THE RATE OF OXYGEN UTILIZATION BY YEAST AS RELATED TO TEMPERATURE

Suspensions of the yeast Saccharomyces cerevisiae gave reproducible rates of O₂ uptake over a period of 6 months. The relation of rate of consumption of O₂ to temperature was tested over a wide range of temperatures, and the constant in the formulation of the relationship is found to be reproducible. The values of this constant (µ) have been obtained for five separate series of experiments by three methods of estimation. The variability of µ has the following magnitudes: the average deviation of a single determination expressed as per cent of the mean is ±2 per cent in the range 30–15°, and ±0.8 per cent in the range 15–3°C. This constancy of metabolic activity measured as a function of temperature can then be utilized for more precise investigations of processes controlling the velocity of oxidations of substrates, and of respiratory systems controlled by intracellular respiratory pigments. The data plotted according to the Arrhemus equation give average values of the constant µ as follows: for the range 35–30°, µ = 8,290; 30–15°, µ = 12,440 ±290; 15–3°, µ = 19,530 ±154. The critical temperatures are at 29.0° and 15.7°C. A close similarity exists between these temperature characteristics (µ) and values in the series usually obtained for respiratory activities in other organisms. This fact supports the view that a common system of processes controls the velocities of physiological activities in yeast and in other organisms.     

ON THE QUANTA OF LIGHT PRODUCED AND THE MOLECULES OF OXYGEN UTILIZED DURING CYPRIDINA LUMINESCENCE

A study of the oxygen consumed per lumen of luminescence during oxidation of Cypridina luciferin in presence of luciferase, gives 11.4 x 10^–5 gm. oxygen per lumen or 88 molecules per quantum of λ = 0.48µ, the maximum in the Cypridina luminescence spectrum. For reasons given in the text, the actual value is probably somewhat less than this, perhaps of the order of 6.48 x 10^–5 gm. per lumen or 50 molecules of oxygen and 100 molecules of luciferin per quantum. It is quite certain that more than 1 molecule per quantum must react. On the basis of a reaction of the type: luciferin + 1/2 O₂ = oxyluciferin + H₂O + 54 Cal., it is calculated that the total efficiency of the luminescent process, energy in luminescence/heat of reaction, is about 1 per cent; and that a luciferin solution containing 4 per cent of dried Cypridina material should rise in temperature about 0.001°C. during luminescence, and contain luciferin in approximately 0.00002 molecular concentration.     

Temperature Dependence of Photosynthesis in Agropyron smithii Rydb. : III. Responses of Protoplasts and Intact Chloroplasts

Protoplasts and intact chloroplasts isolated from Agropyron smithii Rybd. were utilized in an effort to determine the limiting factor(s) for photosynthesis at supraoptimal temperatures. Saturated CO₂-dependent O₂ evolution had a temperature optimum of 35°C for both protoplasts and intact chloroplasts. A sharp decline in activity was observed as assay temperature was increased above 35°C, and at 45°C only 20% of the maximal rate remained. The temperature optimum for 3-phosphoglycerate reduction by intact chloroplasts was 35°C. Above this temperature, 3-phosphoglycerate reduction was more stable than CO₂-dependent O₂ evolution. Reduction of nitrite in coupled intact chloroplasts had a temperature optimum of 40°C with only slight variation in activity between 35°C and 45°C. Reduction of nitrite in uncoupled chloroplasts had a temperature optimum of 40°C, but increasing the assay temperature to 45°C resulted in a complete loss of activity. Reduction of p-benzoquinone by protoplasts and intact chloroplasts had a temperature optimum of 32°C when measured in the presence of dibromothymoquinone. This photosystem II activity exhibited a strong inhibition of O₂ evolution as assay temperature increased above the optimum. It is concluded that, below the temperature optimum, ATP and reductant were not limiting photosynthesis in these systems or intact leaves. Above the temperature optimum, photosynthesis in these systems is limited in part by the phosphorylation potential of the stromal compartment and not by the available reductant.     

TEMPERATURE CHARACTERISTICS FOR METABOLISM OF CHLORELLA : I. THE RATE OF O2 UTILIZATION OF CHLORELLA PYRENOIDOSA WITH ADDED DEXTROSE

The temperature characteristic for the rate of O₂ consumption by Chlorella pyrenoidosa suspended in Knop solution containing 1 per cent glucose was studied between 1° and 27°C. with the Warburg technic. The value of µ was found to be about 19,000 ±1,000 cal. There is some indication of a critical temperature at 20°C., with shift to a lower µ above this temperature. The effect of sudden changes in temperature on the rate of respiration and the variation of the latter with time at constant temperatures are discussed. It is concluded that the "normal" respiration (in absence of external glucose) does not appear in the determination of this temperature characteristic.     

