Determination of p-chloronitrobenzene in plasma by reversed-phase high-performance liquid chromatography

A simple, accurate and precise isocratic reversed-phase high-performance liquid chromatographic method was developed and validated for the determination of p-chloronitrobenzene (p-CNB) in rat plasma. A plasma sample was deproteinized with methanol containing the internal standard (p-bromonitrobenzene). The resulting methanol eluate obtained after centrifugation was filtered and injected into a high-performance liquid chromatograph (50 μl each). A column packed with 5 μm octadecylsilane (ODS) spherical particles was used with isocratic elution of methanol—water (45:55, v/v) at a flow-rate of 1.0 ml/min. The compounds were detected by ultraviolet absorbance at 280 nm. The retention times of p-CNB and the internal standard were 12.5 and 15.5 min, respectively, at a column oven temperature of 30°C. The results were linear from 0.05 to 100 μg/ml (r = 0.999), and the detection limit was 0.01 μg/ml. The relative error and the coefficient of variation on replicate assays were less than 7 and 10%, respectively, for all concentrations studied. The overall recoveries of p-CNB were between 97 and 105%. Plasma samples could be stored for up to one month at −20°C.     

Homogeneous amperometric immunoassay for theophylline in whole blood

A homogeneous spectrophotometric EMIT immunoassay kit for the quantitation of theophylline in serum or plasma has been modified to produce a rapid, amperometric immunoassay requiring a 50 μl whole blood sample. The basis of the detection system for the assay is the electrochemical oxidation of NADH produced by G6PDH-labelled theophylline at a potential of + 150 mV vs Ag/AgCl using platinised activated carbon (PACE) electrodes. Comparison of the amperometric whole blood method with the conventional spectrophotometric plasma assay produced a reasonable correlation: Y = 0·90x − 1·01, (r = 0·98, N = 12). The advantage of the new method is that simple and robust instrumentation can rapidly determine theophylline in whole blood with no sample pre-treatment or separation steps.     

Freezing resistance improvement of Lactobacillus reuteri by using cell immobilization

Lactobacillus reuteri shows certain beneficial effects to human health and is recognized as a probiotic. However, its application in frozen foods is still not popular because of its low survival during freezing and frozen storage. Cell immobilization technique could effectively exert protection effects to microbial cells in order to enhance their endurance to unfavorable environmental conditions as well as to improve their viability and cell concentration. Ca-alginate and κ-carrageenan were used to immobilize L. reuteri in this research, and the immobilized cells were exposed to different freezing temperatures, i.e. − 20 °C, − 40 °C, − 60 °C, − 80 °C, and stored at − 40 °C and − 80 °C for 12 weeks. The objectives were to study the protection effects of cell immobilization against the adverse conditions of freezing and frozen storage, and the effects of freezing temperatures to the immobilized cells. Cell immobilization was used to raise the survival of L. reuteri during freezing and frozen storage in order to develop frozen foods with the probiotic effects of L. reuteri. Results indicated that immobilized L. reuteri possessed better survival in both freezing and frozen storage. The survival of immobilized L. reuteri was higher than that of free cells, and the effects of lower freezing temperature were better than higher freezing temperature. The immobilization effects of Ca-alginate were found to be superior to κ-carrageenan. Cell immobilized L. reuteri exhibits potential to be used in frozen foods.     

Temperature dependence of delayed light emission in spinach chloroplasts

1. Delayed light of chlorophyll emitted at 0.1–3.9 ms after cessation of repetitive flash light was studied at temperatures between +40 and −196 °C in isolated spinach chloroplasts.

2. Induction kinetics of delayed light varied depending on temperature. It was found to be composed of two phases; one was an initial rapid rise followed by a rather fast decline to a low steady state level (fast phase), and the other was a slow increase after the initial rapid rise to the maximum followed by an insignificant slow decrease to a high steady state level (slow phase). The fast phase existed between −175 and 40 °C with the maximum at −40 °C, while the slow phase, between 0 and 40 °C with the maximum at 25 °C.

3. The intensity of delayed light at −175 °C was found to be less than one fiftieth that at 0 °C, and no delayed light emission was observed at −196 °C within experimental accuracy. This is in contrast to the results reported by Tollin, G., Fujimori, E. and Calvin, M. ((1958) Proc. Natl. Acad. Sci. U.S. 44, 1035–1047) in which the intensity of delayed light measured at −170 °C was about a half that at 0 °C.

