Dithionite-difference spectra

An obvious difference was also noted between control and induced skate hepaticdnicrosomal AHH activity in the presence of a-naphthoflavone (10 M). This compound, when added in vitro at this or higher concentrations, caused significant stimulation of AHH activity in control animals (about 3-fold) but inhibition (80%) was found in DBA-pretreated skates. Similar results were earlier reported for control and 3-methylcholanthrene-treated rats (23), where it appears that the response is due to differential effects of a-naphthoflavone on hepatic microsomal cytochrome P-450 (stimulated) and cytochrome P-448 (inhibited) (24). Our data suggests that there may be a novel form of cytochrome P-450 synthesized in skate liver in response to polycyclic hydrocarbon administration, even though there was no hypsochromic shift in the carbon monoxide difference spectrum of dithionite reduced hepatic microsomes from DBA-treated skates (relative to hepatic microsomes from control fish). 

Figure 4. Carbon monoxide difference spectrum of partially purified hepatic microsomal Cytochrome P-448 from DBA-treated little skates. The cuvettes contained dithionite-reduced cytochrome (0.10 mg protein/mL) in lOmM phosphate buffer, pH 7.7, containing 20% glycerol, O.lmM EDTA and O.JtnM dithiothreitol.

Fig. 3. EPR spectra of 100 pairs of Rhodnius salivary glands homogenized in 125 xl of phosphate-buffered saline at pH 7.2 (A) before argon equilibration (B) after equilibration in an argon atmosphere for 4 h (C) after equilibration of (B) with NO for 2 min. (D) difference spectrum, that is B — C. (E) homogenate as in (B) treated with dithionite (DT) to reduce Fe(III) to Fe(II), followed by equilibration with NO for 2 min. (The small signal at g = 2 in A-C is due to copper oxide in the liquid helium which had been condensed at the University of Arizona in a copper-plumbed helium liquiflcation apparatus ) All spectra are plotted on the same scale except (E), which is reduced in amplitude by a factor of 3. Reproduced with permission from Ref 24).

Fig. 10. Absorption spectrum of purified tubular membrane of Rb. sphaeroides at 77 K. The inset shows the dithionite-minus-ferricyanide difference spectrum of the same sample in the cytochrome a-band region. The right side shows results of gel-electrophoresis measurements. See text for discussion. Figure and data source Jungas, Ranck, Rigaud, Joliot and Vermdglio (1999) Supramoiecular organization of the photosynthetic apparatus of Rhodobacter sphaeroides. EMBO J 18 536.

Fig. 7. (A) Absorbance difference spectra of PS-II particles measured at 0 ps (a), 200 ps (b) and 1 ns (c) after excitation with 35-ps, 532-nm pulses. (B) the solid-dot absorbance-difference spectrum of the PS-II particles was obtained by subtracting the 1-ns spectrum (c) from the 200-ps spectrum (b) in (A) the empty-circie spectrum was obtained by subtracting the 1 -ns spectrum (c) in (A) from a 200-ps difference spectrum of PS-II particle containing dithionite [not shown], (C) kinetics of absorbance changes at 655 nm in the presence of ferricyanide (solid dots) or dithionite (empty circles). See text for discussion. Figure source Nuijs, van Gorkom, Plijter and Duysens (1986) Primary-charge separation and excitation of chlorophyll a in photosystem II particles from spinach as studied by picosecond absorbance-difference spectroscopy. Biochim BiophysActa 848 170,171.

Fig. 2. (A) Flash-induced AA in the SDS-fractionated PS-1 core complex (CPI) at 5 K [ with and o without DCIP] (B) Flash-induced AA in TSF-I particles containing dithionite and neutral red at pH 10 and frozen while being illuminated (C) left AA induced by 300-ns, dye laser flashes [710 nm for the blue and green region 590 nm for the red region] insets show individual AA transients at 696 and 480 nm (C) right The difference between the difference spectrum in the left panel and that of P700. (D) Plot of the rate constant vs. reciprocal temperature. Figure source (A) Mathis, Sauer and Remy (1978) Rapidly reversible flash-induced electron transfer on a P-700 chlorophyll-protein complex isolated with SDS. FEBS Lett 88 277 (8) Sauer, Mathis, Acker and van Best (1979) Absorption changes of P-700 reversible in milliseconds at low temperature in Triion-solubilized photosystem I particles. Biochim Biophys Acta 545 469 (C and D) Shuvalov, Dolan and Ke (1979) Spectral and kinetic evidence for two eariy electron acceptors in phoiosystem I. Proc Nat Acad Sci, USA 76 771,773.

