Steric effects on activation energy for the electrochemical oxidation of organocobaloximes

Heterogeneous electron transfer rate constants were determined as a function of electrode potential for one-electron oxidation in acetonitrile (AN) at O °C of a series of organocobaloximes [R-Co(DH)₂L] bearing widely different organic groups. Reaction entropies were determined by voltammetric half-wave potential (E^r_{$\text{1}\text{2}$}) measurements in a non-isothermal cell. The electron transfer coefficients and reorganization parameters were calculated following the Marcus theory. The reaction free energies relative to a reference couple ΔG° are linearly correlated with the polar Taft constant of the organic substituent R.The steric effects on ΔG° are shown by the correlation of E^r_{$\text{sol1}\text{2}$} with the CoC bond distance.Assuming constancy of double layer effects along the series in the given solution composition, the trends of the apparent rate constants k_app were considered in order to evaluate the effects of the nature of the organic ligand on the activation energy ΔG³ of the electron transfer. The steric effects on ΔG³ are pointed out i.a. by consideration of the relationship between ΔG³ and ΔG°.     

The extrasplanchnic origin of blood dihydrotestosterone in elderly men

The splanchnic extraction and interconversion of testosterone (T) and dihydrotestosterone (DHT) were studied in 5 elderly men undergoing cardiac catheterization using a constant Infusion of [1,2-³H] testosterone and [4-¹⁴C] DHT. Metabolic clearance rate (MCR), splanchnic extraction (SE), splanchnic clearance (SC), extrasplanchnic clearance (ESC), transfer constant In blood ([P]_BB^T-DHT) and transfer constant across the liver ([P]_BB^T-DHT) were calc?ulated. The MCR^T was 675 ± 108 (mean ± SC) L/day and MCR^DHT was 409 ± 68 L/day. SE^T was 45.9 ± 7.0% and SE^DHT was 18.5 ± 5.4%. When these values are compared with those recently reported by us for normal men, there is a $\text{1}\text{3}$ reduction in SE^T and $\text{1}\text{2}$ reduction for SE^DHT in elderly men. The calculated SC^T and ESC^T were 355 ± 72 L/day and 320 ± 86 L/day, respectively. SC^DHT and ESC^DHT were 145 + 48 L/day and 263 ± 77 L/day respectively, suggesting that a major fraction of DHT is metabolized in extrasplanchnic organs. No evidence for a net appearance of DHT by either mass or specific activity analysis in hepatic vein blood was observed indicating that the splanchnic compartment does not contribute DHT into the circulation either by $\text{de novp}$ synthesis or via conversion from testosterone. This work indicates that conversion of testosterone to DHT in elderly men occurs entirely in extrasplanchnic tissue.     

Analysis of the kinetics of electron transfer reactions of hemoglobin and myoglobin with inorganic complexes.

The kinetics of methemoglobin reduction by Fe(EDTA)^2? have been studied and found to follow a second order rate law with k = 29.0 $\text{M}$^?1 s^?1 [25°C, μ = 0.2 $\text{M}$, pH 7.0 (phosphate)], $\text{ΔH}{}^3\text{= 5.5 ± 0.7 kcal/mol}$, and $\text{ΔS}{}^2\text{= ?33 ± 2 e.u.}$. The electrostatics-corrected self-exchange rate constant (k₁₁^corr) for hemoglobin based on the Fe(EDTA)^2? cross-reaction is 2.79×10^?3$\text{M}$^?1 s^?1. This rate constant is compared with others reported for a water-soluble iron porphyrin and calculated from published data for the reactions of myoglobin and hemoglobin with Fe(EDTA)^2? and Fe(CDTA)^2?/?. The k₁₁^corr values for these systems range over ten orders of magnitude with heme ? myoglobin > hemoglobin.     

Kinetic parameters for the binding of p-nitrophenyl alpha-d-mannopyranoside to concanavalin A.