Temperature dependence of the enzymic carboxylation and oxygenation of ribulose 1,5-bisphosphate in relation to effects of temperature on photosynthesis

Carboxylase and oxygenase activities of ribulose bisphosphate carboxylase purified from wheat were measured over the temperature range 5 to 35°C either at constant O₂ and CO₂ concentrations or where the O₂ and CO₂ simulated the concentrations in water equilibrated at each temperature with the same gaseous phase. At constant CO₂ (14 micromolar) and O₂ (0.34 millimolar), the oxygenase to carboxylase ratio remained constant at 0.21 between 5 and 25°C but increased to 0.26 at 35°C. At O₂ and CO₂ concentrations near those expected in water equilibrated with air (21% [v/v] O₂) containing 300 μl/l CO₂ at the various temperatures, the ratio of oxygenase to carboxylase activity increased 2.2-fold between 15 and 35°C. At CO₂ and O₂ concentrations expected in water in equilibrium with subatmospheric concentrations of CO₂ in air (21% [v/v] O₂, 210 μl/l CO₂), the oxygenase to carboxylase ratio increased from 0.25 at 10°C to 0.56 at 35°C. Between 20 and 30°C, the apparent Q₁₀ value for the oxygenase reaction was 1.78 and that for the carboxylase was 1.26. Hence, the different responses of photosynthesis and photorespiration to temperature are due more to changes in the relative solubilities of CO₂ and O₂ (the solubility ratio) than to changes in kinetic parameters of the reactions catalyzed by ribulose bisphosphate carboxylase.     

Heat-induced formation of reactive oxygen species and 8-oxoguanine, a biomarker of damage to DNA

Heat-induced formation of 8-oxoguanine was demonstrated in DNA solutions in 10^–3 M phosphate buffer, pH 6.8, by enzyme-linked immunosorbent assays using monoclonal antibodies against 8-oxoguanine. A radiation-chemical yield of 3.7 × 10^–2 µmol J^–1 for 8-oxoguanine production in DNA upon γ-irradiation was used as an adequate standard for quantitation of 8-oxoguanine in whole DNA. The initial yield of heat-induced 8-oxoguanine exhibits first order kinetics. The rate constants for 8-oxoguanine formation were determined at elevated temperatures; the activation energy was found to be 27 ± 2 kcal/mol. Extrapolation to 37°C gave a value of k₃₇ = 4.7 × 10^–10 s^–1. Heat-induced 8-oxoguanine formation and depurination of guanine and adenine show similarities of the processes, which implies that heat-mediated generation of reactive oxygen species (ROS) should occur. Heat-induced production of H₂O₂ in phosphate buffer was shown. The sequence of reactions of thermally mediated ROS formation have been established: activation of dissolved oxygen to the singlet state, generation of superoxide radicals and their dismutation to H₂O₂. Gas saturation (O₂, N₂ and Ar), D₂O, scavengers of ¹O₂, O₂^–• and OH^• radicals and metal chelators influenced heat-induced 8-oxoguanine formation as they affected thermal ROS generation. These findings imply that heat acts via ROS attack leading to oxidative damage to DNA.     

Low growth temperature effects a differential inhibition of photosynthesis in spring and winter wheat

In vivo room temperature chlorophyll a fluorescence coupled with CO₂ and O₂ exchange was measured to determine photosynthetic limitation(s) for spring and winter wheat (Triticum aestivum L.) grown at cold-hardening temperatures (5°C/5°C, day/night). Plants of comparable physiological stage, but grown at nonhardening temperatures (20°C/16°C, day/night) were used in comparison. Winter wheat cultivars grown at 5°C had light-saturated rates of CO₂ exchange and apparent photon yields for CO₂ exchange and O₂ evolution that were equal to or greater than those of winter cultivars grown at 20°C. In contrast, spring wheat cultivars grown at 5°C showed 35% lower apparent photon yields for CO₂ exchange and 25% lower light-saturated rates of CO₂ exchange compared to 20°C grown controls. The lower CO₂ exchange capacity is not associated with a lower efficiency of photosystem II activity measured as either the apparent photon yield for O₂ evolution, the ratio of variable to maximal fluorescence, or the level of reduced primary quinone electron acceptor maintained at steady-state photosynthesis, and is most likely associated with carbon metabolism. The lower CO₂ exchange capacity of the spring cultivars developed following long-term exposure to low temperature and did not occur following over-night exposure of nonhardened plants to 5°C.     

