Uptake of water from a Kandosol subsoil: I. Determination of soil water diffusivity

Aims

To determine soil water diffusivity, D(θ), on undisturbed field soil at medium to low water content (suction range from 10 to 150 m of water), for the purpose of modeling the uptake of water by plant roots.

Methods

The method is based on the analysis of one-step outflow induced by a turbulent stream of dry air over the exposed end of a soil core, with the other end of the core enclosed. The outflow is measured through time as the change in the weight of the core as it sits on a recording balance. D(θ) is calculated by deconvoluting the measured outflow function.

Results

Over the suction range of 10 to 150 m of water, D(θ) calculated on the undisturbed soil ranged from 20?×?10^?9 to 10?×?10^?9 [m²?s^?1], substantially higher than other published estimates over this range in suction.

Conclusions

These unusually large values cast doubt on the view that flow of water to roots limits uptake of water from the targeted subsoil.     

Changes in apparent water permeability during the moulting cycle in the western rock lobster

The kinetics of water exchange, as measured by ³H₂O fluxes, were examined in the western rock lobster, Panulirus longipes (Milne Edwards) during stages C₄, D₃, D₄, D₄-E and B₁ of the moulting cycle. A series of samples of blood and external water was taken during each experiment and the inward and outward rate constants found using the SAAM 25 computer program, assuming a simple 2-compartment model with reversible exchange. Both inward and outward turnover rates remained constant up to the swelling prior to ecdysis when there was an increase of 203% inwards compared with 133% outwards. The latter increased in stage B₁, so that both rates were comparably high, probably due to the increased permeability of the soft integument.Further analysis showed that the rock lobster does not behave as a single compartment with respect to tritiated water. Two compartments were resolved by graphical analysis, one with fast and the other with slow exchange. It is suggested that blood and tissue ‘free’ water comprise one compartment and chemically ‘bound’ water the other, with fast exchange between the free and bound water and relatively slow exchange between free body water and the external medium.     

The effect of nutrient concentrations,nutrient ratios and temperature on photosynthesis and nutrient uptake by Ulva prolifera: implications for the explosion in green tides

The green-tide macroalga, Ulva prolifera, was tested in the laboratory to determine its nutrient uptake and photosynthesis under different conditions. In the nutrient concentration experiments U. prolifera showed a saturated uptake for nitrate but an escalating uptake in the tested range for phosphorus. Both N/P and NO₃ ^?/NH₄ ⁺ ratios influenced nutrient uptake significantly (p?<?0.05) while the PSII quantum yield [Y(II)] (p?>?0.05) remained unaffected. The maximum N uptake rate (33.9?±?0.8 μmol g^?1 DW h^?1) and P uptake rate (11.1?±?4.7) was detected at N/P ratios of 7.5 and 2.2, respectively. U. prolifera preferred NH₄ ⁺-N to NO₃ ^?-N when the NO₃ ^?-N/NH₄ ⁺-N ratio was less than 2.2 (p?<?0.05). But between ratios of 2.2 and 12.9, the uptake of NO₃ ^?-N surpassed that of NH₄ ⁺-N. In the temperature experiments, the highest N uptake rate and [Y(II)] were observed at 20 °C, while the lowest rates were detected at 5 °C. P uptake rates were correlated with increasing temperature.     

Effect of two nutrient solution temperatures on nitrate uptake,nitrate reductase activity,NH4+ concentration and chlorophyll a fluorescence in rose plants

The effect of two nutrient solution temperatures (cold (10 °C) and warm (22 °C)) during two flowering events of rose plants (Rosa × hybrida cv. Grand Gala) were examined by measuring chlorophyll (Chl) a fluorescence, ammonium (NH₄⁺) content and nitrate reductase (NR) activity in four different leaf types, that is, external and internal leaves of bent shoots and lower and upper leaves of flowering stems. Besides, nitrate (NO₃^?) uptake and water absorption, total nitrogen (N) concentration in the plant, dry biomass, and the ratios of shoot/root and thin-white roots/suberized-brown roots were determined. Generally, cold solution increased NO₃^? uptake and thin-white roots production but decreased water uptake, so plants grown at cold solution had to improve their NO₃^? uptake mechanisms to obtain a higher amount of nutrient with less water absorption than plants grown at warm solution. The higher NO₃^? uptake can be related to an increase in NR activity, NH₄⁺ content and total N concentration at cold solution. Nutrient solution temperature also had an effect on the photosynthetic apparatus. In general terms, the effective quantum yield (?_PSII) and the fraction of open PSII reaction centres (q_L) were higher in rose plants grown at cold solution. These effects can be associated to a higher NO₃^? uptake and total N concentration in the plants and were modulated by irradiance throughout all the experiment. Plants could adapt to cold solution by enhancing their metabolism without a decrease in total dry biomass. Nevertheless, the effect of nutrient solution temperature is not simple and also affected by climatic factors.     