Fig. 3. (A) Difference spectrum constructed from absorbance change transients in PS-1 particles poised at —625 mV [in the presence of dithionite] in a pH 10 buffer. Each transient was induced by a 3-s iliumination (B) AA obtained directiy with a P700-enriched, PS-i particie in a conventionai, commerciai spectrophotometer in the light-minus-dark mode (C) AA between 350 and 750 nm measured at 200 K with a Triton-fractionated, PS-1 particle from pea chloroplasts the spectrum shown was obtained by first illuminating at 200 K for 45 m and then at 215 K for three 30-m incremental periods. Figure source (A) Swarthoff, Gast, Amesz and Buisman (1982) Photoaccumulation of reduced primary electron acceptors of photosystem I of photosynthesis. FEBS Lett 146 131 (B) Ikegami and Ke (1984) A 160-kilodalton photosystem-l reaction-center complex. Low temperature absorption and EPR spectroscopy of the early electron acceptors. Biochim Biophys Acta 764 75 (C) Mansfield and Evans (1985) Optical difference spectrum of the electron acceptor Ao in photosystem I. FEBS Lett 190 239.

Fig. 7. Difference spectra of PS-1 particles containing ascorbate and PMS (A) and containing dithionite and PMS plus background illumination to maintain all secondary acceptors chemically reduced (B). Spectral changes induced by 35-ps, 710-nm pulses and recorded 5 ns and 860 ps after the pulse are shown in (A, a) and (A, b), respectively. Spectrum recorded 21 ps after the center of the 35-ps pulse is shown in (A, c) [solid trace]. The dashed trace was similarly measured as the solid trace, except the 35-ps, 710 nm pulse was applied 2 ns after the sample was pre-flashed by a 35-ps, 632-nm pulse. Difference spectra of pre-reduced PS-1 particles induced by 35-ps, 710-nm pulses and recorded 370 ps, 5 ns, and 55 ns after the pulse are shown in (B-a, -b and -c), respectively. The dashed spectrum in (B, a) represents the difference between the solid and dashed traces in (A, c). Difference spectrum for [A --AJ obtained by subtracting the difference spectrum in A, a) from the solid spectrum in (B, a) after normalizing at 700 nm. Figure source Shuvalov, Nuijs, van Gorkom, Smit and Duysens (1986) Picosecond absorbance changes upon selective excitation of the primary electron donor P-700 in photosystem /. Biochim Biophys Acta 850 320-322.

Biotransformation and Identification of Metabolites. The study of fish biotransformation reactions in vitro require a number of modifications from those commonly used with mammalian tissues. In conducting studies with cellular components from fish optimization of enzymatic conditions will often require lower incubation temperatures and because of lower specific activities higher enzymatic fortification. Assay procedures such as those using reduced CO versus reduced difference spectrum for cytochrome P450 also require modification. In this case conventional P-450 assays with fish microsomes may experience detection difficulties due to hemoglobin interferences. Modifications in which both reference and sample cuvettes are equilibrated with CO followed by dithionite additions in the sample cuvette can address this issue (52). 

Figure 4. Difference spectrum of P-450 CO measured with the Cary model 14 split-beam recording spectrophotometer. A suspension of P-3 particles containing 0.95 mg. of protein per ml. of 10 mM phosphate buffer (pH 74) was equally divided between two anaerobic optical cuvettes having Na2S20 in the sidearms. Both cuvettes were gassed for 5 min. with Ng, and a base line of equal optical density was established. Experimental cuvette was then gassed with CO for 2 min., dithionite was added to both cuvettes from the sidearms, and the change in optical density was recorded...

Figure 7 Reduced-oxidized difference spectrum of the membrane fraction from Desulfomicrobium sp. str. Ben-RB in 50 mM MBS (pH 6.5 0.54 mg protein/ml). The reference cuvette contained oxidized membrane fraction. The experimental cuvette contained the membrane fraction in an anoxic cuvette under an atmosphere of 100% nitrogen. Cytochromes were reduced with sodium dithionite (0.1 mg/ml). Thereafter, 10 mM arsenate was added to the experimental anaerobic cuvette to oxidize the cytochromes sequential scans were done (each scan 1.1 min). Arrow indicates the direction in which the absorbance is changing. (From Ref. 9.)...

Fig. 2 Light-minus-dark difference spectrum of a sample reduced with 30 mM sodium dithionite frozen in the dark. Inset enlargement of the low-field triplet peak (points) with a simulation (drawn line) using two gaussian lines with midfield, peak-to-peak width and amplitude 304.95 mT, 1.47 mT,...