4. The induction of delayed light measured at −96 °C was found to be significantly suppressed by the preillumination at −196 °C. This finding suggests that the primary photochemical event still survives at −196 °C without emission of delayed light.

5. Decay kinetics of delayed light after the flash excitation revealed the presence of at least two decay components. A slow decay component with a half decay time of several tens of milliseconds was observed at temperatures higher than 0 °C. A fast decay component with a half decay time of about 0.2 ms was observed at temperatures between −120 and 25 °C. The decay rate of this component was slightly retarded on cooling.

6. The System II particles derived from spinach chloroplasts with digitonin treatment showed a temperature dependence of delayed light similar to that of the chloroplasts. System I particles, on the other hand, scarcely emitted the delayed light at any temperature between 40 and −196 °C.     

Development of a high-performance liquid chromatographic–electrospray mass spectrometric assay for the specific and sensitive quantification of the novel immunosuppressive macrolide 40-O-(2-hydroxyethyl)rapamycin

It was our objective to develop a rapid, sensitive and specific assay to quantify the immunosuppressive macrolide 40-O-(2-hydroxyethyl)rapamycin (SDZ-RAD) in blood of transplant patients. SDZ-RAD was extracted from blood by solid–liquid extraction. SDZ-RAD and its internal standard 28,40-diacetyl rapamycin were quantified using HPLC–electrospray MS. The assay was linear from 0.1 to 100 μg/l (r²=0.99). The mean recovery was 83% for SDZ-RAD and 80.5% for the internal standard. The mean day-to-day precision was 8.0%. Extracted samples were stable at 20°C for at least 48 h and SDZ-RAD blood samples at −80°C for at least six months.     

An automated differential thermal and potentiometric titration apparatus for binding studies

A differential pH-termal titration apparatus is described which can detect pH differences with a sensitivity of ±0.0001 pH units and a thermal sensitivity of ±0.00002°C at a time constant of 0.1 s. With a reaction which yields 1 kcal mol⁻¹, the current system can detect concentrations as low as 4×10⁻⁶ M or, in a 2 ml volume, a total amount of 40 nmol. With a time constant of 0.1 s, the sensitivity is 20±4 μ°C. The experimental protocol is specified by a microprocessor and three modes of operation are possible: titration at constant rate of reagent addition, titration at variable rates of addition so that the contents of both cells are at either constant pH or at a constant temperature and variable rate when a rate of change is specified. Experimental data are collected in files, corrected for heat loss, initial baseline drift, and changes in volume. The final corrected from the standardized run of 0.01338 M HCl in 0.2 M KCl at 25°C calibrate the pH scale yielded the calorimetric conversion constants and pK_w which are calculated and stored for subsequent corrections for the titration of an unknown acid or the measurement of bindin constants and heats.     

Effect of dietary versus pharmacological correction of hypertriglyceridemia on red blood cell membrane sodium/lithium countertransport activity

An elevated red blood cell Na/Li countertransport (Na/Li CT) is often associated with high blood pressure and metabolic abnormalities. Recent studies suggested that a reduction in serum TG levels is associated with a decrease in Na/Li CT activity. However, it is still unclear if this phenomenon could be originated from systemic metabolic alterations or from modifications of the membrane dynamic properties. Aim of the present study was to investigate whether dietary or pharmacological TG lowering therapy might have a different effect on Na/Li CT activity and related metabolic parameters. Twenty normotensive hyper-TG patients were recruited from the Lipid outpatient Clinic: they had a baseline Na/Li CT activity significantly higher compared with age- and BMI-matched normolipidemic controls (386 ± 33 vs 274 ± 39 umol/l RBC/h, p < 0.05). The patients were randomly prescribed one of the following two-months treatment: Group 1)-triglyceride lowering diet; Group 2)-lipid lowering drug (Gemfibrozil 600 mg b.i.d.). Na/Li CT and metabolic and anthropometric variables were measured at baseline and after 1 and 2 months of treatment. At the end of intervention, there was in both groups a significant and comparable fall in plasma triglyceride (group 1: −2.61 ± 0.73 mmol/l p < 0.01; group 2: −4.29 ± 1.20 mmol/l p < 0.01). In the diet-treated group there were, in addition small but significant reductions in body weight (−3.7 ± 0.8 kg p < 0.01), fasting glucose (−0.36 ± 0.14 mmol/l p < 0.05) and insulin levels (−2. ± 0.5 mUl/l, p < 0.01), while no such changes were observed in the fibrate treated patients. Na/Li CT activity was significantly and comparably reduced at the end of treatment in both groups (group 1: −97 ± 28 umol/l cell/h, p < 0.01; group 2: −89 ± 30 umol/l cell/h, p < 0.01). In conclusion, these results indicate that the decrease in Na/Li CT associated with both dietary and drug treatment of hypertriglyceridemia is to be traced to a direct effect of plasma TG concentration on this transport system (probably as a result of modification in the membrane lipid environment) rather than to changes in plasma insulin levels or insulin resistance.     