Action of two juvenile hormone antagonists on lepidopteran epidermis

The juvenile hormone antagonist ETB (ethyl-4-2(t-butylcarbonyloxy)-butoxybenzoate) caused formation of precocious larval-pupal intermediates after the 4th (penultimate)-larval instar of the tobacco hornworm, Manduca sexta, when 50 μg were applied to any 3rd stage larvae or to 4th stage larvae within 12 hr after ecdysis. This dose was most effective within 12 hr after ecdysis to the 3rd stage. In the black mutant larval assay for juvenile hormone, ETB had activity, 0.75 μg per larva giving half-maximal score. In vitro ETB acted as a juvenile hormone to prevent the ecdysteroid-induced change in commitment at concentrations above 0.1 μg/ml with an ED₅₀ at 2.8 μg/ml and as a partial juvenile hormone antagonist to 0.1 μg/ml juvenile hormone I at concentrations between 10^?3 and 10^?2 μg/ml. By contrast, EMD (ethyl-E-3-methyl-2-dodecenoate) had little juvenile hormone-like activity in vitro up to its limits of solubility (100 μg/ml) and exhibited sporadic partial juvenile hormone antagonistic activity in vitro at concentrations between 1 and 100 μg/ml. Since these concentrations were 10–1000 times that of juvenile hormone I in the medium, EMD apparently is not an efficient competitor.     

Specific light uptake rates can enhance astaxanthin productivity in <Emphasis Type="Italic">Haematococcus lacustris</Emphasis>

Lumostatic operation was applied for efficient astaxanthin production in autotrophic Haematococcus lacustris cultures using 0.4-L bubble column photobioreactors. The lumostatic operation in this study was performed with three different specific light uptake rates (q _e) based on cell concentration, cell projection area, and fresh weight as one-, two- and three-dimensional characteristics values, respectively. The q _e value from the cell concentration (q _e1D) obtained was 13.5 × 10^?8 μE cell^?1 s^?1, and the maximum astaxanthin concentration was increased to 150 % compared to that of a control with constant light intensity. The other optimum q _e values by cell projection area (q _e2D) and fresh weight (q _e3D) were determined to be 195 μE m^?2 s^?1 and 10.5 μE g^?1 s^?1 for astaxanthin production, respectively. The maximum astaxanthin production from the lumostatic cultures using the parameters controlled by cell projection area (2D) and fresh weight (3D) also increased by 36 and 22 % over that of the controls, respectively. When comparing the optimal q _e values among the three different types, the lumostatic cultures using q _e based on fresh weight showed the highest astaxanthin productivity (22.8 mg L^?1 day^?1), which was a higher level than previously reported. The lumostatic operations reported here demonstrated that more efficient and effective astaxanthin production was obtained by H. lacustris than providing a constant light intensity, regardless of which parameter is used to calculate the specific light uptake rate.     

Effects of saline root environment (NaCl) on nitrate and potassium uptake kinetics for rose plants: a Michaelis–Menten modelling approach