Binding of the chromogenic ligand p-nitrophenyl α-d-mannopyranoside to concanavalin A was studied in a stopped-flow spectrometer. Formation of the protein-ligand complex could be represented as a simple one-step process. No kinetic evidence could be obtained for a ligand-induced change in the conformation of concanavalin A, although the existence of such a conformational change was not excluded. The entire change in absorbance produced on ligand binding occurred in the monophasic process monitored in the stopped-flow spectrometer. The value of the apparent second-order rate constant (k_a) for complex formation (k_a = 54,000 s^?1m^? at 25 °C, pH 5.0, Γ/2 0.5) was independent of the protein concentration when the protein was in the range of 233–831 μm in combining sites and in excess of the ligand. The apparent first-order rate constant (k_?a) for dissociation of the complex was obtained from the rate constant for the decomposition of the complex upon the addition of excess methyl α-d-mannopyranoside (k_?a = 6.2 s^?1 at 25 °C, pH 5.0, Γ/2 0.5). The ratio $\text{k}{}_{\mathrm{<mtext>a</mtext>}}{}_{\mathrm{<mtext>?</mtext><mtext>a</mtext>}}$ (0.9 × 10₄m^?1) was in reasonable agreement with value of 1.1 ± 0.1 × 10⁴m^?1 determined for the equilibrium constant for complex formation by ultraviolet difference spectrometry. Plots of ln($\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{T}$) and ln($\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{T}$) vs $\text{1}\text{T}$ were linear (T is temperature) and were used to evaluate activation parameters. The enthalpies of activation for formation and dissociation of the complex are 9.5 ± 0.3 and 16.8 ± 0.2 kcal/mol, respectively. The unitary entropies of activation for formation and dissociation of the complex are 2.8 ± 1.1 and 1.3 ± 0.7 entropy units, respectively. These entropy changes are much less than those usually associated with substantial changes in the conformation of proteins.     

Kinetics of testosterone metabolism in normal postmenopausal women and women with breast cancer

The constant infusion and single injection techniques were utilized to study the kinetics of ³H-testosterone (T) metabolism in postmenopausal women with and without breast cancer. The metabolic clearance rates (mean ± SEM) for normal postmenopausal women were $\text{578 ± 82}\text{and}\text{644 ±128}\text{1}\text{24}\text{h}$ as obtained by the constant infusion and single injection techniques, respectively. The corresponding results for the women with breast cancer (patients) are $\text{644 ± 25}\text{and}\text{617 ± 106}\text{1}\text{24}\text{h}$. The single injection technique yielded values for rate constants (units) and volumes of distribution (1); k₁ = 37.5 ± 1.6 for the normals and 34.5 ±1.9 for the patients. K₂ = 76.6 ± 5.1 for the normals and 71.1 ± 1.6 for the patients, V₁ = 7.9 ± 2.2 for the normals and 8.7 ± 1.4 for the patients and V₂ = 7.0 ± 1.5 for the normals and 6.4 ± 1.2 for the patients. The constant infusion technique yielded values for the conversion ratios for the transformation of T to several products; 4-androstene-3,17-dione/T of 0.02 ± 0.003 for normals and 0.03± 0.002 for patients, 5α-dihydrotestosterone/T of 0.02 ± 0.002 for normals and 0.03 ± 0.002 for patients, estrone/T of 0.04 ± 0.01 for normals and 0.04 ± 0.01 for patients, estradiol-17β/T of 0.02 ± 0.005 for normals and 0.03 ± 0.005 for patients and estrone sulfate/T of O.16 ± 0.02 for normals and 0.24 ± 0.06 for patients. The T plasma concentrations and production rates were similar for the two groups of subjects. Hence there were no significant differences between the normals and the patients for all the kinetic parameters. It was determined that all the estradiol being produced in postmenopausal women could be coming from circulating T.     