A sexually dimorphic response in supercooling temperature, enhanced by starvation, in the lesser mealworm Alphitobius diaperinus (Coleoptera: Tenebrionidae)

The effect of starvation on supercooling temperature (SCP) distribution was investigated in adults and larvae of Alphitobius diaperinus (Coleoptera: Tenebrionidae).

The mean values for SCPs of adults fed at 20°C were −14.5±2.4°C (31 males) and −10.3±1.3°C (29 females). The distribution of the SCPs of these control adults was unimodal. No significant differences were observed in either mean wet weight or mean dry weight between males and females.

The mean values for SCPs of adults starved for 1 month at 20°C were found to be bimodal due to sexual dimorphism. The mean SCPs for males was lower (−17±2.6°C; 28) than that for females (−11.2±1.8°C; 26). No significant differences were observed in either mean dry weight or wet weight between males and females.

The SCPs of both fed and starved larvae, kept for 1 month at 20°C were −12.3±2.4°C (fed) and −18.0±2.6°C (starved).     

Analysis of Ecteinascidin 743, a new potent marine-derived anticancer drug, in human plasma by high-performance liquid chromatography in combination with solid-phase extraction

A reversed-phase high-performance liquid chromatographic method has been developed and validated for the quantification of the novel anticancer drug Ecteinascidin 743 in human plasma. The sample pretreatment of the plasma samples involved a solid-phase extraction (SPE) on cyano columns. Propyl-p-hydroxybenzoate was added after the sample pretreatment to correct for variability in injection volumes. The separation was performed on a Zorbax SB-C₁₈ column (75×4.6 mm I.D., particle size 3.5 μm) with acetonitrile–25 mM phosphate buffer, pH 5.0 (70:30, v/v) as the mobile phase. The flow-rate was 1.0 ml/min and the eluent was monitored at 210 nm. The accuracies and precisions of the assay fall within ±15% for all quality control samples and within ±20% for the lower limit of quantitation, which was 1.0 ng/ml using 500 μl of plasma. The overall recovery of the sample pretreatment procedure for Ecteinascidin 743 was 87.0±5.9%. The drug was found to be stable in human plasma at −30°C for at least 2 months. At room temperature Ecteinascidin 743 was stable in human plasma for 5 h at most.     

Determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in human and animal plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A highly sensitive HPLC method with automated column switching was developed for the simultaneous determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in plasma samples from man, Cynomolgus monkey, rabbit, rat and mouse. Plasma (0.4 ml) was deproteinated by adding ethanol (1.5 ml) containing the internal standard acitretin. After centrifugation, 1.4 ml of the supernatant were directly injected onto the precolumn packed with LiChrospher 100 RP-18 (5 μm). 1.25% ammonium acetate and acetic acid-ethanol (8:2, v/v) was used as mobile phase during injection and 1% ammonium acetate and 2% acetic acid-ethanol (102:4, v/v) was added, on-line, to decrease the elution strength of the injection solution. After backflush purging of the precolumn, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm. Two coupled Superspher 100 RP-18 endcapped columns (both 250×4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid: (A) 600:300:60:10 (v/v/v/v), (B) 950:20:5:20 (v/v/v/v), and (C) 990:5:0:5 (v/v/v/v). The method was linear in the range 0.3–100 ng/ml, at least, with a quantification limit of 0.3 ng/ml. The mean recoveries from human plasma were 93.2%–94.4% and the mean inter-assay precision was 2.8%–3.2% (range 0.3–100 ng/ml). Similar results were obtained for animal plasma. The analytes were found to be stable in the plasma of all investigated species stored at −20°C for 4.3 months and at −80°C for 9 months, at least. At this temperature, human plasma samples were even stable for 2 years. The method was successfully applied to more than 6000 human and 1000 animal plasma samples from clinical and toxicokinetic studies. Endogenous levels determined in control patients and pregnant women were similar to published data from volunteers.     