Greenhouse-grown cut flower roses are often irrigated with moderately saline irrigation water. The salt/ballast ions are either present initially in poor quality raw water or reclaimed municipal water, or accumulated in greenhouse irrigation water that is captured and reused. Such ions can inhibit root absorption of essential nutrients. The objective of this work was to quantify the influence of NaCl concentration on the uptake of nitrate and potassium by roses and develop a predictive model of uptake inhibition based on NaCl, NO₃ ^?, and K⁺ concentration. One year-old rose plants (Rosa spp. ‘Kardinal’ on ‘Natal Briar’ rootstock) were moved into growth chambers where nitrogen and potassium depletion were monitored during 6 days. Eight different initial NaCl treatments varying from zero to 65 mol m^?3 were used and within these there were two initial NO₃ ^? and K⁺ concentrations: high concentration (HC, 7.0 mol m^?3 and 2.6 mol m^?3 NO₃ ^? and K⁺ respectively) or low concentration (LC, 3.5 mol m^?3 and 1.3 mol m^?3 NO₃ ^? and K⁺ respectively). Plant NO₃ ^? uptake was negatively affected by NaCl concentration. NO₃ ^? maximum influx (I_max) declined from 5.1 µmol to 2.5 µmol per gram of plant dry weight per hour as NaCl concentration increased from zero to 65 mol m^?3. A modified Michaelis–Menten (M–M) equation taking into account inhibition by NaCl provided the best fit for NO₃ ^? uptake in response to varying NaCl concentration. K⁺ uptake was unaffected by NaCl concentration. A M–M equation that did not include inhibition was suitable for describing K⁺ uptake at varying NaCl concentration. The resulting empirical models could assist with decision making, such as: adjustment of NO₃ ^? fertilization based on NaCl concentration, necessity of water desalinization, or determination of the desired leaching fraction.     

Carbon assimilation during larval development of the marine teleost Leiostomus xanthurus Lacépède

In larval spot, Leiostomus xanthurus Lacépède, ¹⁴C labeled food was rapidly digested and assimilated with up to 20% respired as ¹⁴CO₂ within 10 h from ingestion. Two indices of carbon assimilation, total carbon utilization (range 9.4 to 64.7%) and total carbon absorption (range 67.2 to 99.2%) were calculated from the amount of ¹⁴C ingested, retained, and defecated. Carbon utilization and absorption were not significantly (P > 0.10) related to development as measured by age (7 to 47 days from hatching), length (1.8 to 11.4 mm), or dry weight (0.022 to 4.425 mg).     

Filtration and ingestion rates of Salpa fusiformis Cuvier (Tunicata : Thaliacea): Effects of size,individual weight and algal concentration

Filtration and ingestion rates of Salpa fusiformis Cuvier were determined while fed Phaeodactylumtricomutum Bohlin at concentrations of 2?64 × 10³ cells·ml^?1. Filtration and ingestion rates increase exponentially with increasing length and body protein. The relations between protein content and body length, and between filtration rate and weight are similar for blastozooids and oozooids. A capture efficiency of the order of 6–32% is calculated: Salpa fusiformis seems to have a low retention efficiency, but its very high filtration rate gives it pride of place amongst filter-feeders. Specific filtration rates are independent of weight; specific ingestion rates are independent of weight for blastozooids, but for oozooids they seem to diminish with increasing weight. The mean daily ration (μg C ingested · μg body C^?1) is 107% for a blastozooid and 117% for an oozooid. Specific filtration rates decrease exponentially as particle concentration rises, as for many other filter-feeders, and the specific ingestion rate follows an Ivlev relation.     

Changes in lipid and protein during starvation and the moulting cycle in the tiger prawn, Penaeus esculentus Haswell

Tiger prawns, Penaeus esculentus Haswell (mean wt 20.8 ± 0.3 g, range 13.9–27.7 g) contained 1–2% extractable lipid, 13% protein (biuret method) and 71–74% water (wet wt). In 21 days, the weight of fed prawns increased by 3% and that of starved prawns decreased by 4.4%. Protein was the major energy source during 14 days of starvation, with a loss of 550 mg of total protein compared with a loss of 84 mg of total lipid. The absolute amount of water present remained constant. Of three different tissue compartments, abdomen, cephalothorax, and digestive gland, the abdomen contributed the most protein (330 mg) and lipid (35 mg) during 14 days of starvation. Digestive gland, although containing the largest percentage wet wt of lipid, accounted for only 8.3% of the total lipid in the prawn, and contributed only 18 mg of lipid in 14 days of starvation. Lipid concentration in the digestive gland increased during early premoult (stage D₄) and dropped in late premoult (stage D₄). Resting oxygen consumption rate remained constant at ≈0.1 ml · g^?1 · h^?1 at 25°C during 21 days of starvation.     