New experimental approach to the estimation of rate of electron transfer from the primary to secondary acceptors in the photosynthetic electron transport chain of purple bacteria

A method for calculating the rate constant ($\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$) for the oxidation of the primary electron acceptor (A₁) by the secondary one (A₂) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: $\text{(4.5±1.4) · 10}{}^3\text{s}{}^{\mathrm{?1}}$ and $\text{(6.9±1.2) · 10}{}^3\text{s}{}^{\mathrm{?1}}$, respectively.The proposed method has also been used for the estimation of the $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P⁺ at a rate exceeding this for the transfer of electron from A₁ to A₂. The method of Parson cannot be used in this case. The value of $\text{K}{}_{\mathrm{<mtext>A</mtext><mtext>1</mtext><mtext>A</mtext><mtext>2</mtext>}}$ has been found to be $\text{(2.7±0.8) · 10}{}^3\text{s}{}^{\mathrm{?1}}$.The activation energies for the A₁ to A₂ electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively.     

Acid dissociation of cyclohexaamylose and cycloheptaamylose

Acid dissociation constants of aqueous cyclohexaamylose (6-Cy) and cycloheptaamylose (7-Cy) have been determined at 10–47 and 25–55°C, respectively, by pH potentiometry. Standard enthalpies and entropies of dissociation derived from the temperature dependences of these pK_a's are ΔH⁰ = 8.4 ± 0.3 kcal mol^?1, $\text{Δ}\text{S}{}^0\text{= ?28. ± 1}\text{cal mol}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$ for 6-Cy and ΔH⁰ = 10.0 ± 0.1 kcal mol^?1, $\text{Δ}\text{S}{}^0\text{= ?22.4 ±0.3}\text{cal mol}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$ for 7-Cy. Intrinsic ¹³C nmr resonance displacements of anionic 6- and 7-Cy were measured at 30°C in 5% D₂O ($\text{v}\text{v}$). These results indicate that the dissociation of 6- and 7-Cy involves both C2 and C3 2⁰-hydroxyl groups. The thermodynamic and nmr parameters are discussed in terms of interglucosyl hydrogen bonding.     

Hydrogen bonding requirements for the insulin-sensitive sugar transport system of rat adipocytes

(1) The $\text{t}\text{1}\text{2}$ for 1.3 mM D-allose uptake and efflux in insulin-stimulated adipocytes is 1.7 ± 0.1 min. In the absence of insulin mediated uptake of D-allose is virtually eliminated and the uptake rate ($\text{t}\text{1}\text{2}\text{= 75.8 ± 4.99}\text{min}$) is near that calculated for nonmediated transport. The kinetic parameters for D-allose zero-trans uptake in insulin-treated cells are K_zt^oi = 271.3 ± 34.2 mM, V_zt^oi = 1.15 ± 0.12 mM · s^?1. (2) A kinetic analysis of the single-gate transporter (carrier) model interacting with two substrates (or substrate plus inhibitor) is presented. The analysis shows that the heteroexchange rates for two substrates interacting with the transporter are not unique and can be calculated from the kinetic parameters for each sugar acting alone with the transporter. This means that the equations for substrate analogue inhibition of the transport of a low affinity substrate such as D-allose can be simplified. It is shown that for the single gate transporter the K_i for a substrate analogue inhibitor should equal the equilibrium exchange K_m for this analogue. (3) Analogues substituted at C-1 show a fused pyranose ring is accepted by the transporter. 1-Deoxy-D-glucose is transported but has low affinity for the transporter. High affinity can be restored by replacing a fluorine in the β-position at C-1. The K_i for $\text{d}\text{-glucose}\text{= 8.62}\text{mM}$; the K_i for $\text{β-}\text{fluoro-}\text{d}\text{-glucose}\text{= 6.87}\text{mM}$. Replacing the ring oxygen also results in a marked reduction in affinity. The K_i for $\text{5-}\text{thio-}\text{d}\text{-glucose}\text{= 42.1}\text{mM}$. (4) A hydroxyl in the gluco configuration at C-2 is not required as 2-deoxy-d-galactose (K_i = 20.75 mM) has a slightly higher affinity than d-galactose (K_i = 24.49 mM). A hydroxyl in the manno configuration at C-2 interferes with transport as d-talose (K_i = 35.4 mM) has a lower affinity than d-galactose. (5) d-Allose (K_m = 271.3 mM) and 3-deoxy-d-glucose (K_i = 40.31 mM) have low affinity but high affinity is restored by substituting a fluorine in the gluco configuration at C-3. The K_i for $\text{3-}\text{fluoro-}\text{d}\text{-glucose}\text{= 7.97}\text{mM}$. (6) Analogues modified at C-4 and C-6 do not show large losses in affinity. However, 6-deoxy-d-glucose (K_i = 11.08 mM) has lower affinity than d-glucose and 6-deoxy-d-galactose K_i = 33.97 mM) has lower affinity than d-galactose. Fluorine substitution at C-6 of d-galactose restores high affinity. The K_i for $\text{6-}\text{fluoro-}\text{d}\text{-galactose}\text{= 6.67}\text{mM}$. Removal of the C-5 hydroxymethyl group results in a large affinity loss. The K_i$\text{d}\text{-xylose}\text{= 45.5}\text{mM}$. The K_i for $\text{l}\text{-arabinose}\text{= 49.69}\text{mM}$. (7) These results indicate that the important hydrogen bonding positions involved in sugar interaction with the insulin-stimulated adipocytes transporter are the ring oxygen, C-1 and C-3. There may be a weaker hydrogen bond to C-6. Sugar hydroxyls in non-gluco configurations may sterically hinder transport.     

