The role of oxygen availability in the embryonation of Heterakis gallinarum eggs.

The importance of oxygen availability in the embryonation of the infective egg stages of the gastrointestinal nematode parasite Heterakis gallinarum was studied in the laboratory. Unembryonated H. gallinarum eggs were kept under either aerobic conditions by gassing with oxygen, or anaerobic conditions by gassing with the inert gas nitrogen, under a range of constant temperatures. Oxygenated eggs embryonated at a rate influenced by temperature. Conversely, eggs treated with nitrogen showed no embryonation although when these eggs were transferred from nitrogen to oxygen gas after 60 days of treatment, embryonation occurred. This demonstrated that oxygen is an essential requirement for H. gallinarum egg development, although undeveloped eggs remain viable, even after 60 days in low oxygen conditions. The effects of climate on the biology of free-living stages studied under constant laboratory conditions cannot be applied directly to the field where climatic factors exhibit daily cycles. The effect of fluctuating temperature on development was investigated by including an additional temperature group in which H. gallinarum eggs were kept under daily temperature cycles between 12 and 22°C. Cycles caused eggs to develop significantly earlier than those in the constant mean cycle temperature, 17°C, but significantly slower than those in constant 22°C suggesting that daily temperature cycles had an accelerating effect on H. gallinarum egg embryonation but did not accelerate to the higher temperature. These results suggest that daily fluctuations in temperature influence development of the free-living stages and so development cannot be accurately predicted on the basis of constant temperature culture.     

Laboratory culture of the Nudibranch Tritonia diomedea bergh (Tritoniidae: Opisthobranchia) and some aspects of its behavioral development

Tritonia diomedea Bergh was reared from oviposition, through metamorphosis to reproductive maturity in the laboratory. The larvae of T. diomedea are planktotrophic and undergo considerable shell growth (from 144.6–329 μ average maximum shell length). Metamorphosis does not require induction, but there may be a preference to metamorphose in the presence of the probable adult prey, a small Virgularia sp. Larvae in cultures fed no food, Dunaliella tertiolecta Butcher Isochrysis galbana Parke, or Monochrysis lutheri Droop did not achieve metamorphic competence at near ambient sea-water temperatures (11.9±1.3 and 13.0±0.8°C). Larvae from cultures fed Monochrysis at room temperature (20.8±1.5°C) or fed a 1 : 1 mixture of Isochrysis and Monochrysis at near ambient sea-water temperatures did metamorphose. Even so, only those larvae fed the 1 : 1 mixture survived more than a few days following metamorphosis. Adult behavioral patterns developed gradually, feeding being first observed at 5 days, swimming in response to NaCl crystals at about 60 days, copulation at about 272 days, and oviposition at about 277 days after metamorphosis. Growth rates were determined for field collected Tritonia diomedea; smaller animals gained and lost weight relatively faster than larger animals.     

Synchronous division and the pattern of diel vertical migration of Heterosigma akashiwo (Hada) Hada (Raphidophyceae) in a laboratory culture tank

Heterosigma akashiwo (Hada) Hada (Raphidophyceae) causes red tides in Osaka Bay (Japan). A clonal culture of the alga was grown in a 2 m tall culture tank on a 12: 12 LD cycle to determine patterns of vertical migration and cell division. A specific growth rate of 0.43 ln unit · day⁻¹ was obtained during complete mixing conditions. Under weakly stratified conditions (≈ΔT = 3–4°C/1.5 m), H. akashiwo in the tank grew and showed a similar pattern of vertical migration to that observed in the field for at least 6 days. Cell concentration, mean cell volume, and photosynthetic capacity, estimated by DCMU-induced fluorescence increase of H. akashiwo, were monitored in the stratified tank at 2-h intervals over 24 h at three levels in the water column. Cell ascent began shortly before the light period and vertically swimming cells were smaller in size than those sampled near the bottom of the tank. The cell division cycle and the pattern of vertical migration were phased individually by the light regime and were well synchronized with each other. This synchrony must be due to the interrelation between these two processes or the existence of a clock which controlled endogenous rhythms of both processes and was entrained by a light: dark cycle. The relative increase of fluorescence with DCMU was higher for migrating cells than for non-migrating cells.     