Hydrolysis of p-nitrotrifluoroacetanilide catalyzed by water and imidazole

The hydrolyses of p-nitrotrifluoroacetanilide catalyzed by water and imidazole were examined at 70°C. The pH-rate constant profile of the hydrolysis in H₂O was examined in the pH range 0.0–11.4. The hydrolysis was independent of pH in the region from pH 1.0 to 4.5, presumably a water-catalyzed reaction. The rate constant and the D₂O solvent isotope effect for this reaction were 1.0 × 10^?4 sec^?1 and 3.7, respectively. Both natural imidazole and imidazolium cation catalyzed hydrolysis. The rate constant of the hydrolysis catalyzed by neutral imidazole was determined to be 5.4 × 10^?3M^?1 sec^?1 and the D₂O solvent isotope effect was 1.8.     

UV-Vis absorption study of some tetragonal chromium (III) complexes

A UV-Vis absorption study was performed in order to elucidate the electronic energy levels of three tetragonal chromium (III) complexes, namely trans-[Cr(en)₂(CN)₂]ClO₄, trans-[Cr(cyclam)(CN)₂]ClO₄, and trans-[Cr(NH₃)₄(CN)₂]ClO₄. The absorption spectra of the preceding complexes have been analyzed via Gaussian analysis to locate the quartet band maxima of the tetragonal components. The deconvoluted band maxima were then fitted with the tetragonal energy matrices of d³ configuration with full configuration interaction, neglecting spin-orbit interaction. The ligand field parameters D_q, D_t, and D_s along with the electron correlation parameters have been extracted via the fitting procedure. The significance of these parameters and the translated angular overlap model parameters has been discussed. We have also uncovered in the spectrum of the ethylenediamine complex the low intensity doublet absorption bands and a high intensity charge transfer band which have been tentatively assigned.     

Enhanced efficacy of nitrifying biomass by modified PVA_SB entrapment technique

In this study, we developed a novel technique for preparing polyvinyl alcohol (PVA) hydrogel as an immobilizing matrix by the addition of sodium bicarbonate. This resulted in an increase in the specific surface area of PVA_sodium bicarbonate (PVA_SB) hydrogel beads to 65.23 m² g^?1 hydrogel beads, which was approximately 85 and 14 % higher than those of normal PVA and PVA_sodium alginate (PVA_SA) hydrogel beads, respectively. The D _e value of PVA_SB hydrogel beads was calculated as 7.49 × 10^?4 cm² s^?1, which was similar to the D _e of PVA_SA hydrogel beads but nearly 38 % higher than that of the normal PVA hydrogel beads. After immobilization with nitrifying biomass, the oxygen uptake rate and the ammonium oxidation rate of nitrifying biomass entrapped in PVA_SB hydrogel beads were determined to be 19.53 mg O₂ g MLVSS^?1 h^?1 and 10.59 mg N g MLVSS^?1 h^?1, which were 49 and 43 % higher than those of normal PVA hydrogel beads, respectively. Scanning electron microscopy observation of the PVA_SB hydrogel beads demonstrated relatively higher specific surface area and revealed loose microstructure that was considered to provide large spaces for microbial growth. This kind of structure was also considered beneficial for reducing mass transfer resistance and increasing pollutant uptake.     

Transition duration of ingested deuterium oxide to eccrine sweat during exercise in the heat

The time necessary for the initial appearance of ingested water as sweat during exercise in the heat remains unknown. Based on the current literature, we estimated fluid transition through the body, from ingestion to appearance as sweat, to have a minimum time duration of approximately three minutes. The purpose of this study was to test this prediction and identify the time necessary for the initial enrichment of deuterium oxide (D₂O) in sweat following ingestion during exercise in the heat. Eight participants performed moderate intensity (40% of maximal oxygen uptake) treadmill exercise in an environmental chamber (40 °C, 40% rH) to induce active sweating. After fifteen minutes, while continuing to walk, participants consumed D₂O (0.15 ml kg⁻¹) in a final volume of 50 ml water. Scapular sweat samples were collected one minute prior to and ten minutes post-ingestion. Samples were analyzed for sweat D₂O concentration using isotope ratio mass spectrometry and compared to baseline. Mean±SD ∆ sweat D₂O concentration at minutes one and two post-ingestion were not significantly higher than baseline (0 min). Minutes three (9±3 ppm) through ten (23±11 ppm) post-ingestion had ∆ sweat D₂O concentrations significantly (P<0.05) higher than baseline. Such results suggest that ingested water rapidly transports across the mucosal membrane of the alimentary canal into the vasculature space, enters the extravascular fluid, and is actively secreted by the eccrine sweat glands onto the surface of the skin for potential evaporation in as little as three minutes during exercise in the heat.     