Stopped-flow kinetics of the interaction of the fluorescent calcium indicator Quin 2 with calcium ions

The kinetics of the Quin 2-Ca²⁺ interaction have been studied using stopped-flow fluorimetry. Mixing the Quin 2-Ca²⁺ complex with a large excess of EGTA, EDTA or MgCl₂ resulted in first order dissociation kinetics. The observed dissociation rate increased slightly with increasing EGTA concentration yielding a limiting value of 83±4 s^?1 for the dissociation rate constant (k_?) at pH 7.2, 37°C, ± 3mM Mg²⁺. The temperature dependence of the dissociation was weak (activation energy = 22±1 kJ/mol) and around neutral pH the pH dependence was negligible. The association reaction was too fast to be monitored directly. From this and the instrument dead-time, the second order rate constant k₊ was estimated to be ≥10⁹ M^?1s^?1, in agreement with the calculation from $\text{k}{}_{\mathrm{+}}\text{=}\text{k}{}_{\mathrm{?}}\text{K}$. These data should be useful in evaluating the potential of Quin 2 to measure fast intracellular Ca²⁺ transients.     

Calorimetric measurement of stepwise enthalpy changes for the binding of four oxygens to adult human hemoglobin

The successive enthalpy changes for the four steps of oxygen binding by diphosphoglycerate-free adult human hemoglobin have been measured by direct calorimetry at pH 7.4 and 6°. Average results in kcal/(mole O₂) are: ΔH₁ = ?25.1 ± 2.8; ΔH₂ = ?12.6 ± 3.0, ΔH₃ = ?12.5 ± 3.0, and ΔH₄ = ?10.1 ± 1.4. These results imply a substantial temperature dependence for the cooperativity of O₂ binding by the protein and generally resemble the van't Hoff results by Roughton $\text{et al}$. [Roy. Soc. of London Proc., $\text{B 144}$, 29 (1955)] for sheep hemoglobin at pH 9.1 and a temperature range of 2° to 19°.     

Properties of [3H]diazepam binding sites on rat blood platelets

Intact rat blood platelets are shown to possess benzodiazepine binding sites of the peripheral type, binding of [³H]diazepam being strongly inhibited by Ro5-4864 (K_i = 3.6 ± 0.5 nM) but only weakly inhibited by clonazepam (K_i = 35.1 ± 18.2 μM). Binding of [³H]diazepam is specific and saturable. Scatchard analysis reveals a single class of binding sites with K_D = 14.7 ± 1.0 nM and B_max = 564 ± 75 fmoles/10⁸ platelets. The Hill coefficient is 0.94, indicating a lack of binding site heterogeneity or negative cooperativity. Binding reaches equiliibrium at 6 min, with k₊₁ = 2.9 × 10⁷ M^?1 min^?1, and is rapidly reversible ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>\; =\; 2.2\; </mtext><mtext>min</mtext>}}$ with K_?1 = 0.315 min^?1. K_D derived from the rate constants agrees with that estimated by Scatchard analysis. K_D of the crude membrane fraction of platelets is also close to that of intact platelets. Binding of [³H]diazepam is linear with platelet number (between 0.25–2 × 10⁸ platelets), is temperature sensitive with maximum binding at 0°C, and has a broad optimal pH range between pH 5–9.     