The Influence of Host Species on Some Biological and Behavioural Aspects of Dibrachys boarmiae (Hymenoptera: Pteromalidae), Parasitoid of Kermania pistaciella (Lepidoptera: Tineidae)

Dibrachys boarmiae was recently found as a parasitoid of the pistachio twig borer moth, Kermania pistaciella, an important pistachio pest in Iran. Several biological and behavioural aspects of the wasp were studied under controlled conditions using three different lepidopterous host species, K. pistaciella, Sitotroga cerealella and Galleria mellonella. Development time from egg to adult decreases from 54 days at 15°C to 12.6 days at 32.5°C. The thermal constant was estimated 270°D, and the lower development threshold was at 9.9°C. Longevity of females that had the opportunity to oviposit and access to food (honey) was on average 23 days, and 134 offspring were produced during 13 days of oviposition. Ninety percent of ovipositions occur during the first 10 days of the parasitoid's life, although no pre-oviposition period was found. If females were deprived of food, longevity as well as fecundity, were drastically reduced to 8 days and only 79 offspring respectively. Females were able to mate immediately after emergence and the sex ratio of the progeny was strongly female biased on all examined hosts, especially on the largest host G. mellonella (0.08 male). When females were kept with a male throughout their life, the total number of progeny decreased to 92 wasps on average but the sex ratio was unaffected. The parasitoid significantly preferred to attack the pre-pupal stage, and this influenced the size of clutches allocated to different host stages. The parasitoid laid larger clutches on bigger hosts.     

Nitrogen and energy loss in the marine teleost Lithognathus mormyrus (Linnaeus)

Nitrogen excretion and assimilation efficiencies in Lithognathus mormyrus (Linnaeus), a marine teleost present in high-energy surf zones in Algoa Bay, South Africa, were determined under controlled laboratory conditions at 15, 20 and 25 °C. Ammonia was the major form of nonfaecal nitrogen excreted by starved and fed L. mormyrus. Urea and amino acids were secondary excretory products. Ammonia excretion rates were temperature independent and the excretion rates of fed fish were significantly higher than starved fish at 20 and 25 °C but not at 15 °C. The mass component (b) of the mass/ammonia excretion equation was temperature independent and ranged from 0.590 to 0.669 and from 0.670 to 0.767 for starved and fed fish, respectively. The mean percentage of food energy lost via the dissolved nonfaecal excretory products (exogenous plus endogenous) was 4.12%. Assimilation efficiencies ranged from 71.89 to 98.78% for dry matter and from 96.89 to 99.88% on an energy basis. The combined nonfaecal and faecal energy loss was calculated at 10.11% of the ingested energy. The omnivorous ichthyofauna present in the surf zone ecosystem recycle 33 g N · m strip · yr⁻¹. This constitutes < 1 % of total phytoplankton nitrogen requirements.     

The respiratory chain of Azotobacter vinelandii II. The effect of cyanide on cytochrome d

1. Cyanide causes a slow disappearance of the oxidized band (648 nm) of cytochrome d in particles of Azotobacter vinelandii and inhibits the appearance of the reduced band (631 nm). No effect of cyanide is found on the reduced band of cytochrome d.

2. The kinetics of the disappearance of the 648-nm band of cytochrome d with excess cyanide deviates from first-order kinetics at lower temperatures (22 °C) indicating that at least two conformations of the enzyme are involved. At higher temperatures (32 °C) the observed kinetics of the cyanide reaction are first order with a k_on = 0.7 M⁻¹·s⁻¹ and with an estimated k_off of approximately 5·10⁻⁵ s⁻¹.

3. The value of the k_off (7·10⁻⁴−14·10⁻⁴ s⁻¹ at 32 °C) determined from the rate of reduction of cyanocytochrome d by Na₂S₂O₄ or NADH is one order of magnitude larger than the k_off value found when the enzyme is in its oxidized state.