Effect of Temperature on Composting of Sewage Sludge

The effect of temperature on the composting reaction of sewage sludge was investigated at 50, 60, and 70°C. The total amount of CO₂ evolved and the final conversion of volatile matter were maximum at 60°C., suggesting that the optimal temperature for composting was around 60°C. The specific CO₂ evolution rate (moles of CO₂ evolved per hour per viable cell) was maximum at 70°C. The isolated thermophilic bacterium which was dominant at 60°C but did not grow at 70°C showed that the rate of O₂ consumption measured on the agar plate at 70°C was four times higher than that at 60°C. This showed that the energy yielded from catabolism is rather uncoupled with the anabolism at 70°C in the metabolism of microorganisms indigenous in the compost. A higher respiratory quotient was observed at 70°C than at any other temperature.     

Dual action of respiratory inhibitors: inhibition of germination and prevention of dormancy induction in lettuce seeds

`Grand Rapids' lettuce Lactuca sativa L. seeds germinate readily at 15°C but poorly at 25°C in darkness. When held in dark at 25°C for an extended period, the ungerminated seeds become dormant as shown by their inability to germinate or transfer to 15°C in darkness. Induction of dormancy at 25°C was prevented by exposure to CN<sup>−</sup>, azide, salicylhydroxamic acid (SHAM), dinitrophenol, and pure N<sub>2</sub> as determined by subsequent germination at 15°C on removal of inhibitors. The effectiveness of inhibitors to break dormancy declined as dormancy intensified. At relatively low levels, CN<sup>−</sup>, SHAM, and azide promoted dark germination at 25°C while at high levels they were inhibitory. Uptake of O<sub>2</sub> by seeds held at 25°C for 4 days in 1.0 millimolar KCN was inhibited by 67% but was promoted 61% when KCN was removed. Correspondingly greater inhibition (79%) and promotion (148%) occurred when 1.0 millimolar SHAM was added to KCN solution. When applied alone, SHAM had little effect on O<sub>2</sub> uptake. These data indicate that Cyt pathway of respiration plays a dominant role in the control of both dormancy induction and germination of lettuce seeds, and `alternative pathway' is effectively engaged in presence of CN⁻. The channeling of respiratory energy use for processes governing germination or dormancy is subject to control by physical and chemical factors.

A scheme is proposed that illustrates compensatory use of energy for processes controlling dormancy induction and germination. A block of germination, e.g. by low water potential polyethylene glycol solution or a supraoptimal temperature spares energy to be utilized for dormancy induction while a block of dormancy induction by low levels of CN⁻ (similar to GA and light effects) drives germination. Blocking both processes by inhibitors (e.g. CN⁻, CN⁻ + SHAM) presumably leads to accumulation of `reducing power' with consequent improvement in O₂ uptake and oxidation rates of processes controlling germination or dormancy induction upon removal of the inhibitors.

     

Subtropical mouse-tailed bats use geothermally heated caves for winter hibernation

We report that two species of mouse-tailed bats (Rhinopoma microphyllum and R. cystops) hibernate for five months during winter in geothermally heated caves with stable high temperature (20°C). While hibernating, these bats do not feed or drink, even on warm nights when other bat species are active. We used thermo-sensitive transmitters to measure the bats’ skin temperature in the natural hibernacula and open flow respirometry to measure torpid metabolic rate at different ambient temperatures (T_a, 16–35°C) and evaporative water loss (EWL) in the laboratory. Bats average skin temperature at the natural hibernacula was 21.7 ± 0.8°C, and no arousals were recorded. Both species reached the lowest metabolic rates around natural hibernacula temperatures (20°C, average of 0.14 ± 0.01 and 0.16 ± 0.04 ml O₂ g⁻¹ h⁻¹ for R. microphyllum and R. cystops, respectively) and aroused from torpor when T_a fell below 16°C. During torpor the bats performed long apnoeas (14 ± 1.6 and 16 ± 1.5 min, respectively) and had a very low EWL. We hypothesize that the particular diet of these bats is an adaptation to hibernation at high temperatures and that caves featuring high temperature and humidity during winter enable these species to survive this season on the northern edge of their world distribution.     