Structure of a bacteriochlorophyll-protein from the green photosynthetic bacterium Chlorobium limicola: crystallographic evidence for a trimer

Crystals of the bacteriochlorophyll-protein from the green photosynthetic bacterium Chlorobium limicola, previously described by J. M. Olson et al. (1969), have been re-examined by X-ray diffraction. The space group is P6₃, as reported in the earlier work, but revised cell dimensions, $\text{a = b = 112.4 ± 0.4}\text{A}\text{?}\text{, c = 98.4 ± 0.4}\text{A}\text{?}$, were obtained, leading to a unit cell volume one third of that reported previously. Correction of this error leads to the conclusion that the bacteriochlorophyll-protein complex must be a trimer consisting of three identical subunits arranged about a crystallographic symmetry axis. Also a new trigonal crystal form of the bacteriochlorophyll-protein has been obtained, and is consistent only with a molecule composed of three identical or near-identical subunits. Models of the molecular packing for both crystal forms are presented.The molecular weight of the bacteriochlorophyll-protein complex, determined from crystal density measurements, is (1.53 ± 0.23) × 10⁵, and the overall molecular dimensions are about 55 Å along the trimer axis, and 83 Å at right angles to this. There are probably seven bacteriochlorophyll molecules in each subunit.     

Spatial requirements for insulin-sensitive sugar transport in rat adipocytes

(1) Alkyl sugar inhibition of d-allose uptake into adipocytes has been used to explore the spatial requirements of the external sugar transport site in insulin-treated cells. α-methyl and β-methyl glucosides show low affinity indicating very little space around C-1. The high affinity of d-glucosamine (K_i = 9.05 ± 0.66 mM) is lost by N-acetylation. $\text{N-}\text{Acetyl-}\text{d}\text{-glucosamine}$ shows no detectable affinity, indicating that a bulky group at C-2 is not accepted. Similarly $\text{2,3-}\text{di}\text{-O-}\text{methyl-}\text{d}\text{-glucose}$ (K_i = 42.1 ± 7.5 mM) has lower affinity than $\text{3-O-}\text{methyl-}\text{d}\text{-glucose}$ (K_i = 5.14 ± 0.32 mM) indicating very little space around C-2 but much more around C-3. A reduction in affinity does occur if a propyl group is introduced into the C-3 position. The K_i for $\text{3-O-}\text{propyl-}\text{d}\text{-glucose}$ is 11.26 ± 2.12 mM. $\text{6-O-}\text{Methyl-}\text{d}\text{-galactose}$ (K_i = 87.2 ± 17.9 mM) and $\text{6-O-}\text{propyl-}\text{d}\text{-glucose}$ (K_i = 78.07 ± 12.6 mM) show low affinity compared with d-galactose and d-glucose, indicating steric constraints around C-6. High affinity is restored in $\text{6-O-}\text{pentyl-}\text{d}\text{-galactose}$ (K_i = 4.66 ± 0.23 mM) possibly indicating a hydrophobic binding site around C-6). (2) In insulin treated cells $\text{4,6-O-}\text{ethylidene-}\text{d}\text{-glucose}$ (K_i = 6.11 ± 0.5 mM) and maltose (K_i = 23.5 ± 2.1 mM) are well accommodated by the site but trehalose shows no detectable inhibition. These results indicate that the site requires a specific orientation of the sugar as it approaches the transporter from the external solution. C-1 faces the inside while C-4 faces the external solution. (3) To determine the spatial and hydrogen bonding requirements for basal cells 40 μM $\text{3-O-}\text{methyl-}\text{d}\text{-glucose}$ was used as the substrate. Poor hydrogen bonding analogues and analogues with sterically hindering alkyl groups showed similar K_i values to those determined for insulin-treated cells. These results indicate that insulin does not change the specificity of the adipocyte transport system.     

