Separation of solubilized alpha and beta adrenergic receptors by affinity chromatography.

Isoelectric focusing in the presence of Nonidet P-40 splits human chromatographically pure γ globin chains into two bands of isoelectric points 6.95 and 6.85, respectively. The comparison of the relative proportions of the two bands with the ratios between the G_γ and A_γ non allelic chains of human fetal hemoglobin suggests that the band at pI 6.95 corresponds to G_γ and the band at pI 6.85 corresponds to the A_γ chain; the latter is the only band present in a patient with Greek type hereditary persistence of fetal hemoglobin, producing only A_γ chains. Fluorography of electrofocusing-separated radioactive γ globin chains synthesized by thalassemic reticulocytes indicates that the relative $\text{G}{}_{\mathrm{\gamma }}\text{A}{}_{\mathrm{\gamma }}$ synthetic ratios are similar to the relative amounts of G_γ and A_γ chains accumulated in the erythrocytes, suggesting that the activities for the G_γ and A_γ mRNAs decay at roughly similar rates.     

Gibberellin biosynthesis in a cell-free system from immature seeds of Pisum sativum

A cell-free system from immature pea seeds converts ¹⁴C-labelled $\text{ent}$-kaurene to $\text{ent}$-kaurenol, $\text{ent}$-kaurenal, $\text{ent}$-kaurenoic acid, $\text{ent}$-7α-hydroxykaurenoic acid, and gibberellin A₁₂-aldehyde. The latter becomes converted further to 13-hydroxygibberellin A₁₂, gibberellin A44, gibberellin A₁₂-alcohol, and several unidentified products. Thus the biosynthesis of gibberellins $\text{via ent}$-kaurene is now established for a member of the $\text{Leguminosae}$. It is the first time that 13-hydroxylation of gibberellins has been observed in a cell-free system and that gibberellin A₁₂-alcohol has been obtained in any biological system.     

Crystal structure analysis of sea lamprey hemoglobin at 2 Å resolution

The crystal structure of the predominant hemoglobin component of blood from the sea lamprey, Petromyzon marinus, has been determined by X-ray diffraction analysis. Crystals for this analysis were grown from cyanide methemoglobin V as crystal type D₂. These crystals are in space group P2₁2₁2₁ and have unit cell dimensions of $\text{a = 44.57}\text{A}\text{?}\text{, b = 96.62}\text{A}\text{?}\text{and}\text{c = 31.34}\text{A}\text{?}$. Isomorphous heavyatom derivatives were prepared by soaking crystals in solutions of Hg(CN)₂, K₂Hg(CNS)₄ and KAu(CN)₂. Diffracted intensities to as far as 2 Å spacings were measured on a diffractometer. Phases were found by means of the isomorphous replacements and anomalous scattering, with supplementary information provided by the tangent formula. An atomic model was fitted to the final electron density map in a Richards optical comparator. The lamprey hemoglobin molecule is generally similar in structure to other globins, but differs in many details. Each molecule is in contact with ten neighboring molecules in the crystal lattice. The nature of the binding of the heavy atoms to lamprey hemoglobin has been interpreted.     

Preliminary crystallographic study of omega-amino acid: pyruvate aminotransferase from Pseudomonas sp. F-126.

Large single crystals of ω-amino acid: pyruvate aminotransferase, were prepared by dialysis of the enzyme solution against 2.2 m-ammonium sulphate solution at pH 7.8. X-ray diffraction patterns show that the crystals belong to the orthorhombic space group I222 or I2₁2₁2₁ with unit cell dimensions $\text{a = 124.1}\text{A}\text{?}\text{, b = 137.9}\text{A}\text{?}\text{, and}\text{c = 61.2}\text{A}\text{?}$. The asymmetric unit consists of one monomer of molecular weight 43,000.     