Effect of temperature on electrolyte metabolism of isolated frog skin

A study is presented on the effect of temperature on unidirectional active ion transport, resting electrolyte equilibrium (electrolyte composition), and oxygen consumption in isolated frog skin. The aims were twofold: first, to find out whether the rate of active transport can be changed without affecting the Na⁺ and K⁺ balance of skin itself; second, to arrive at minimal ΔNa/ΔO₂ values by correlating quantitatively inhibition of active ion transport with inhibition of O₂ consumption. NaCl transport was maximal at 20°C. At 28° and at temperatures below 20°, rate of NaCl transport was diminished. In many instances NaCl transport was diminished in skins which maintained their normal Na⁺ and K⁺ content. In several cases, however, neither rate of transport nor resting electrolyte equilibrium was affected; in other cases, both were. O₂ consumption decreased when lowering the temperature over the range from 28 to 10°C. From a plot of log Q_OO2 against 1/T an activation energy of µ 13,700 cal. was calculated, valid for the range from 10 to 20°C. It appeared that µ was smaller for temperatures above 20°C. Working between 10 and 20°, it was found that, on the average, 4 to 5 equivalents of Na⁺ were transported for one mole of O₂ consumed in skins with undisturbed resting electrolyte equilibrium.     

Effect of growth temperature and temperature shifts on spinach leaf morphology and photosynthesis

The growth kinetics of spinach plants (Spinacia oleracea L. cv Savoy) grown at 5°C or 16°C were determined to allow us to compare leaf tissues of the same developmental stage rather than chronological age. The second leaf pairs reached full expansion at a plant age of 32 and 92 days for the 16°C and 5°C plants, respectively. Growth at 5°C resulted in an increased leaf area, dry weight, dry weight per area, and leaf thickness. Despite these changes, pigment content and composition, room temperature in vivo fluorescence, and apparent quantum yield and light-saturated rates of CO₂ exchange or O₂ evolution were not affected by the growth temperature. Furthermore, 5°C expanded leaves were found to be more resistant to photoinhibition at 5°C than were 16°C expanded leaves. Thus, it is concluded that spinach grown at low temperature is not stressed. However, shifting spinach leaves from 5°C to 16°C or from 16°C to 5°C for 12 days after full leaf expansion had occurred resulted in a 20 to 25% reduction in apparent quantum yields and 50 to 60% reduction in light saturated rates of both CO₂ exchange and O₂ evolution. This was not accompanied by a change in the pigment content or composition or in the room temperature in vivo fluorescence. It appears that leaf aging during the temperature shift period can account for the reduction in photosynthesis. Comparison of cold-hardened and non-hardened winter rye (Secale cereale L. cv Muskateer) with spinach by in vivo fluorescence indicated that rye is more sensitive to both short term and longer duration temperature shifts than is spinach. Thus, susceptibility to an abrupt temperature shift appears to be species dependent.     

THE VIOLOGEN INDICATORS

The tabulation gives the normal potentials of the various indicators at 30°C.; referred to the normal hydrogen electrode, the accuracy is estimated to be ±0.002 volt. Normal potentials of the viologens at 30°C.: Methyl viologen –0.446 volts Ethyl viologen –0.449 volts Betaine viologen –0.444 volts Benzyl viologen –0.359 volts Supposing some solution brings about a coloration of one of these indicators to the extent of A per cent of the maximum color, the oxidation-reduction potential of this solution is E = E_o – 0.06 log See PDF for Equation where E_o is the normal potential according to the above tabulation. This normal potential is independent of pH.     

Metabolic Depression in Cunner (Tautogolabrus adspersus) Is Influenced by Ontogeny,and Enhances Thermal Tolerance

