristic absorption band centred at 628 nm. The visible difference spectrum of increasing concentration of the dye and the same concentrations of the dye plus a fixed concentration of hexokinase exhibited a maximum, a minimum and an isobestic point at 683, 585, and 655 nm respectively. The visible difference spectrum of the blue dye and the dye in 50% ethylene glycol showed a maximum and a minimum at 660 and 570 nm respectively. The visible difference spectrum of the blue dye in the presence of the dye and hexokinase modified at the active site by pyridoxal phosphate, iodoacetamide and o-phthalaldehyde was devoid of bands characteristic of the hexokinase-blue dye complex. Size-exclusion-chromatographic studies in the absence or presence of guanidinium chloride showed that the enzyme inactivated by the blue dye was co-eluted with the unmodified enzyme. The dialysis residue obtained after extensive dialysis of the gel-filtered complex, against a buffer of high ionic strength, showed an absorption maximum at 655 nm characteristic of the dye-enzyme complex. Inactivation data when analysed by 'Kitz-Wilson'-type kinetics for an irreversible inhibitor, yielded values of 0.05 min-1 and 92 microM for maximum rate of inactivation (k3) and dissociation constant (Kd) for the enzyme-dye complex respectively. Sugar and nucleotide substrates protected hexokinase against inactivation by the blue dye. About 2 mol of the blue dye bound per mol of hexokinase after complete inactivation. The inactivated enzyme could not be re-activated in the presence of 1 M NaCl. These results suggest that Cibacron Blue 3G-A inactivated hexokinase by an irreversible adduct formation at or near the active-site. Spectral and kinetic studies coupled with an analysis of the 3D representations of model compounds corresponding to the substructures of the blue dye suggest that 1-amino-4-(N-phenylamino)anthraquinone-2-sulphonic acid part of the blue dye may represent the minimum structure of Cibacron Blue 3G-A necessary to bind hexokinase.(ABSTRACT TRUNCATED AT 400 WORDS)

Inactivation of Yeast Hexokinase by Cibacron Brilliant Red 3B-A

Procion or Cibacron blue dyes, containing polynuclear aromatic rings and mono- and dichlorotriazine nuclei, immobilized on dextran matrices, have been used for over a decade to purify diverse groups of enzymes by dye-ligand chromatography. Comparatively less attention has been paid to investigating the nature of molecular interactions between similarly constituted red dyes and various enzymes so as to ascertain their potential and thus justify their use in the purification of enzymes by dye-ligand chromatography. We investigated and found that Cibacron brilliant red 3B-A, a monochlorotriazine dye, inhibited phosphotransferase activity of yeast hexokinase. The dissociation constant, KD, and the rate of dye-enzyme complex formation,were 120 μM and 0.1 min−1, respectively. The enzyme was protected from inactivation by sugar and nucleotide substrates. About 2 mol of the dye bound per mole of the enzyme. The chromophore of the dye showed absorption at 524 nm. The visible difference spectrum of increasing concentration of the dye and same concentrations of the dye plus a fixed concentration of hexokinase exhibited a maximum, a minimum, and an isosbestic point at 569, 501, and 512 nm, respectively. The difference spectrum of the dye and dye in 60% ethylene glycol showed a maximum and a minimum at 556 and 495 nm, respectively. The dye showed no visible difference spectrum in the presence of hexokinase modified at the active site by iodoacetamide, pyridoxal phosphate, and o-phthalaldehyde. Hexokinase modified by the dye coeluted with the unmodified enzyme during size-exclusion chromatography in the absence or presence of guanidinium hydrochloride. These results suggest that the dye interacts with the hydrophobic environment of the active site of the enzyme. Analysis of the kinetics of inhibition of hexokinase by model compounds and comparison of their computer-assisted three-dimensional representations with that of Cibacron brilliant red 3B-A suggest that 1-amino-8-naphthol-3,6-disulfonic acid may represent the minimum structure for the dye to bind.

Tissue-specific regulation of bovine heart cytochrome-c oxidase activity by ADP via interaction with subunit VIa.