Experimental determination of the redox potential of the superoxide radical O 2

The redox potential of ^?O₂^? was determined based on the dependence of the electron transfer reaction from ^?O₂^? upon the known redox potential of various acceptors A (including a range of quinones, dyes and ferricyanide). The efficiencies and the rates of these electron transfer processes were determined, using the technique of pulse radiolysis, by monitoring the formation kinetics of the semiquinone radical anions at the appropriate wavelength. From the percentage efficiency versus E^o′ plot, an E^o′ value of + 0.15 ± 0.01 V at pH 7.0 and 22°C, or E^o = + 0.57 ± 0.01 V, for the ${}^{\mathrm{?}}\text{O}{}_2{}^{\mathrm{?}}\text{O}{}_2$ couple was obtained. The rate k(^?O₂^? + A → O₂ + ^?A^?) = 9.8 × 10⁸ M^?1 sec^?1 where A = p-benzoquinone. The E^o value for the ^?HO₂ radical is > 1.0 V. It was also found that hydroquinone can quantitatively reduce ^?O₂^? to H₂O₂, k(^?O₂^? + QH₂ → QH^? + HO₂^?) = 1.6 ± 0.1 × 10⁷ M^?1 sec^?1 at pH 7.0 and 22°C.     

Direct electrochemical oxidation of Clostridium pasteurianum ferredoxin: Identification of facile electron-transfer processes relevant to cluster degradation

Rapid oxidation processes relevant to the degradation of [4Fe4S] clusters in Clostridium pasteurianum ferredoxin were studied via direct (unmediated) heterogeneous electron transfer at a pyrolytic graphite electrode. Differential-pulse voltammograms of native [4Fe4S] ferredoxin showed two well-defined oxidation peaks corresponding to apparent E-values of +793 and +1120 mV at 5°C. Direct involvement of the cluster was established through parallel experiments with the 2[4Fe4Se] derivative for which peak positions were shifted. Square-wave voltammetry showed that the product of the first electron transfer, which may correspond to the ‘super-oxidised’ [4Fe4S]³⁺ oxidation level, undergoes rapid degradation (t_{$\text{1}\text{2}$} < 1.6 ms at 5°C). The second oxidation process, as characterised by a significant (?100 mV) negative shift upon selenium substitution, very likely represents oxidation of S(Se) still associated with the protein and possibly contained within the remaining FES(Se) substructure.     

Phase transition properties of aqueous dispersions of homologues of all-trans 2,3-dipalmitoylcyclopentano-1-phosphocholine

In a previous publication, (Singer, M.A., Jain, M.K., Sable, H.Z., Pownall, H.H., Mantulin, W.W., Lister, M.D. and Hancock, A.J. (1983) Biochim. Biophys. Acta, 731, 373–377), we reported the properties of aqueous dispersions of the six diastereo-isomers of cyclopentanoid analogues of dipalmitoylphosphatidylcholine. Two of these isomers displayed unusually high enthalpies of transition, about double that of dipalmitoylphosphatidylcholine. One of the high enthalpy isomers whose configuration is all-trans has now been modified by the insertion of extra methylene residues ($\text{n = 3}$ through 9) between the nitrogen and phosphorus atoms of the headgroup. Vesicles were formed from these lipids and studied by ²²Na permeability measurements, differential scanning calorimetry, fluorescence polarization, ³¹P-NMR, and freeze-fracture electron microscopy. Vesicles composed of lipids with $\text{n = 2}$ or 3 exhibit a sharp transition at 46°C or 49°C, respectively, and a high enthalpy with no detectable sub- or pretransitions. Lipids with $\text{n > 3}$ exhibit a main transition between 38 and 43°C with enthalpies $\text{< 10}\text{kcal/mol}$ and after prolonged cooling (more than 3 days at 4°C) a broad endotherm at about $\text{20 ± 3°}\text{C}$ with enthalpies $\text{> 4}\text{kcal/mol}$. These same dispersions display a permeability peak at 20–25°C and a second increase in ²²Na efflux in the temperature range 30–40°C. The results of ³¹P-NMR measurements suggest that the acyl chains in 2,3-dipalmitoylcyclopentanol-phosphocholine ($\text{n = 2}$) bilayers have restricted rotation below the main phase transition temperature.     