Evaluation of the water environments in deoxygenated sickle cells by longitudinal and transverse water proton relaxation rates

The longitudinal and transverse water proton relaxation rates of oxygenated and deoxygenated erythrocytes from both normal adults and individuals with sickle cell disease were measured as a function of temperature at two different frequencies. The simplest model which fits all of the data consists of three different environments for water molecules. The majority of the water (98%) has a correlation time indistinguishable from bulk water (3 × 10^?11 sec). Secondly, there is a small amount of water (1.3–1.5%) present which has a correlation time of 2–4 × 10 ^?9 sec and is apparently independent of the erythrocyte sample studied. Presumably this water is the hydration sphere around the hemoglobin molecules and its correlation time is significantly slower than bulk water. The third environment contains approximately 0.2% of the water present and has a correlation time≥ 10^?7 sec. This third environment is considered tightly bound to the hemoglobin because the water proton correlation time is very similar to the expected rotational correlation time for the hemoglobin molecules. The value of the transverse relaxation rate, f_b(T_2b)^?1, for the tightly bound water fraction decreases in oxy ($\text{S}\text{S}$), deoxy ($\text{A}\text{A}$), and oxy ($\text{A}\text{A}$) erythrocyte samples as the temperature is increased as expected for a rotational correlation time process. In dramatic contrast,f_b (T_2b)^?1 increases almost linearly as the temperature is increased over the whole 4 ° to 37 °C temperature range in samples of deoxy ($\text{S}\text{S}$) erythrocytes. The observation suggests a continual increase in the formation of deoxyhemoglobulin S polymers rather than a sudden transition from a homogeneous solution of deoxyhemoglobin S molecules to a solid gel.     

The reduction of the oxygen-evolving system in chloroplasts by thylakoid components

Using thoroughly dark-adapted thylakoids and an unmodulated Joliot-type oxygen electrode, the following results were obtained. (i) At high flash frequency (4 Hz), the oxygen yield at the fourth flash (Y₄) is lower compared to Y₃ than at lower flash frequency. At 4 Hz, the calculated S₀ concentration after thorough dark adaptation is found to approach zero, whereas at 0.5 Hz the apparent $\text{S}{}_0\text{(}\text{S}{}_0\text{+}\text{S}{}_1\text{)}$ ratio increases to about 0.2. This is explained by a relatively fast donation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.0–1.5}\text{s}$) of one electron by an electron donor to S₂ and S₃ in 15–25% of the Photosystem II reaction chains. The one-electron donor to S₂ and S₃ appears to be rereduced very slowly, and may be identical to the component that, after oxidation, gives rise to ESR signal II_s. (ii) The probability for the fast one-electron donation to S₂ and S₃ has nearly been the same in triazine-resistant and triazine-susceptible thylakoids. However, most of the slow phase of the S₂ decay becomes 10-fold faster ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 5–6}\text{s}$) in the triazine-resistant ones. In a small part of the Photosystem II reaction chains, the S₂ decay was extremely slow. The S₃ decay in the triazine-resistant thylakoids was not significantly different from that in triazine-susceptible thylakoids. This supports the hypothesis that S₂ is reduced mainly by Q^?_A, whereas S₃ is not. (iii) In the absence of CO₂/HCO^?_A and in the presence of formate, the fast one-electron donation to S₂ and S₃ does not occur. Addition of HCO^?₃ restores the fast decay of part of S₂ and S₃ to almost the same extent as in control thylakoids. The slow phase of S₂ and S₃ decay is not influenced significantly by CO₂/HCO^?₃. The chlorophyll a fluorescence decay kinetics in the presence of DCMU, however, monitoring the Q^?_A oxidation without interference of Q_B, were 2.3-fold slower in the absence of CO₂/HCO^?₃ than in its presence. (iv) An almost 3-fold decrease in decay rate of S₂ is observed upon lowering the pH from 7.6 to 6.0. The kinetics of chlorophyll a fluorescence decay in the presence of DCMU are slightly accelerated by a pH change from 7.6 to 6.0. This indicates that the equilibrium Q^?_A concentration after one flash is decreased (by about a factor of 4) upon changing the pH from 7.6 to 6.0. When direct or indirect protonation of Q^?_B is responsible for this shift of equilibrium Q^?_A concentration, these data would suggest that the pK_a value for Q^?_B protonation is somewhat higher than 7.6, assuming that the protonated form of Q^?_B cannot reduce Q_A.     

Separation and comparison of primary structures of three formylmethionine tRNAs from E. coli K-12 MO

Three chromatographically distinct tRNAs^fMet from $\text{E. coli}$ K-12 MO were separated by reversed-phase chromatography and designated tRNA_A^fMet, tRNA_B^fMet, and tRNA₃^fMet. The tRNA_A^fMet corresponds to the published sequence for tRNA^fMet ($\text{E. coli}$). The tRNA_B^fMet differs from tRNA_A^fMet in that the 4-thiouridine in nucleotide position 8 has interacted with cytidine in position 13 to form a cross-linked product. The tRNA₃^fMet differs from tRNA_A^fMet in that 7-methyl-guanosine (in position 47) has been replaced by adenosine.     