4. No effect of cyanide is found on the spectrum of cytochrome a₁.     

Purification and kinetics of two novel thermophilic extracellular proteases from Lactobacillus helveticus, from kefir with possible biotechnological interest

Two thermophilic extracellular proteases, designated Lmm-protease-Lh (29 kDa) and Hmm-protease-Lh (62 kDa), were purified from the Lactobacillus helveticus from kefir, and found active in media containing dithiothreitol; the activity of Lmm-protease-Lh was increased significantly in media containing also EDTAK₂. Both novel proteases maintained full activity at 60 °C after 1-h incubation at 10 °C as well as at 80 °C, showing optimum k_cat/K_m values at pH 7.00 and 60 °C. Only irreversible inhibitors specific for cysteine proteinases strongly inhibited the activity of both novel enzymes, while they remained unaffected by irreversible inhibitors specific for serine proteinases. Both enzymes hydrolyzed the substrate Suc-FR-pNA via Michaelis–Menten kinetics; conversely, the substrate Cbz-FR-pNA was hydrolyzed by Lmm-protease-Lh via Michaelis–Menten kinetics and by Hmm-protease-Lh via substrate inhibition kinetics. Valuable rate constants and activation energies were estimated from the temperature-(k_cat/K_m) profiles of both enzymes, and useful results were obtained from the effect of different metallic ions on their Michaelis–Menten parameters.     

Germination, Radial Growth, and Sporulation of Beauveria bassiana and Metarhizium anisopliae Isolates and Their Virulence to Chilo partellus (Lepidoptera: Pyralidae) at Different Temperatures

The effect of temperature regimes on conidia germination, radial growth and virulence of Beauveriabassiana and Metarhizium anisopliae against the spotted stalk borer Chilo partellus was investigated with Ethiopian isolates. Conidia germination, radial growth and sporulation of all isolates were retarded at 15 and 35°C. A suitable temperature range for the isolates was between 20 to 30°C. Conidia germination was less tolerant of low temperature (15°C) than radial growth. Radial growth and sporulation reacted differently to temperature. At both 25 and 30°C, all isolates induced 100% mortality to C. partellus larvae in six days. The LT₅₀ decreased for the isolates with increasing temperature.     

Biology and prey range of Cryptognatha nodiceps (Coleoptera: Coccinellidae), a potential biological control agent for the coconut scale, Aspidiotus destructor (Hemiptera: Diaspididae)

Cryptognatha nodiceps Marshall is an important natural enemy used in biological control programmes against Aspidiotus destructor (Signoret), an introduced pest of coconut in many countries. In order to increase our understanding of C. nodiceps, the present study to quantify aspects of the developmental, reproductive and feeding biology under constant environmental conditions (26±2°C, 55-65% RH) was carried out. In addition, methods for culturing the scale and the beetle were developed. The average duration of development was 19.2±0.1 days for males and 19.6±0.2 days for females. Female longevity and lifetime fecundity was 35.6±5.1 days and 141.0±35.6 eggs, respectively. Life table statistics were as follows: reproductive rate, 9.99, intrinsic rate of increase, 0.09, finite rate of increase, 1.1, doubling time, 7.8 days and generation time, 41.1 days. Studies on feeding biology showed that C. nodiceps was oligophagous. The coccinellid fed and reproduced on prey species in two families, Diaspididae and Coccidae. Adults also fed on the coconut whitefly Aleurodicus cocois (Curtis) (Aleyrodidae) but no reproduction occurred on this prey. Aphididae, Psyllidae and Pseudococcidae were not fed upon.     

Grafting tomato (Solanum lycopersicum) onto the rootstock of a high-altitude accession of Solanum habrochaites improves suboptimal-temperature tolerance