To examine the effect of ontogeny on metabolic depression in the cunner (Tautogolabrus adspersus), and to understand how ontogeny and the ability to metabolically depress influence this species'' upper thermal tolerance: 1) the metabolic rate of 9°C-acclimated cunner of three size classes [0.2–0.5 g, young of the year (YOY); 3–6 g, small; and 80–120 g, large (adult)] was measured during a 2°C per day decrease in temperature; and 2) the metabolic response of the same three size classes of cunner to an acute thermal challenge [2°C h⁻¹ from 10°C until Critical Thermal Maximum, CT_Max] was examined, and compared to that of the Atlantic cod (Gadus morhua). The onset-temperature for metabolic depression in cunner increased with body size, i.e. from 5°C in YOY cunner to 7°C in adults. In contrast, the extent of metabolic depression was ∼80% (Q₁₀ = ∼15) for YOY fish, ∼65% (Q₁₀ = ∼8) for small fish and ∼55% (Q₁₀ = ∼5) for adults, and this resulted in the metabolic scaling exponent (b) gradually increasing from 0.84 to 0.92 between 9°C to 1°C. All size classes of cunner had significantly (approximately 60%) lower routine metabolic rates at 10°C than Atlantic cod. However, there was no species'' difference in the temperature-induced maximum metabolic rate, and this resulted in factorial metabolic scope values that were more than two-fold greater for cunner, and CT_Max values that were 6–9°C higher (∼21 vs. 28°C). These results: 1) show that ontogeny influences the temperature of initiation and the extent of metabolic depression in cunner, but not O₂ consumption when in a hypometabolic state; and 2) suggest that the evolution of cold-induced metabolic depression in this northern wrasse species has not resulted in a trade-off with upper thermal tolerance, but instead, an enhancement of this species'' metabolic plasticity.     

Temperature Effects on Day Old Drosophila Pupae

Day old Drosophila pupae were subjected to a variety of closely controlled temperature shocks. Twenty-five hours after puparium formation (at 23°), temperatures from 39.5–41.5° (Q₁ = 2.3) differentially disturb the formation of the posterior crossvein. Three other separate treatments disturb posterior crossvein formation: treatments in the range 36.0–37.0° at 25 hours; 37.3–37.8° at 25 hours; and 39.5–41.5° at 19 hours. Certain qualitative effects are associated with certain temperatures: elliptical holes are seen in wings of flies exposed 25 hours after puparium formation to temperatures from 37.3–37.8°. Anterior crossvein defects ensue if animals are similarly exposed to temperatures from 37.9–38.2°. Within the physiological range, animals raised at higher temperatures are more resistant to the effects of temperatures at 39.5–41.5°. An extremely rapid temperature adaptation by exposures to temperatures in the range 31–38° results in markedly greater resistance to heat shock; here resistance to production of crossvein defects increases faster than to death. The association between qualitative effects and treatment temperatures is modified by changing the temperature at which the animals spend their first day of pupal life. Summation experiments support conclusions drawn from the simpler experiments. Genetic variation and interspecific variation are discussed in the present context, as well as implications of the role of protein denaturation in the biological effects of high temperatures and further, more general experiments.     

Parallel Changes in H2O2 and Catalase during Thermotolerance Induced by Salicylic Acid or Heat Acclimation in Mustard Seedlings

Spraying mustard (Sinapis alba L.) seedlings with salicylic acid (SA) solutions between 10 and 500 μm significantly improved their tolerance to a subsequent heat shock at 55°C for 1.5 h. The effects of SA were concentration dependent, with higher concentrations failing to induce thermotolerance. The time course of thermotolerance induced by 100 μm SA was similar to that obtained with seedlings acclimated at 45°C for 1 h. We examined the hypothesis that induced thermotolerance involved H₂O₂. Heat shock at 55°C caused a significant increase in endogenous H₂O₂ and reduced catalase activity. A peak in H₂O₂ content was observed within 5 min of either SA treatment or transfer to the 45°C acclimation temperature. Between 2 and 3 h after SA treatment or heat acclimation, both H₂O₂ and catalase activity significantly decreased below control levels. The lowered H₂O₂ content and catalase activity occurred in the period of maximum thermoprotection. It is suggested that thermoprotection obtained either by spraying SA or by heat acclimation may be achieved by a common signal transduction pathway involving an early increase in H₂O₂.     