Transport of thiamine and 4-methyl-5-hydroxyethylthiazole by Salmonella typhimurium

The transport of thiamine and 4-methyl-5-hydroxyethylthiazole (MHET), its thiazole moiety, was studied using whole cells of Salmonella typhimurium. It was found that the bacteria possessed an active transport system for thiamine that had K_m 0.21 μM and V_max 33 nmol·min^?1·(mg dry wt. cells)^?1. Transport of thiamine was glucose dependent, whereas MHET uptake was dependent on both glucose and 2-methyl-4-amino-5-hydroxymethylpyrimidine (MAHMP), the pyrimidine moiety of thiamine. Uptake of both thiamine and MHET was severely curtailed by cyanide, azide, N-ethylmaleimide and carbonyl cyanide m-chlorophenylhydrazone. Oxythiamine inhibited thiamine, but not MHET, uptake and thiamine slightly inhibited MHET uptake. 2-Methyl-4-amino-5-methoxymethylpyrimidine and 4-amino-5-hydroxymethylpyrimidine were unable to replace MAHMP as stimulators of MHET uptake, but 2-methyl-4-amino-5-aminomethylpyrimidine was marginally effective in this regard. Similar results were obtained with attempts to replace MAHMP as a growth requirement for a purD mutant of Salmonella typhimurium. MHET uptake showed saturation kinetics only in the presence of MAHMP, and is not otherwise actively transported.     

The diffusional and osmotic water permeability of the hindgut cuticle in larval Sarcophaga bullata

The diffusional water permeability (P_D) and the pressure filtration coefficient (L_P) of isolated larval hindgut cuticle of the fleshfly, Sarcophaga bullata, were measured using tracer techniques coupled with a simple mathematical model system based on equations of non-equilibrium thermodynamics. Data obtained from the model system were matched to experimental tracer data by means of a mathematical optimization scheme. The following parameter values were obtained: P_D for tritiated water = 1.02 × 10^?6 cm-sec^?1, and L_P = 9.18 × 10^?11 cm³-dyn^?1-sec^?1. These results are now being used to determine reflection coefficients (σ's) and solute mobilities (ω's) for the cuticle system in an attempt to gain an understanding of the mechanisms controlling solute and water movements across the hindgut wall.     

The bioavailability of26Al-labelled aluminium citrate and aluminium hydroxide in volunteers

A study was undertaken to determine the fraction of ingested aluminium taken up by two male volunteers, following their ingestion of either aluminium citrate or aluminium hydroxide. In addition, the effects of simultaneous citrate ingestion on the gastrointestinal absorption of aluminium from its hydroxide was studied. Volunteers received three oral doses of²⁶Al-labelled aluminium compound in water. The doses were administered directly into the stomach using a paediatric feeding tube. Blood samples were collected from the volunteers at 1, 4 and 24 h after administration, and their daily output of urine and faeces was collected for 6 days. These samples were analysed for their²⁶Al content using either coincidence gamma-counting or accelerator mass spectrometry. The uptake of aluminium was greatest following its administration in the citrate form and was least following intake as the aluminium hydroxide suspension. The co-administration of citrate, with the aluminium hydroxide suspension, was found to enhance the levels of²⁶Al uptake in both volunteers. Using a urinary excretion factor based on the results of previous studies, the fractional aluminium uptake from each of the species was calculated: aluminium citrate, 5.23 × 10^–3; aluminium hydroxide, 1.04 × 10^–4; aluminium hydroxide with citrate, 1.36 × 10^–3.     

Bimodal oxygen uptake in a freshwater air-breathing fish,Notopterus chitala

Measurements of bimodal oxygen uptake have been made in a freshwater air-breathing fish,Notopterus chitala at 29.0±1(S.D.)°C. xhe mean oxygen uptake from continuously flowing water without any access to air, was found to be 3.58±0.37 (S.E.) ml O₂ · h^?1 and 56.84+4.29 (S.E.) ml O₂ · kg^?1 · h^?1 for a fish weighing 66.92 + 11.27 (S.E.) g body weight. In still water with access to air, the mean oxygen uptake through the gills were recorded to be 2.49 ± 0.31 (S.E.) ml O₂ · h^?1 and 38.78 ± 1.92 (S.E.) ml O₂ · kg^?1 · h^?1 and through the accessory respiratory organs (swim-bladder) 6.04±0.87 (S.E.) ml O₂ · h^?1 and 92.32±2.91 (S.E.) ml O₂ · kg^?1 · h^?1 for a fish averaging 66.92±11.27 (S.E.) g. Out of the total oxygen uptake (131.10 ml O₂ · kg^?1 · h^?1), about 70% was obtained through the aerial route and the remainder 30% through the gills.     