Grafting is regarded as a promising tool to broaden the temperature optimum of elite tomato cultivars. However, suitable low-temperature tolerant tomato rootstocks are not yet available and its breeding is hampered by a lack of variation in low-temperature tolerance within the cultivated tomato. In this study, therefore, the impact of grafting tomato (Solanum lycopersicum Mill. cv. Moneymaker, Sl) onto the rootstock of a cold-tolerant high-altitude accession of a related wild species (Solanum habrochaites LA 1777 Humb. & Bonpl., Sh) was examined at different combinations of optimal (25 °C) and/or suboptimal (15 °C) air/root-zone temperatures (RZT), i.e. 25/25, 25/15, 15/25 and 15/15 °C. Self-grafted tomato plants were used as controls. Both scion/rootstock combinations, Sl/Sl and Sl/Sh, were grown hydroponically and compared for biomass production and partitioning, plant morphology, carbohydrate partitioning and leaf C and N status. Grafting tomato onto Sh increased the relative growth rate of shoots with 26 and 11% at 25/15 and 15/15 °C, respectively. This increase could be attributed to stimulation of the leaf expansion rate. Graft combinations with Sh rootstocks were characterized by higher root mass ratios, particularly at 15 °C RZT. Suboptimal RZT strongly reduced the relative growth rate of Sl roots but not of Sh. This was correlated to differences in inhibition of root elongation. In contrast to tomato grafted onto Sh, leaf total C and total N concentrations increased in self-grafted tomato plants in response to 15 °C RZT. The increase in leaf total C concentration of Sl/Sl graft combinations at 15 °C RZT could be ascribed largely to starch accumulation. This study illustrates that growth of vegetative tomato plants at suboptimal temperature is for a significant part inhibited by its poor root development. Grafting tomato onto a low-temperature rootstock provides an alternative tool to reduce, in part, the grow-limiting effects of suboptimal RZ temperature on the shoot. To improve the low-temperature tolerance of existing commercial tomato rootstocks, S. habrochaites LA 1777 appeared to be a valuable germplasm pool.     

Temperature-mediated characteristics of the dusky salamander (Desmognathus fuscus) of southern Appalachia

Thermal preference of the salamander Desmognathus fuscus was measured in a linear thermal gradient with floor temperatures ranging from 10 to 30°C. Salamanders were acclimated to 21±1°C and a 12 : 12 photoperiod with photophase centered at 1200 h for 8 weeks prior to being placed in the gradient. Substrate temperatures were measured under the salamanders’ stomachs from 1200 to 2400 h at 2 h intervals immediately after feeding and after seven days fasting. We found no selection for temperature in fasting or postprandial D. fuscus. We compared the rate at which D. fuscus cooled and heated with that of a control and found no significant difference. We determined the desiccation rate of D. fuscus at 16 and 26°C and found a significantly more rapid desiccation at 26°C.     

Photosynthetic responses of seven tropical seagrasses to elevated seawater temperature

This study uses chlorophyll a fluorescence to examine the effect of environmentally relevant (1–4 h) exposures of thermal stress (35–45 °C) on seagrass photosynthetic yield in seven tropical species of seagrasses. Acute response of each tropical seagrass species to thermal stress was characterised, and the capacity of each species to tolerate and recover from thermal stress was assessed. Two fundamental characteristics of heat stress were observed. The first effect was a decrease in photosynthetic yield (F_v / F_m) characterised by reductions in F and F_m′. The dramatic decline in F_v / F_m ratio, due to chronic inhibition of photosynthesis, indicates an intolerance of Halophila ovalis, Zostera capricorni and Syringodium isoetifolium to ecologically relevant exposures of thermal stress and structural alterations to the PhotoSystem II (PSII) reaction centres. The decline in F_m′ represents heat-induced photoinhibition related to closure of PSII reaction centres and chloroplast dysfunction. The key finding was that Cymodocea rotundata, Cymodocea serrulata, Halodule uninervis and Thalassia hemprichii were more tolerant to thermal stress than H. ovalis, Z. capricorni and S. isoetifolium. After 3 days of 4 h temperature treatments ranging from 25 to 40 °C, C. rotundata, C. serrulata and H. uninervis demonstrated a wide tolerance to temperature with no detrimental effect on F_v / F_m′ qN or qP responses. These three species are restricted to subtropical and tropical waters and their tolerance to seawater temperatures up to 40 °C is likely to be an adaptive response to high temperatures commonly occurring at low tides and peak solar irradiance. The results of temperature experiments suggest that the photosynthetic condition of all seagrass species tested are likely to suffer irreparable effects from short-term or episodic changes in seawater temperatures as high as 40–45 °C. Acute stress responses of seagrasses to elevated seawater temperatures are consistent with observed reductions in above-ground biomass during a recent El Niño event.     