The effect of partial dehydration on the developmental capacity of mouse embryos stored in the supercooled state

The effect of partial dehydration on the ability of mouse blastocysts to withstand storage at subzero temperatures without freezing was studied. The embryos were equilibrated with a supercooling medium developed at the Centre for Food and Animal Research, containing 3% (Medium A) or 6% (Medium B) methanol and propanediol, and then with the same medium, A or B, containing 0–0.5 mol sucrose. The embryos were placed in 0.25 ml straws, cooled to −5°C or −10°C and stored for up to 3 days. After storage, the embryos were cultured for 48 h in M16 and their ability to develop into expanded blastocysts was used to gauge their survival in supercooled storage.

The maximal beneficial effect of partial dehydration occurred in media supplemented with 0.3–0.5 mol sucrose: the proportions of dehydrated embryos surviving 24 h storage at −5°C and −10°C were 84–85% and 91–100%, respectively, compared with only 58% and 52% of non-dehydrated, supercooled embryos. The corresponding figures for dehydrated embryos after 48 or 72 h storage at −5°C were 86–92% and 38–58% compared with 13% and 4% of non-dehydrated embryos. Similarly, 75–85% and 47–55% of partially dehydrated embryos survived storage for 48 h or 72 h, respectively, at −10°C, compared with 5% and 0% of non-hydrated embryos. Thus, reducing the water content of early mouse blastocysts improved their ability to withstand subzero storage.     

Methane production from organic acid-rich wastewaters by immobilized thermophilic methane-producing bacteria

Thermophilic methane-producing bacteria isolated from a wastewater treatment facility have been immobilized in acetylcellulose filter with agar. The immobilized cells produced methane from wastewaters in rich organic acid (acetic, propionic and butyric acids) at the rate of 1.4 μmol mg protein⁻¹ h⁻¹. The optimum conditions for methane production by immobilized whole cells were 52–55°C and pH 7.0–8.0. The immobilized cells retained 80% of the initial activity after exposure to air. The immobilized thermophilic bacteria produced methane continuously over 10 days at 52°C.     

Finger temperatures during military field training at 0 to −29 °C

1. Skin and rectal temperatures were recorded continuously in 70 measurements during typical tasks of infantry and artillery training at 0 to −29 °C. The duration of the measurements varied from 55 min to 9.5 h.

2. The distribution of finger skin temperatures was quite similar at ambient temperature ranges 0 to −10 °C and −10 to −20 °C, while at −20 to −30 °C the finger temperatures were clearly lower.

3. At different ambient temperature ranges, 20–69% of finger temperatures were low enough to cause cold thermal sensations.

4. Sensation of cold was experienced at a finger temperature of 11.6±3.7 °C (mean±SD).     

Characteristics of thermoluminescence bands of intact leaves and isolated chloroplasts in relation to the watersplitting activity in photosynthesis

Plant materials (intact leaves, chloroplasts or subchloroplast particles) preilluminated at a low temperature (e.g. −60°C) were rapidly cooled to −196°C and then the luminescence emitted from the sample on raising the temperature was measured as a function of temperature, by means of a sensitive photo-electron counting technique. Mature spinach leaves showed five luminescence bands at different temperatures which were denoted as Z_v, A, B₁, B₂ and C bands. The A, B₁, B₂ and C bands appeared at constant temperatures, −10, +25, +40 and +55°C, respectively, being independent of the illumination temperature, but the Z_v band appeared at a variable temperature slightly higher than the illumination temperature. The B₁ and B₂ bands were absent in the thermoluminescence profiles of samples devoid of the oxygenevolving activity, such as heat-treated spinach leaves, wheat leaves greened under intermittent illumination and photosystem-II particles prepared with Triton X-100. It was deduced that these luminescence bands arise from the energy stored by the electron flow in photosystem II to evolve oxygen, and other bands were ascribed to charge-separation in some other sites not related to the oxygen evolving system.     