Influence of temperature on Photosystem II electron transfer reactions

The influence of temperature on the rate of reduction of P-680⁺, the primary donor of Photosystem II, has been studied in the range 5–294 K, in chloroplasts and subchloroplasts particles. P-680 was oxidized by a short laser flash. Its oxidation state was followed by the absorption level at 820 nm, and its reduction attributed to two mechanisms: electron donation from electron donor D₁ and electron return from the primary plastoquinone (back-reaction).Between 294 and approx. 200 K, the rate of the back-reaction, on a logarithmic scale, is a linear function of the reciprocal of the absolute temperature, corresponding to an activation energy between 3.3 and 3.7 kcal · mol^?1, in all of the materials examined (chloroplasts treated at low pH or with Tris; particles prepared with digitonin). Between approx. 200 K and 5 K the rate of the back-reaction is temperature independent, with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.6}\text{ms}$. In untreated chloroplasts we measured a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.7 ms for the back-reaction at 77 and 5 K.The rate of electron donation from the donor D₁ has been measured in darkadapted Tris-treated chloroplasts, in the range 294–260 K. This rate is strongly affected by temperature. An activation energy of 11 kcal · mol^?1 was determined for this reaction.In subchloroplast particles prepared with Triton X-100 the signals due to P-680 were contaminated by absorption changes due to the triplet state of chlorophyll a. This triplet state has been examined with pure chlorophyll a in Triton X-100. An Arrhenius plot of its rate of decay shows a temperature-dependent region (292–220 K) with an activation energy of 9 kcal · mol^?1, and a temperature-independent region (below 200 K) with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1}\text{ms}$.     

Photoaffinity labelling by S-(p-azidophenacyl)-glutathione of glyoxalase II and glutathione S-transferase

S-(p-azidophenacyl)-glutathione, $\text{l}$, is a linear competitive inhibitor at pH 7.40 of beef liver glyoxalase II with K_i = 7.96 × 10^?4 M. On irradiation at 340 nm it covalently inhibits glyoxalase II to a level of 42 ± 5% inhibition. This photoaffinity labelling is prevented by the presence of a glyoxalase II competitive inhibitor (the hemimercaptal of glutathione and methylglyoxal). A crude preparation of sheep liver glutathione S-transferases is also irreversibly inactivated (86% ± 5% inhibition) by irradiation at 320 nm in the presence of $\text{l}$.     

Laser-light scattering investigation of the micellar properties of gangliosides

The micellar properties of gangliosides in water solutions were investigated by quasielastic light scattering measurements. G_M1 and G_D1a gangliosides were isolated from calf brain, purified to more than 99% and dissolved in 0.025 M Tris—HCI buffer (pH 6.8) at 37°C. The average intensity of scattered light and the intensity correlation function were measured by an apparatus including a 5145 Å argon laser and a real-time digital correlator. The scattered intensity data allowed the derivation of an upper limit to the critical micelle concentration (c₀) and the evaluation of the molecular weight (M) of the micelle. The intensity correlation function gave the diffusion coefficient D, and hence the hydrodynamic radius R_H, and also contained information on the polydispersity of the sample. We find c_o < 1 × 10^?6 M for both G_M1 and G_D1a, M = 532 000 ± 50 000 and $\text{R}{}_{\mathrm{<mtext>H</mtext>}}\text{= 63.9 ± 2}\text{A}\text{?}$ for G_M1, and M = 417 000 ± 40 000 and $\text{R}{}_{\mathrm{<mtext>H</mtext>}}\text{= 59.5 ± 2}\text{A}\text{?}$ for G_D1a. The mixture 3:1 of the two gangliosides gave intermediate values for all examined parameters. The presence of cations, within the physiological concentration range. and, in particular of Ca²⁺, did not influence significantly the values of c_o and the main features of the micelle.