Suppression of Meloidogyne hapla populations by Hirsutella minnesotensis

The effects of the endoparasitic fungus Hirsutella minnesotensis on populations of Meloidogyne hapla from Michigan (MI), Rhode Island (RI), Connecticut (CT), Lyndonville, New York (NYL), Geneva, New York (NYG), and Wisconsin (WI) were studied in the greenhouse. Twenty-day-old tomato (cv. Rutgers) seedlings were inoculated with either 0 or 600 eggs of each nematode population mixed with either 0, 0.02, or 0.1 g of fresh H. minnesotensis mycelium 0.1 L^-1 of soil in pots containing 0.5 L of soil, and maintained at 25±2°C for 2 months. No effect of the fungal treatments and nematode treatments on tomato plant heights and shoot dry weights was observed. While all M. hapla populations were suppressed by H. minnesotensis, the degree to which each population was affected varied slightly. Across fungal treatments and nematode populations, the fungus reduced total number of nematodes in roots by 61-98%, with the highest for NYG and RI, intermediate for NYL and CT, and lowest for MI and WI populations. The study demonstrated that H. minnesotensis may be used as a potential suppressor of M. hapla in vegetable production systems in the Great Lakes Region.     

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.     

Direct biosynthesis of poly(3-hydroxyalkanoates) bearing epoxide groups

The biosynthesis of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas cichorii YN2 cultured with C6–C12 1-alkenes was studied. PHAs containing repeating units with terminal epoxide groups were obtained when C7–C12 1-alkenes were fed separately as the only carbon source, but no epoxidized unit was detected when 1-hexene was fed. The content of epoxidized units in the polymers was in the range of 4–20 mol%, which was not dependent on the C atom length of the 1-alkene used as a substrate. The polymers produced undergo a glass transition at around −40 °C, and number average molecular weights were in the range of 1 50 000–2 00 000 as determined by GPC relative to polystyrene, with M_w/M_n ratios of 1.9–2.5. As an intermediate, the corresponding 1,2-epoxyalkane was found in the culture medium. According to this result, the epoxidation of the 1-alkene is the initial step in the synthetic pathway of the epoxy unit in the polymer.     

Morphology and germination characteristics of the cysts of Chattonella ovata (Raphidophyceae), a novel red tide flagellate in the Seto Inland Sea, Japan

To elucidate the mechanism of bloom outbreaks of Chattonella ovata (Raphidophyceae), we investigated the cysts of C. ovata and succeeded in finding them from the bottom sediments of Hiroshima Bay. The morphology of the cysts was mostly hemispherical in shape, with a diameter of ca. 30 μm and height of ca. 20 μm. The cysts were usually adhering to solid materials, such as diatom frustules, yellow-greenish in color and had several dark brown grains. The cyst wall was smooth and had no ornamentation. Because the morphological characteristic of the cysts was in general agreement with those of Chattonella antiqua and Chattonella marina, it was difficult to differentiate the cysts of these three species. Germination of the cysts of C. ovata was observed at temperatures from 17.5 to 30 °C, but not at 15 °C or below. The number of the germinated cysts increased with increasing temperature and the optimum temperature for germination was 30 °C. Although cysts of C. antiqua and C. marina germinated at temperatures from 15 to 30 °C, optimum temperature of germination was 22.5 °C. The lower limit and optimum temperatures for germination of C. ovata cysts was higher than for C. antiqua and C. marina. The role of cysts in the population dynamics of C. ovata is discussed.     

Uracil-DNA N-glycosylase (UNG) from the marine, psychrophilic bacterium Vibrio salmonicida shows cold adapted features: A comparative analysis to Vibrio cholerae uracil-DNA N-glycosylase

The genes encoding uracil-DNA N-glycosylase (UNG) from the marine, psychrophilic bacterium Vibrio salmonicida and the mesophilic counterpart Vibrio cholerae have been cloned and expressed in Escherichia coli. The purified proteins have been characterized in order to reveal possible cold adapted features of the V. salmonicida UNG (vsUNG) compared to the V. cholerae UNG (vcUNG). Characterization experiments demonstrated that both enzymes possessed the highest activities at pH 7.0–7.5 and at salt concentrations in the range of 25–50 mM NaCl. Temperature optima for activity were determined to approximately 30 °C for vsUNG and 50 °C for vcUNG. Temperature stability of the enzymes was compared at 4 °C and 37 °C, and vsUNG was found to be more temperature labile than vcUNG. Kinetic studies performed at three different temperatures, 15 °C, 22 °C and 37 °C, demonstrated higher catalytic efficiency for vsUNG compared to vcUNG due to lower K_M-values. The increased substrate affinity of vsUNG is probably caused by an increased number of positively charged residues in the DNA-binding site of the enzyme compared to vcUNG. Thus, activity and stability measurements reveal typical cold adapted features of vsUNG.     