The activity of reconstituted cytochrome-c oxidase (EC 1.9.3.1) from bovine heart is stimulated by intraliposomal ADP but not by NaCl of the same ionic strength. A monoclonal antibody which reacts with subunits VIa-H (heart-type) and VIc, due to the evolutionary relationship between these subunits, also stimulates the activity of the enzyme from bovine heart but not from bovine liver. The antibody induces a conformational change in the heart enzyme but not in the liver enzyme, as shown by the visible difference spectrum. Preincubation of heart cytochrome-c oxidase with the antibody prevents stimulation of activity by intraliposomal ADP after reconstitution in liposomes. Reconstituted liver cytochrome c oxidase is not stimulated by intraliposomal ADP. The data suggest tissue-specific regulation of the activity of cytochrome-c oxidase by ADP via interaction with the matrix domain of subunit VIa-H.

Time-resolved Fourier transform infrared spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: Asp-96 reprotonates during O formation; Asp-85 and Asp-212 deprotonate during O decay.

The protonation state of key aspartic acid residues in the O intermediate of bacteriorhodopsin (bR) has been investigated by time-resolved Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis. In an earlier study (Bousché et al., J. Biol Chem. 266, 11063-11067, 1991) we found that Asp-96 undergoes a deprotonation during the M-->N transition, confirming its role as a proton donor in the reprotonation pathway leading from the cytoplasm to the Schiff base. In addition, both Asp-85 and Asp-212, which protonate upon formation of the M intermediate, remain protonated in the N intermediate. In this study, we have utilized the mutant Tyr-185-->Phe (Y185F), which at high pH and salt concentrations exhibits a photocycle similar to wild type bR but has a much slower decay of the O intermediate. Y185F was expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. Time-resolved FTIR difference spectra and visible difference spectra of this mutant were measured from hydrated multilayer films. A normal N intermediate in the photocycle of Y185F was identified on the basis of characteristic chromophore and protein vibrational bands. As N decays, bands characteristic of the all-trans O chromophore appear in the time-resolved FTIR difference spectra in the same time range as the appearance of a red-shifted photocycle intermediate absorbing near 640 nm. Based on our previous assignment of the carboxyl stretch bands to the four membrane embedded Asp groups: Asp-85, Asp-96, Asp-115 and Asp-212, we conclude that during O formation: (i) Asp-96 undergoes reprotonation. (ii) Asp-85 may undergo a small change in environment but remains protonated. (iii) Asp-212 remains partially protonated. In addition, reisomerization of the chromophore during the N-->O transition is accompanied by a major reversal of protein conformational changes which occurred during the earlier steps in the photocycle. These results are discussed in terms of a proposed mechanism for proton transport.

Effects of amino acid substitutions in the F helix of bacteriorhodopsin. Low temperature ultraviolet/visible difference spectroscopy.

Site-specific mutagenesis in combination with low temperature UV/visible difference spectroscopy has been used to investigate the role of individual amino acids in the structure and function of bacteriorhodopsin (bR). We examined the effects of eight single amino acid substitutions, all in the putative F helix, on the absorption of bR as well as formation of the K and M intermediates. Both the absorbance spectra and the photocycle difference spectra of Escherichia coli expressed bR as well as the mutants S183A, P186G, and E194Q all closely resembled the corresponding purple membrane spectra. In contrast the Pro-186----Leu substitution resulted in the loss of the normal photocycle and a large blue shift in the bR state lambda max. Thus, Pro-186 appears to play a critical role in maintaining the normal protein-chromophore interactions, although the pyrrolidine ring is not essential since proline could be replaced by glycine at this position. The mutants W182F, W189F, and S193A did not appear to be directly involved in the bathochromic shift of bR since they all had lambda max's close to that of purple membrane and produced intermediates similar to K and M. However, alterations in the UV and visible difference spectra as well as the appearance of some irreversibility in the photoreactions indicate that these mutants have altered protein-chromophore interactions during the photocycle. Unlike the other mutants examined, Y185F exhibited a red-shifted form of bR and K raising the possibility that Tyr-185 is directly involved in color regulation. In addition, UV difference peaks previously associated with a tyrosine deprotonation were absent in Y185F indicating that Tyr-185 undergoes protonation changes during the photocycle in agreement with recent Fourier transform infrared difference measurements (Braiman, M.S., Mogi, T., Stern, L. J., Hackett, N., Chao, B. H., Khorana, H.G., and Rothschild, K. J. (1988) Proteins: Structure, Function, and Genetics 3, 219-229). Our results suggest that Trp-182, Tyr-185, Pro-186, Trp-189, and Ser-193, all of which are within a 100 degrees segment of the F helix, are part of a retinal-binding pocket.