Diffusion of tetracycline across the outer membrane of Escherichia coli K-12: involvement of protein Ia.

The possibility that passage of tetracycline across the outer membrane of $\text{E.}\text{coli}$ K-12 is controlled by one or more of the proteins I_a, I_b and II¹ (Henning's nomenclature) was investigated. A mutant lacking protein I_a (obtained by selection for resistance to phage TuI_a) was more resistant to tetracycline than wild-type strains or those lacking only proteins I_b or II¹. The envelope protein composition of a tetracycline-resistant mutant ($\text{cmlB}$) was altered in several respects, but the major change involved loss of protein I_a. These data support our previous suggestion [12] that tetracycline diffuses across the outer membrane through hydrophilic regions. Furthermore, they imply that only protein I_a plays a significant role in the passage of this antibiotic across the outer membrane.     

Induction of erythroid differentiation in Friend murine erythroleukemic cells by inorganic selenium compounds.

Inorganic selenium compounds are shown to be inducers of hemoglobin synthesis in malignant murine erythroleukemia (MEL) cells. SeO₂ can induce hemoglobin synthesis at $\text{1}\text{20}$ the concentration of butyric acid and $\text{1}\text{5000}$ the concentration of dimethylsulfoxide (DMSO), two potent inducers of erythroid differentiation in MEL cells. SeO₂ and H₂SeO₃ showed an equivalent capacity to stimulate hemoglobin synthesis in three different MEL cell lines. The incorporation of ³H-glycine into hemoglobin was demonstrated in lysates of SeO₂-induced MEL cells.     

Synthesis of 3,4-13C2 steroids

A-ring enollactones $\text{1a}$, $\text{1b}$ or $\text{9}$ derived from 4-cholesten-3-one, testosterone benzoate or 3-oxo-4-estren-17β-yl benzoate were condensed with [1,2-¹³C₂]acetyl chloride to give intermediates $\text{2a}$, $\text{2b}$ or $\text{10}$. $\text{2a}$ and $\text{2b}$ were cyclized by acid or base to give 3,4-¹³C₂-labeled 4-cholesten-3-one and testosterone, respectively. [3,4-¹³C₂]4-Cholesten-3-one was converted via reduction of its trimethylsilyl enol ether to [3,4-¹³C₂]cholesterol. Acetyl enollactone $\text{10}$ was cyclized in acetic acid to [3,4-¹³C₂]3-oxo-4-estren-17β-yl benzoate followed by aromatization and hydrolysis to produce [3,4-¹³C₂]estradiol-17β. Alternatively, cyclization of $\text{10}$ with base afforded [3,4-¹³C₂]3-oxo-4-estren-17β-ol directly, which was then oxidized and aromatized to yield [3,4-¹³C₂]estrone. Ozonolysis of progesterone, conversion to the diketal ester $\text{16}$ and acylation followed by acid hydrolysis furnished [3,4-¹³C₂]progesterone.     

Preliminary crystallographic data for cross-linked (lysine7-lysine41)-ribonuclease A

A cross-linked derivative of ribonuclease A, N^ε,N^ε′-(2,4-dinitrophenylene-1,5)-(lysine⁷-lysine⁴¹)-RNase A, has been crystallized by dialysis against 30% ($\text{v}\text{v}$) ethanol/water mixtures buffered at high pH. Single crystals belong to the orthorhombic space group P2₁2₁2₁, $\text{a = 37.2}\text{A}\text{?}\text{, b = 41.2}\text{A}\text{?}\text{, b = 41.2}\text{A}\text{?}$, with one molecule in the Crystallographic asymmetric unit.     