Investigation of the transport of intact glutathione in human and rat type II pneumocytes

The aim of the study was to investigate whether there is transmembrane transport of intact glutathione ([³H]-GSH, 0.1 μCi) in rat and human type II pneumocytes (T2P), and if this transport might be dependent on the redox state of the extracellular fluid. The T2P were pretreated with acivicin (250 μM) to inhibit γ-glutamyl-transferase activity and with L-buthionine-[SR]-sulfoximine (1 mM) to inhibit intracellular GSH synthesis. After 48 h in culture, initial GSH influx rate was 0.70 ± 0.20 nmol/min/mg protein (37°C) and 0.35 ± 0.04 nmol/min/mg protein (4°C) during the first 5 min in rat T2P. In human T2P, the initial GSH influx rate was 0.36 ± 0.30 nmol/min/mg protein (37°C) and 0.32 ± 0.06 nmol/min/mg protein (4°C) during the first 10 min. Thereafter no further influx was found. The influx of 1 mM GSH in freshly isolated rat and human T2P in suspension was 2.3 ± 0.3 and 1.2 ± 0.3 nmol/mg protein after 15 min at 37°C, and 2.8 ± 0.2 and 1.0 ± 0.3 nmol/mg protein at 4°C, respectively. When GSH influx was studied at different concentrations between 0 and 40 mM, a linear increase without saturation or difference between 37°C and 4°C was found. Preexposure to ouabain had no effect on GSH influx. Efflux of GSH was stimulated and influx inhibited by preexposure of the cells to reduced thiols, while disulphides inhibited efflux and favoured inward uptake. Thus, in human and rat T2P a GSH-carrier exists which operates as an effluxer. At GSH concentrations in the physiological range no uptake is seen, but some uptake can be observed at GSH concentrations above normal physiological levels. The uptake appears to be energy-independent and non-saturable. Efflux of GSH is stimulated and influx inhibited by reduced thiols, while disulphides inhibit the efflux and favour inward uptake. GSH uptake in T2P thus may depend on concentration gradients and driving forces, such as the redox state of the extracellular fluid.     

The effect of acclimation on the acid-base status of pigeons exposed to high ambient temperatures

Arterial pH, PCO₂ (P_aCO₂), plasma bicarbonate [HCO₃⁻ and respiratory frequency were measured in pigeons exposed to ambient temperatures (T_aS) of 30–60°C. Acclimated, nonpanting birds regulated acid-base balance at normal levels, when exposed to T_as) between 30 and 53°C T_a. At higher T_as (55–60°C), both nonpanting and panting acclimated pigeons regulated pH at normal levels, 7.544 ± 0.011 (SD) and 7.531 ± 0.022 (SD), respectively, accompanied by a slight hypocapnia, 24.8 ± 4.0 Torr and 23.8 ± 2.49 Torr (P_aCO₂), respectively. Nonacclimated birds, exposed to 50°C T_a, endured a severe hypocapnia (P_aCO₂ of 9.1 ± 2.52 Torr) and alkalosis (pH of 7.702 ± 0.048). Thirteen exposures to > 50°C T_a, 4–6 h a day, resulted in a significant improvement in the capacity of the panting pigeon to maintain an almost normal acid-base balance, i.e. actual and standard [HCO₃⁻ of 22.6 ± 1.22 and 25.7 ± 1.10 mM/l, respectively, and only a slight hypocapnia (P_aCO₂ of 23.6 ± 3.9 Torr) and alkalosis (pH of 7.589). The suggestion that acclimation to high T_as (50–60°C) is needed for fine adjustment between the competing needs for heat dissipation, pulmonary gas exchange, and acid-base regulation in the heat-exposed pigeon is discussed.     

Continuous sucrose hydrolysis by an immobilized whole yeast cell biocatalyst

An immobilized biocatalyst with invertase activity prepared by immobilization of whole yeast cells without use of any insoluble carrier was tested in tubular fixed-bed reactors from the point of view of possible application for continuous full-scale sucrose hydrolysis. At inlet sucrose concentration above 60% (w/w) and reaction temperature 60–70°C, total sucrose hydrolysis was achieved at a flow rate of 0.6–1.5 bed volumes per hour. At a flow rate about 10 bed volumes per hour, the conversion was still 0.5. The specific productivity of the biocatalyst was 3–25 h⁻¹; the productivity of the reactor was 1–9 kg l⁻¹ h⁻¹. The half-life of the biocatalyst invertase activity was 815 h at 70°C. The specific pressure drop over the biocatalyst bed was less than 23 kPa m⁻¹. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors.     