Metal ion binding to D-xylose isomerase from Streptomyces violaceoruber.

The binding of two activating cations, Co2+ and Mg2+, and of one inhibitory cation, Ca2+, to D-xylose isomerase from Streptomyces violaceoruber was investigated. Equilibrium-dialysis and spectrometric studies revealed that the enzyme binds 2 mol of Co2+/mol of monomer. Difference absorption spectrometry in the u.v. and visible regions indicated that the environment of the first Co2+ ion is markedly different from that of the second Co2+ ion. The first Co2+ appears to have a six-co-ordinate. The conformational change induced by binding of Co2+ to the first site is maximum after the addition of 1 equivalent of Co2+ and yields a binding constant greater than or equal to 3.3 x 10(6) M-1. Binding of Co2+ to the second, weaker-binding, site caused a visible difference spectrum. The association constant estimated from Co2+ titrations at 585 nm agrees satisfactorily with the value of 4 x 10(4) M-1 obtained from equilibrium dialysis. Similarly, the enzyme undergoes a conformational change on binding of Mg2+ or Ca2+, the binding constants being estimated as 1 x 10(5) M-1 and 5 x 10(5) M-1 respectively. Competition between the activating Mg2+ and Co2+ and the inhibitory Ca2+ ion for both sites was further evidenced by equilibrium dialysis and by spectral displacement studies.

The Mechanism of Anticoccidial Action of Arprinocid-1-N-Oxide

Neither arprinocid nor its liver microsomal metabolite arprinocid-1-N-oxide binds to calf thymus DNA. Neither compound showed an effect on the synthesis of DNA, RNA, or proteins in HeLa cells or on the rate of respiration by Eimeria tenella mitochondria. The 1-N-oxide differs from the parent compound by its toxic effect on HeLa cells (ID50 = 5.0 ppm) reflected primarily through cellular vacuole formation from dilation of rough endoplasmic reticulum structures. Similar vacuole formation was observed in E. tenella merozoites pulse-treated with the drug. This effect on both types of cells is prevented by SKF-525A, an inhibitor of microsomal drug metabolism. Drug-induced visible absorption difference spectra and alterations in the electron paramagnetic signal of rat liver microsomal cytochrome P-450 indicate direct bindings of arprinocid-1-N-oxide to cytochrome P-450. These findings suggest cytochrome P-450 mediated microsomal metabolism involving arprinocid-1-N-oxide as part of the mechanism of antococcidial action of the drug. This metabolism may cause destruction of endoplasmic reticulum leading to cell death.

Non-covalent association of heavy and light chains of human immunoglobulin G: Studies using light chain labelled with a fluorescent probe