The ferritin content of human red blood cells during the replacement of embryonic cells by fetal cells

Mature human embryonic erythrocytes (hemoglobin is ≥ 90% of the cellular protein) contained at least 20 times as much ferritin as human adult erythrocytes, suggesting the possibility that the embryonic red cells participate in iron storage as they do in other embryonic or larval vertebrates. The ferritin content of mature red cells in the circulation declined when fetal red cells replaced embryonic red cells; the cell replacement was monitored by the disappearance of embryonic ε-chains and the appearance of the fetal globin chains, γ^A and γ^G. A constant ratio of 0.67 was obtained for $\text{γ}{}^{\mathrm{<mtext>G</mtext>}}\text{γ}{}^{\mathrm{<mtext>A</mtext>}}\text{+ γ}{}^{\mathrm{<mtext>G</mtext>}}$ from the first detectable appearance (4 weeks after conception) until 13 weeks, a value which is similar to the value previously obtained at 20 weeks gestation and birth but higher than that observable in adults; thus, both γ^G and γ^A chains are produced in similar amounts throughout gestation. The high levels of ferritin in normal human embryonic erythrocytes emphasize the similarity of erythropoiesis in human embryos and other vertebrates. In addition, the results show that red cell ferritin can be used as a marker for studying the mechanism of induction of embryonic erythropoiesis in cultured cell lines, such as K562 from human chronic myelocytic leukemia, and that ferritin content may also serve as a marker for cellular transformations involving reversions to embryonic erythropoiesis.     

Osmotic pressure method to measure salt induced folding/unfolding of bovine serum albumin

A new approach has been developed to monitor protein folding by utilizing osmotic pressure and a range of salt concentrations in a well characterized protein, bovine serum albumin (BSA). It is hypothesized that both the ‘effective’ osmotic molecular weight, A_e, and the solute/solvent interaction parameter, I, in the empirical relation $\text{M}{}_{\mathrm{solvent}}\text{M}{}_{\mathrm{solute}}\text{= (}\text{RTϱ}\text{A}{}_{\mathrm{e}}\text{)}\text{1}\text{gp}\text{+ I}$ [1] can be used as measures of protein folding. I is a measure of solvent perturbed by the solute and is thought to depend directly upon the solvent accessible surface area (ASA). It is reasoned that larger solvent accessible surface area of an unfolded or denatured protein should perturb more water and produce larger I-values. Thus I-values allow calculation of a unfolded protein fraction, f_u^a, due to changes in relative solvent accessible surface area. It has been observed that A_e decreases for filamentous, denatured proteins due to segmental motion of the molecule [2]. This allows calculation of unfolded protein fraction from the effective molecular weight, f_u^m. Colloid osmotic pressure of BSA was measured in a range of salt concentrations at 25°C, and pH = 7 (above the isoelectric point of BSA at pH = 5.4). Both S and I were used to monitor protein folding as the salt concentration was varied. In general, larger and variable I-values and smaller A_e were observed at salt concentrations less than 50 mmolal NaCl (I_max = 8.9), while constant I = 4.1 and A_e = 66,500 were observed above 50 mmolal NaCl. The two expressions for fractional unfolding (f_u^a and f_u^m) are in general agreement. Small differences in the parameters below 50 mmolal salt concentration are explained with well known shifts in the relative amounts of α-helix, β-sheet and random coil in denatured BSA. The relative amounts of these shifts agree with predictions in the literature attributed to continuous BSA expansion rather than an ‘all-or-none’ conversion.     

Electron paramagnetic resonance crystallography of spin-labeled hemoglobin-protein fine structures.

Various hemoglobin derivatives have been labeled at the Cys-β93 residue with a bulky and “strongly immobilized” nitroxide maleimide (I) and a smaller, more flexible and “weakly immobilized” nitroxide iodoacetamide (II) and crystallized. The angular dependence of the paramagnetic resonance of the spin-label was measured for the ab, $\text{ac}{}^1$ and $\text{bc}{}^1$ planes at 298 K and 77 K for spin-labeled crystals of Oxyhemoglobin, methemoglobin fluoride, and methemoglobin azide. In the case of the methemoglobin crystals, the angular variation of the heme resonance was also monitored at 77 K. From the hyperfine splitting data, the spin-label I was found to assume specific orientations at both temperatures with some motional narrowing at 298 K. Spin-label II is specifically oriented only at room temperature but is frozen at 77 K in random orientations. Oxyhemoglobin labeled with I (I-HbO₂²) has the most prominent spin-label orientation (z∥b, x∥a) and the less abundant spin-labels with (z∥b ± 15 °) (Ohnishi et al., 1966). The corresponding spin-label orientations for I-Hb⁺ F^? are ($\text{z∥a, x∥c}{}^1$) and ($\text{z∥c}{}^1\text{, x∥a}$). Crystals of I-Hb⁺ N^?₃ have spin-labels oriented along angular directions similar, but not identical to those of I-Hb⁺F^?. Therefore, there are probably significant peptide segmental displacements when HbO₂ is oxidized to methemoglobins. At 25 °C II-Hb⁺ N^?₃ has spin-label orientations not too different from those in I-Hb⁺ N^?₃, whereas in HbO₂ the two spin-labels show significant differences in their orientations.     