TEMPERATURE ACTIVATION OF THE UREASE-UREA SYSTEM USING CRUDE AND CRYSTALLINE UREASE

1. The hydrolysis of urea catalyzed by jack bean meal has been followed by determining colorimetrically after Nesslerization the ammonia nitrogen, and volumetrically the carbon dioxide liberated at successive intervals during the reaction. During the early part of hydrolysis the rate of ammonia or carbon dioxide liberation is constant for all the urease solutions which were used. 2. When log rate of NH₃ or CO₂ formation was plotted against 1/T, the points fell along a straight line, the slope of which corresponded to an activation energy of either 8,700 or 11,700 calories per gram mol. Frequently urease, when dissolved in sulfite solution, was characterized by an activation energy of 11,700 below and 8,700 above the critical temperature of about 23°C. At high temperatures the plotted points fell off from the curve due to temperature inactivation. 3. Essentially the same results on temperature activation were obtained with crude jack bean meal, Arlco urease, crystalline urease not recrystallized, and crystalline urease once recrystallized. The temperature characteristic which was obtained depended in part upon the composition of the medium. When dissolved in water, or aqueous solutions of glycerine, KCN, Na₂S₂O₂, cystine, Na₂SO₄, and K₄Fe(CN)₆, the temperature characteristic or µ of urease is 8,700. On the other hand, when urease is dissolved in solutions of K₃Fe(CN)₆ or H₂O₂ the µ value is 11,700. When dissolved in a solution containing Na₂SO₃ and NaHSO₃ the µ value may be either 8,700 or 11,700 over the whole temperature range, or 11,700 below and 8,700 above 23°C. 4. When crystalline urease is dissolved in varying mixtures of K₄Fe(CN)₆ and K₃Fe(CN)₆, the temperature characteristic depends upon the oxidation-reduction potential of the digest. When E_h is greater than +0.46 volt µ = 11,700, when less than +0.42 volt µ = 8,700, when between +0.42 – +0.46 µ = 11,700 below and 8,700 above the critical temperature. 5. It is suggested that in reducing or in indifferent solutions the configuration of the urease molecule (as determined especially by SH groups present) is such that the activation energy is 8,700 calories. In oxidizing solutions the urease molecule has been so altered (perhaps by the oxidation of the SH groups) as to be partly inactivated and now has an activation energy of 11,700. Such changes in the urease molecule are reversible (unless oxidation has proceeded too far) and are accompanied by a corresponding change in the activation energy.     

CARBON DIOXIDE PRODUCTION AND DURATION OF LIFE OF DROSOPHILA CULTURES

The total CO₂ produced by aseptic Drosophila cultures during the entire duration of life has been determined at 15°, 26°, and 30°C. in the dark and at 22–26°C. in the light. The total amount of CO₂ produced is not constant but is greater at 15° than at 26° or 30°, and is much greater in the light than in the dark. The total duration of life, therefore, is not determined by the time required to produce a limiting amount of CO₂.     

Permanganate Fixation of Plant Cells

In an evaluation of procedures explored to circumvent some of the problems of osmium tetroxide-fixation and methacrylate embedding of plant materials, excised segments of root tips of Zea mays were fixed for electron microscopy in potassium permanganate in the following treatment variations: unbuffered and veronal-acetate buffered solutions of 0.6, 2.0, and 5.0 per cent KMnO₄ at pH 5.0, 6.0, 6.7, and 7.5, and temperatures of 2–4°C. and 22°C. After fixation the segments were dehydrated, embedded in epoxy resin, sectioned, and observed or photographed. The cells of the central region of the rootcap are described. The fixation procedures employing unbuffered solutions containing 2.0 to 5.0 per cent KMnO₄ at a temperature of 22°C. gave particularly good preservation of cell structure and all membrane systems. Similar results were obtained using a solution containing 2.0 per cent KMnO₄, buffered with veronal-acetate to pH 6.0, and a fixation time of 2 hours at 22°C. The fixation procedure utilizing veronal-acetate buffered, 0.6 per cent KMnO₄ at 2–4°C. and pH 6.7 also gave relatively good preservation of most cellular constituents. However, preservation of the plasma membrane was not so good, nor was the intensity of staining so great, as that with the group of fixatives containing greater concentrations of KMnO₄. The other fixation procedures did not give satisfactory preservation of fine structure. A comparison is made of cell structures as fixed in KMnO₄ or OsO₄.     

Monomeric Amyloid Beta Peptide in Hexafluoroisopropanol Detected by Small Angle Neutron Scattering

Small proteins like amyloid beta (Aβ) monomers are related to neurodegenerative disorders by aggregation to insoluble fibrils. Small angle neutron scattering (SANS) is a nondestructive method to observe the aggregation process in solution. We show that SANS is able to resolve monomers of small molecular weight like Aβ for aggregation studies. We examine Aβ monomers after prolonged storing in d-hexafluoroisopropanol (dHFIP) by using SANS and dynamic light scattering (DLS). We determined the radius of gyration from SANS as 1.0±0.1 nm for Aβ_1–40 and 1.6±0.1 nm for Aβ_1–42 in agreement with 3D NMR structures in similar solvents suggesting a solvent surface layer with 5% increased density. After initial dissolution in dHFIP Aβ aggregates sediment with a major component of pure monomers showing a hydrodynamic radius of 1.8±0.3 nm for Aβ_1–40 and 3.2±0.4 nm for Aβ_1–42 including a surface layer of dHFIP solvent molecules.