The effects of constant and changing tempertures on the development of eggs of the freshwater snail Lymnaea auricularia (L.)

The rate of development of Lymnaea auricularia eggs was studied at various constant temperatures between 10° and 36°C. Development was accelerated as the temperature increased and at 36°C the eggs failed to develop. Spring eggs showed differences in their rate of development when compared with summer eggs when measured at similar tempertures.

Both spring and summer eggs were more than 90% fertile. Hatching success was high at temperatures between 10° and 30° (100%–82/9%); while at 34°C it was reduced to 60.6% for spring eggs. It was above 87% at temperatures between 10° and 34°C but it dropped to 62.3% at 36°C for summer eggs.

In one regularly changing temperature experiment a significant acceleration (P < .05) was found. In two others there was no significant difference beween predicted and observed egg durations. In one suddenly changing temperature regime (1 day at 20°, 1 day at 30° and so on) a huge retardation of development was found. In the other suddenly changing experiment (1 day at 15°, 1 day at 25°) no significant difference was found.

The exposure of eggs to extreme temperature (4°C, freezing and 4°C caused a retardation in the race of subsequent development of eggs at 25°C.     

Study of the thermal stability of D-amino acid oxidase from Trigonopsis variabilis reveals enzyme inactivation via multiple steps

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (TvDAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20-60°C) and protein concentration (5-100 µmol L^-1). The time courses of loss of enzyme activity in 100 mmol L^-1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (k _a) increased 10⁴-fold in response to an increase in temperature from 20 to 60°C. The rate constants of FAD release (k _b) and binding (k _-b) as well as the irreversible aggregation of the apo-enzyme (k _agg) were less sensitive to changes in temperature, their activation energy (E _a) being about 52 kJ mol^-1 in comparison with an E _a value of 185 kJ mol^-1 for k _a. The rate-determining step of TvDAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. TvDAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5'-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L^-1) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade TvDAO. However, it did not stabilize TvDAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.     

Reduction of ferricytochrome c by hydrogen atoms: Evidence for intramolecular transfer of reducing equivalent

Direct evidence obtained by means of the technique of pulse radiolysis-kinetic spectrometry, with measurements in the time range 10⁻⁶ to 1 s, is presented that, consequent upon reaction of a single H-atom with a single molecule of ferricytochrome c, a reducing equivalent is transmitted via the protein structure to the ferriheme moiety. Such transmission accounts for at least 70% of the total reduction of the ferri to the ferro state of cytochrome c. The remainder of the total reduction takes place without stages resolvable on the time scale of these experiments. Reduction brought about by H atoms appears to follow a different course than reduction by hydrated electrons. In the latter case, intramolecular transmission of reducing equivalents could not be demonstrated (Lichtin, N. N., Shafferman, A. and Stein, G. (1973) Biochim. Biophys. Acta 314, 117–135).

Not every H-atom reacts with ferricytochrome c at a site which results in conversion of the Fe(III) state to the Fe(II) state. Approximately half of reacting H-atoms do not produce reduction.

The following second order rate constants have been determined in solutions of low ionic strength at 20±2 °C: k[H+ferricytochrome c] = (1.0±0.2) · 10¹⁰ M⁻¹ · s⁻¹ at pH 3.0 and 6.7; k[H+ferrocytochrome c] = (1.3±0.2) · 10¹⁰ M⁻¹ · s⁻¹ at pH 3.0; k[e⁻_aq + ferrocytochrome c] = (1.9±0.4) · 10¹⁰ M⁻¹ · s⁻¹ at pH 6.7.