Determination of the maximum and minimum lethal temperatures (LT50) for Loxosceles intermedia Mello-Leitão, 1934 and L. laeta (Nicolet, 1849) (Araneae, Sicariidae)

In this study, the maximum and minimum lethal temperatures (LT₅₀) of L. intermedia and L. laeta were determined in two treatments: gradual heating (25–50°C) and cooling (25°C to −5°C), and 1 h at a constant temperature. In gradual temperatures change, L. intermedia mortality started at 40°C and the LT₅₀ was 42°C; for L. laeta, mortality began at 35°C and the LT₅₀ was 40°C. At low temperatures, mortality was registered only at −5°C for both species. In the constant temperature L. intermedia showed a maximum LT₅₀ at 35°C and L. laeta at 32°C; the minimum LT for both species was −7°C.     

The role of blood platelets in nucleoside metabolism: assay, cellular location and significance of thymidine phosphorylase in human blood

The enzyme thymidine phosphorylase (thymidine: orthophosphate deoxyribosyltransferase, EC 2.4.2.4.), which plays a crucial role in nucleic acid metabolism in both prokaryotic and eukaryotic cells by regulating the availability of thymidine, is present in mammalian blood. Here we describe a simple, rapid HPLC-based micromethod for the assay of blood thymidine phosphorylase. We have arbitarily defined 1 unit of blood thymidine phosphorylase activity as the activity required to produce a 1-nM increment in the plasma concentration of thymine after incubation for 1 h at 37°C with a saturating concentration of exogenous thymidine.

In normal adults, whole (peripheral venous) blood thymidine phosphorylase activity with blood cells intact was 64 ± 11 units (mean ± S.D., n =20, range 45–89). The apparent Michaelis constant for thymidine was of the order to 10⁻⁴ M but varied nearly 5-fold between different individuals. Activity increased when blood cells were permeabilised or lysed with non-ionic detergents, implying that thymidine phosphorylase is an intracellular enzyme which may be influenced by exogenous as well as intracellular factors. When blood from normal donors was fractionated, thymidine phosphorylase activity consistently co-isolated with platelets. Whole-blood thymidine phosphorylase activity correlated well with platelet parameters. Although thymidine phosphorylase activity was also detected in plasma and serum, the small size and notorious fragility of platelets suggest its platelet origin.

Blood from leukaemic donors showed significantly increased thymidine phosphorylase activity compared to normal controls (mean activity ± S.D. was 96 ± 27 units; range 58–140, n = 8).

Thymine formation from thymidine was temperature- and pH-depdendent in whole blood. 2′-Deoxyuridine and 3 of its 5-halogenated analogues (but not 3′-azido-3′-deoxythymidine (AZT), were catabolised by blood thymidine phosphorylase, even during blood clotting at room temperature. Assumptions about in vivo concentrations of these compounds should therefore be interpreted cautiously.

In the presence of high concentrations of thymine and suitable deoxyribose donors, small amounts of thymidine were formed in some blood samples, so it is conceivable that thymidine catabolism may be reversible in vivo under some circumstances.     

Light-induced absorbance changes in chloroplasts mediated by Photosystem I and Photosystem II at low temperature

Stable light-induced absorbance changes in chloroplasts at −196 °C were measured across the visible spectrum from 370 to 730 nm in an effort to find previously undiscovered absorbance changes that could be related to the primary photochemical activity of Photosystem I or Photosystem II. A Photosystem I mediated absorbance increase of a band at 690 nm and a Photosystem II mediated absorbance increase of a band at 683 nm were found. The 690-nm change accompanied the oxidation of P₇₀₀ and the 683-nm increase accompanied the reduction of C-550. No Soret band was detected for P₇₀₀.

A specific effort was made to measure the difference spectrum for the photooxidation of P₆₈₀ under conditions (chloroplasts frozen to −196 °C in the presence of ferricyanide) where a stable, Photosystem II mediated EPR signal, attributed to P₆₈₀⁺ has been reported. The difference spectra, however, did not show that P₆₈₀⁺ was stable at −196 °C under any conditions tested. Absorbance measurements induced by saturating flashes at −196 °C (in the presence or absence of ferricyanide) indicated that all of the P₆₈₀⁺ formed by the flash was reduced in the dark either by a secondary electron donor or by a backreaction with the primary electron acceptor. We conclude that P₆₈₀⁺ is not stable in the dark at −196 °C: if the normal secondary donor at −196 °C is oxidized by ferricyanide prior to freezing, P₆₈₀⁺ will oxidize other substances.