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.     

Interactions and space requirement of the phosphate head group of membrane lipids: The single crystal structures of a triclinic and a monoclinic form of hexadecyl-2-deoxyglycerophosphoric acid monohydrate

The crystal structures of a triclinic form (HPA1) and a monoclinic form (HPA2) of hexadecyl-2-deoxyglycerophosphoric acid monohydrate were determined by single crystal analysis. The unit cell dimensions for HPA1 are $\text{a = 4.75, b = 5.72, c = 44.36}\text{A}\text{?}$ and α = 91.0, β = 101.5, γ = 100.5° (P$\text{1}$) and for HPA2, $\text{a = 4.75, b = 5.72, c = 88.72}\text{A}\text{?}$ and γ = 100.8° (P2₁). In both structures the molecules are fully extended and pack tail-to-tail in bilayers with tilting (47°) hydrocarbon chains. In HPA2, however, the chain tilt alternatingly changes direction in adjacent bilayers, giving rise to a doubled unit cell which spans two bilayers. The dihydrogen phosphate groups interact by hydrogen bonds and are arranged in rows. Laterally between these phosphate rows the water molecules are accommodated producing a compact two-dimensional network of hydrogen bonds. The packing cross-section in the layer plane of the dihydrogen phosphate monohydrate group is 26.7 Ų in both structures. The hydrocarbon chains pack according to the triclinic (T|) chain packing mode. In HPA2, however, the chain packing is somewhat less compact with accounts for a 2% increase in the molecular volume. In both structures the ether oxygen is accommodated into the hydrocarbon matrix without distortion of the chain packing.     

Amiloride stimulation of sodium transport in the presence of calcium and a divalent cation chelator

Amiloride in nM to μM concentrations stimulates the short circuit current ($\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$) of the toad urinary bladder by as much as 120% when applied in conjunction with apical Ca²⁺ and a divalent cation chelator. A significant decrease in transepithelial resistance ($\text{R}{}_{\mathrm{<mtext>t</mtext>}}$) is observed simultaneously. This response is spontaneously reversible and its amplitude is dependent upon apical sodium concentrations. The stimulated $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ persisted when acetazolamide (1 mM) was introduced, HPO₄^2? substituted for HCO₃^? or SO₄^2? replaced Cl^?. Consequently, the increase in $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ is not due to the change of Cl^?, H⁺ or HCO₃^? flux. This behavior in a ‘tight’ epithelium may be related to the mechanism controlling apical sodium permeability.     

Magnetic resonance study of 13C reductively methylated glycophorin and glycophorin glycopeptides

¹³C nuclear magnetic resonance (n.m.r.) spectral data for ¹³C reductively methylated N-terminal tryptic glycopeptides and for ¹³C reductively methylated N-terminal glyco-octapeptides derived from homozygous glycophorins A^M and A^N are presented. Their ¹³C chemical shift data are compared with the previously published ¹³C n.m.r. data for ¹³C reductively methylated homozygous glycophorins A^M and A^N in order to investigate the means of display of the MN blood determinants by these species. The pH dependence of the ¹³C resonances of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ leucine of glyco-octapeptide A^N and of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine of glyco-octapepti A^M indicated that only a slight structural perturbation occurs at the N-terminus when a large portion of the glycoprotein molecule is removed. However, one structural ‘state’ of ¹³C reductively methylated glycophorin A^M is lost when the glyco-octapeptide A^M is produced. The ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ leucine of glycooctapeptide A^N titrated with a $\text{p}\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ of 7.7 (Hill coefficient $\text{～ 1}$). The ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine, on the other hand, exhibited an unusual pH dependence, indicating the existence of some possible steric constraints or hydrogen bonding in this molecule. In comparison to the data obtained for ¹³C-labelled glycooctapeptide A^M molecule, the pH dependence of the chemical shift of the ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine of tripeptide tri-L-serine is also presented. Circular dichroism (c.d.) spectra indicated that the reductive methylation technique does not cause a large perturbation of the glycophorin A molecule.     

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.