The fluorescent probe, 5-iodoacetamidofluorescein (5-IAF), was specifically attached to the COOH-terminal cysteine residue of the L chains of two monoclonal human IgKκ proteins. The induced circular dichroism and fluorescence properties of the bound 5-IAF probe were used to study changes in its microenvironment upon reassociation of autologous or heterologous L chains and their domains with Fd' fragments. Recombination of 5-IAF-L with Fd' at pH 5.4 was accompanied by a red-shifted visible difference spectrum, an increase in the magnitude of the induced optical activity and an enhancement of fluorescence. These results were compatible with the transfer of the 5-IAF moeity to a less polar and more asymmetric environment. Equilibrium and kinetic data obtained from difference spectroscopy and fluorescence studies indicated that 5-IAF-L bound with high affinity to Fd' in a 1:1 ratio with a second-order rate constant of 1618 M −1 sec −1 at 25°C. Using the same approach, it was shown that the labelled constant region fragment (5-IAF-C κ) bound to Fd' with an association constant of 5 × 10 6 M −1 and a forward rate constant of 30 M −1 sec −1. When 5-IAF-C κ was recombined with a preformed Fd'V κ complex, however, the intensity of the visible difference spectrum, the fluorescence emission, the binding affinity and the forward rate constant of the reaction, were significantly increased. In contrast, no spectroscopic changes were observed when V κ was recombined with a preformed Fd'-5-IAF-C κ complex. These data suggest that: (1) the high affinity interaction between Fd' and L results from the summation of relatively weak interactions between paired domains; (2) the binding of Vκ to Fd' modulates the reactivity of Cγl toward Cκ, probably through conformation changes transmitted via V H-Cγl contacts; and (3) conversely, once 5-IAF-Cκ is bound to Fd', the addition of the complementary Vκ domains does not induce any detectable change in the vicinity of the fluorescent probe.

INTERACTION OF PYRIDOXAL AND PYRIDOXAL PHOSPHATE WITH PEPTIDES IN A MODEL FOOD SYSTEM DURING THERMAL PROCESSING

ABSTRACTA soluble model system was utilized to study the interactions of pyridoxal (PL) and pyridoxal phosphate (PLP) with proteins during the thermal processing of foods. Heat stable peptides, rather than intact proteins, were used in the model system to maintain solubility and permit subsequent evaluation of the peptide binding of the B6 vitamers. Ultraviolet and visible difference spectra and fluorescence emission spectra confirmed the binding of PL and PLP to the peptides during processing. Total peptide‐bound PLP (as the sum of Schiff base, pyridoxylamino, and substituted aldamine derivatives) comprised about 21% of the model system PLP. Approximately 60% of the peptide‐bound PLP was bound via nonreducible linkages, suggesting the possible formation of biologically unavailable pyridoxylamino complexes. The acid stability of the PLP complexes, tested to simulate gastric conditions, indicated that most of the nonreducible bound PLP was in the form of pyridoxylamino complexes. Therefore, approximately 10% of the model system PLP was rendered biologically unavailable during these process conditions. The presence of ascorbic acid and/or glucose in the model systems had no significant effect on the extent or manner of peptide‐binding of the B6 vitamers; however, glucose‐induced browning appeared to slightly inhibit pyridoxylamino complex formation. The results of this study indicate that losses of vitamin B6 bio‐availability during thermal processing may be lower than previously reported.

Electronic properties of N-heteroaromatics. XLI. Charge-transfer interactions of the carcinogen 4-nitroquinoline 1-oxide with methylsubstituted benzenes and methylsubstituted aniline derivatives

With the object of examining the π-acceptor property of carcinogenic 4-nitroquinoline 1-oxide, spectrophotometric examination was made on the interaction of the N-oxide compound with a variety of methyl substituted benzenes and anline derivatives. The following results were obtained through analysis of the new absorption bands which appeared in the visible difference spectra of mixed systems of the above derivatives and the N-oxide compound : (1) Absorption maxima of the difference bands shifted toward a longer wavelength region with increasing polarity of the solvents. (2) There was a linear relationship between IP values of the methyl substituted benzenes and aniline derivatives and hv values of the absorption maxima. (3) It was confirmed that complexes of 1 : 1 molar ratio were formed by the interaction. (4) A corresponding relationship was found to exist between K values of the methyl substituted benzene-N-oxide compound complexes and IP values of the substituted benzenes. (5) The K values of the aniline derivative-N-oxide compound complexes were affected markedly by the steric effect of substitutent methyl groups. (6) Analysis of the pH effect indicated that the uonionized species of aniline derivatives were more favorable for the interaction. Based upon these results (1-6), it was presumed that π-π type and n-π type charge transfers were involved in the interaction of 4 -nitroquinoline 1-oxide with methyl substituted benzenes and aniline derivatives. The magnitude of ΔH values was consistent with this presumption. It was also found that the carcinogenic quinoline compound is a stronger π-acceptor than non-carcinogenic 4-nitropyridine 1-oxide.