EPR spectra of the partially purified cytochromes were obtained (Fig. 1.) In addition, the spectra of cyt c549 in both unfractionated thylakoid membranes and in a detergent extract thereof were determined. These are presented as the difference spectrum between samples reduced with anthraquinol-2 sulfonate and with dithionite. All four spectra show peaks characteristic of a low spin heme. The principal -values for the four samples, determined from these spectra, ar shown in Table 2. A weak feature at 1.4 (data not shown) has also been tentatively assigned as the peak of the cytochrome. 

Rgure 16 Attempts to record absorbance spectra in a very turbid ceil suspension. A shows the absoiute spectrum (a scan of a cuvette with the buffer taken as the baseline) of reduced membranes of the bacterium Zymomonas mobilis, acquired with a single-beam instrument (Beckman DU 650). B shows AA acquired with a duai-waveiength instrument (SDB-4) with 500 nm as the reference wavelength. C is the reduced minus as prepared difference spectrum obtained with the same duakwaveiength instrument. The concentration of membrane protein was 10 mg mr sodium dithionite was used as the reductant. 

Fio. 4. Difference spectrum of cytochrome P-4S0 in hepatic microsomes. The dotted line is a base-line difference between two cuvettes containing aliquots of the same microsomal suspension. One cuvette is then bubbled with carbon monoxide for a few minutes, the reducing agent sodium hydrosulfite (dithionite) added to both cuvettes and the difference spectrum (solid line) recorded. (Omura and Sato, J. BioL Chem ... 

Low-temperature absorption spectra (120 K) of dithionite-reduced Rb. sphaeroides RCs in the presence of MV are shown in Fig. 1. Spectrum ll was recorded before illumination and spectrum l2 after 30 min. illumination at room temperature. Spectrum I3 is the computed difference between spectra li and l2. Selective reduction of BPheM during illumination of the RCs at room temperature is ensured by the bleaching of the Qx and Qy absorption bands at 530 nm and 750 nm, respectively (3). Moreover, in the near-infrared region of this difference spectrum, a positive peak at 811 nm and a negative peak at 793 nm are observed. As previously published, the trapped BPheM" state is stable at room temperature in anaerobic conditions over a period of time as long as two days, and relaxes as soon as it is... 

Figure 2 displays the 150 K absorption spectrum of dithionite-reduced Rb sphaeroides RCs in the presence of MV before (2l) and after (22) illumination at 150 K for 30 min. in a nitrogen-cooled cryostat. Spectrum 23 is the computed difference between spectra 2 and 22. This difference spectrum exhibits features characteristic of BPhcL reduction (2) i.e. selective bleachings of the Qx and Qy transitions at 541 nm and 758 nm, accompanied by a downshift affecting one of the major components of the 800-nm transition. Warming the sample to room temperature results in a complete relaxation of the BPheL into neutral BPheL in less than one minute. 

Figure 4a shows an FTIR difference spectrum for dithionite-reduced reaction centers arising from the transition PIQa" PI QA (the difference spectrum represents I" - I). The carbonyl region is dominated by four negative absorptions at 1747, 1732, 1683, and 1655 cm" as well as a broad positive absorption at 1714 cm . Other peaks characteristic for the I" difference spectrum are an intense positive absorption at 1593 cm in addition to smaller positive absorptions at 1552, 1467, and 1371 cm . An FTIR difference spectrum for I" was first recorded by Nabedryk et al. [25] and band assignments could be postulated with the aid of electrochemically-generated BPha spectra [10]. 

FTIR light-induced difference spectra for Q -containing reaction centers, (a) Difference spectrum obtained for dithionite-reduced reaction centers the difference is taken between PIQa" PI"Qa . (b) Difference spectrum obtained for ascorbate-reduced reaction centers the difference is taken between PIQa and PI-Q ", (c) Difference spectrum obtained for ascorbate-reduced reaction centers illuminated for 5 minutes prior to recording the "light" spectrum the difference is taken between PIQa and PI Qa " (T =... 

Fig. 6 UV-vis spectra of LbL films of C54 P450 enz5unes on aminosilane-functionalized fused silica slides a high spin ferric form of purified human cyt P450 1A2 assembled with polyions b CO difference spectrum of human C54 P450 1A2 assembled with polyions after reducing to the ferrous form by sodium dithionite and purging pH 7.0 buffer with CO. c Low spin ferric form of purified bacterial cyt P450cam assembled with pol5dons. Reprinted with permission from the American Chemical Society, Ref. [47], Cop5urght 2009...

FIGURE 6 (right). Solid line absorption difference spectrum measured 10 ms after a flash, in the presence of 25 yM N-methylphenazonium methosulfate (PMS) and 5 mM ascorbate. Ag = 4.5 x 10 . Dashed line in vitro absorption difference spectrum of vitamin Kj (Q - Q) in methanol (E.J. Land, personal communication), normalized at 395 nm. Insert 100 yM PMS, 10 mM dithionite,... 

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