Estimation of leaf area at the level of branch,tree and stand inCryptomeria japonica

The frequency distribution of diameter (x) in foliage shoot segments, ø(x), was examined in 18 branches at different height levels of three trees in a 25-yr-old sugi (Cryptomeria japonica D. Don) plantation. The ø-x relationships were approximated by power-form equations, in which the exponent differed among the branches from ?0.6 to ?4.2. Leaf area (S _B) and leaf weight (W _B) of a branch were estimated on the basis of the ø-x relationship, and the dependency of specific leaf area (SLA ₀) and density (ρ ₀) of a foliage shoot segment on itsx. SLA _B value of a branch defined byS _B/W _B ranged from 27 to 80 cm² g. d.w.^?1 according to the exponent in the function of ø(x). Total leaf area (u) and leaf weight (wl) of a tree were estimated by summation ofS _B andW _B for seven sample trees. TheSLA _T value of a tree defined byu/wl ranged from 65 to 76 cm² g d.w.^?1 and increased with stem diameter at clear length (D _B). By use of the allometric equations betweenu andD _B,LAI of the plot was estimated to be 17.3 ha ha^?1 (half of the total surface area of needles). By a process similar to that used for calculatingLAI, the amount of woody tissues included in sugi foliage was evaluated to be about 10% of the stand foliage biomass.     

Mössbauer, EPR, and MCD studies of the C9S and C42S variants of Clostridium pasteurianum rubredoxin and MCD studies of the wild-type protein

Rubredoxins contain a mononuclear iron tetrahedrally coordinated by four cysteinyl sulfurs. We have studied the wild-type protein from Clostridium pasteurianum and two mutated forms, C9S and C42S, in the oxidized and reduced states, with Mössbauer, integer-spin EPR, and magnetic circular dichroism (MCD) spectroscopies. The Mössbauer spectra of the ferric C42S and C9S mutant forms yielded zero-field splittings, D=1.2?cm^?1, that are about 40% smaller than the D-value of the wild-type protein. The ⁵⁷Fe hyperfine coupling constants were found to be ca. 8% larger than those of the wild-type proteins. The present study also revealed that the ferric wild-type protein has δ=0.24±0.01?mm/s at 4.2?K rather than δ=0.32?mm/s as reported in the literature. The Mössbauer spectra of both dithionite-reduced mutant proteins revealed the presence of two ferrous forms, A and B. These forms have isomer shifts δ=0.79?mm/s at 4.2?K, consistent with tetrahedral Fe²⁺(Cys)₃(O-R) coordination. The zero-field splittings of the two forms differ substantially; we found D=?7±1?cm^?1, E/D=0.09 for form A and D=+6.2±1.3?cm^?1, E/D=0.15 for form B. Form A exhibits a well-defined integer-spin EPR signal; from studies at X- and Q-band we obtained g _z =2.08±0.01, which is the first measured g-value for any ferrous rubredoxin. It is known from X-ray crystallographic studies that ferric C42S rubredoxin is coordinated by a serine oxygen. We achieved 75% reduction of C42S rubredoxin by irradiating an oxidized sample at 77?K with synchrotron X-rays; the radiolytic reduction produced exclusively form A, suggesting that this form represents a serine-bound Fe²⁺ site. Studies in different buffers in the pH?6–9 range showed that the A:B ratios, but not the spectral parameters of A and B, are buffer dependent, but no systematic variation of the ratio of the two forms with pH was observed. The presence of glycerol (30–50% v/v) was found to favor the B form. Previous absorption and circular dichroism studies of reduced wild-type rubredoxin have suggested d-d bands at 7400, 6000, and 3700?cm^?1. Our low-temperature MCD measurements place the two high-energy transitions at ca. 5900 and 6300?cm^?1; a third d-d transition, if present, must occur with energy lower than 3300?cm^?1. The mutant proteins have d-d transitions at slightly lower energy, namely 5730, 6100?cm^?1 in form A and 5350, 6380?cm^?1 in form B.