Metal complexes of cyclic diamines,dissociation kinetics of bis(1,5-diazacyclo-octane)nickel(II) in acidic and basic solution,and electrochemical studies

The preparation of the planar yellow [Ni([8]aneN₂)₂](ClO₄)₂ is described. The complex dissociates in basic solution, with rate = k_OH[NiL][OH^?] (L = 1,5-diazacyclo-octane). At 25 °C, k_OH = 4.5 x 10^?2 M^?1 s^?1 and the corresponding activation parameters are ΔH^≠ = 69.2 kJ mol^?1 and ΔS₂₉₈^≠ = ?38.6 J K^?1 mol^?1. Acid catalysed dissociation in quite slow even in strongly acidic solutions. The kinetic data in this case can be fitted to the expression K_obs = k_o + K_H[H⁺], where k_o relates to a solvolytic pathway and k_H to the acid catalysed pathway. At 60 °C, K_o = 2 x 10^?5 s^?1 and k_H is 2 x 10^?5 M^?1 s^?1. Possible mechanisms for these reactions are considered.The Ni(II)/Ni(III) redox couple for NiLⁿ⁺ is irreversible on Pt using MeCN as solvent.     

Reversible linkage isomerization of pentaamminecobalt(III) complexes of urea and its N-methyl derivatives

The (NH₃)₅CoOC(NH₂)₂³⁺ ion is consumed in water according to the rate law k(obs.) = k₁ + k₂[OH⁻], where k₁ = 4.0 × 10⁻⁵ s⁻¹ and k₂ = 14.2 M⁻¹ s⁻¹ (0–0.1 M [OH⁻];μ = 1.1 M, NaClO₄, 25 °C). A hitherto unrecognized intramolecular O- to N- linkage isomerization reaction has been detected. In strongly acid solution only aquation to (NH₃)₅CoOH₂³⁺ is observed, but in 0.1–1.0 M [OH⁻], 7% of the directly formed products is the urea-N complex (NH₃)₅CoNHCONH₂²⁺ which has been isolated. In the neutral pH region a much greater proportion (25%) of the products is the urea-N species. These results are interpreted in terms of an urea-O to urea-N linkage isomerization reaction competing with hydrolysis for both spontaneous (k₁) and base-catalyzed (k₂) pathways; the rearrangement is not observed in strongly acidic solution (pH ⩽ 1) because the protonated N-bonded isomer (pK′_a ≈ 3) is unstable with respect to the O-bonded form. The appearance of the isomerization pathway as the pH is raised in the 0–6 region is commensurate with a rate increase which cannot be attributed to a contribution from the base catalysis term k₂[OH⁻]. It is argued that this observation establishes, for the spontaneous pathway, that hydrolysis and linkage isomerization are separate reaction pathways — there is no common intermediate. The product distribution and rate data lead to the complete rate law, k(obs.) = k₁ + k₂[OH⁻] = (k_s + k_ON) + (k_OH + k′_ON) [OH⁻] for the reactions of the O-bonded isomers, where k_s, k_OH are the specific rates for hydrolysis, and k_ON, k′_ON are the specific rates for O- to N-linkage isomerization, by spontaneous and base-catalyzed pathways respectively; k_ON = 1.3 × 10⁻⁵ s⁻¹ and k′_ON = 1.1 M⁻¹ s⁻¹ (μ = 1.0 M, NaClO₄, 25 °C). The O- to N- linkage isomerization has been observed also for complexes of N-methylurea, N,N-dimethylurea and N-phenylurea, but not for the N,N′-dimethylurea species. There is an approximately statistical relationship among the data for −NH₂ capture (versus H₂O), while −NHR and −NR₂ do not compete with water as nucleophiles for Co(III) in either the spontaneous or base-catalyzed hydrolysis processes. For each urea-O complex, O- to N-isomerization is a more significant parallel reaction in the spontaneous as opposed to the base-catalyzed pathway. This is interpreted as being indicative of more associative character in the spontaneous route to products, a conclusion supported by other evidence. Some activation parameter data have been recorded and the effect of the N-substitution on the rates of solvolysis (H₂O, Me₂SO) is discussed. The urea-N complexes have been isolated as their deprotonated forms, [(NH₃)₅CoNHCONRR′](ClO₄)₂·xH₂O (R,R′ = H, CH₃). They are kinetically inert in neutral to basic solution but in acid they protonate (H₂O, pK′_a 2–3; μ = 1.0 M, 25 °C) and then isomerize rapidly back to their O-bonded forms. Some solvolysis accompanies this N- to O-rearrangement in H₂O and Me₂SO. Specific rates and activation parameters are reported. The kinetic data follow a rate law of the form k_NO(obs.) = (k + k_NO)[H⁺]/(K′_a + [H⁺]) and the active species in the reaction is the protonated form; k, k_NO are the specific rates for hydrolysis and isomerization, respectively. Proton NMR data establish that the site of protonation (in Me₂SO) is the cobalt-bound nitrogen atom. For the unsubstituted urea species (NH₃)₅CoNH₂CONH₂³⁺, diastereotopic exo-NH₂ protons arising from restricted rotation about the CN bond are observed. The relevance to the mechanism of the linkage isomerization process is considered. ¹³C and ¹H NMR and electronic absorption spectral data are presented, and distinctions between linkage isomers and the solution structures (electronic and conformational) are discussed. The urea-N/urea-O complex equilibrium is governed by the relation K′_NO(obs.) = K′_NO[H⁺]/[H⁺](K′_a), where K′_NO is the equilibrium constant = [(NH₃₅Co(urea-O)³⁺]/[(NH₃)₅Co(urea-N)³⁺]. Values for K′_NO(=k_NO/k_ON = 260 and pK′_a ≈ 3 for the NH₂CONH₂ system are consistent with the stability of the N-isomer in feebly acidic to basic solution (e.g. pH 6, K′_NO(obs.) = 2.6 × 10⁻²) and instability in acid solution (e.g. pH 1, K′_NO(obs.) = 240). The equilibrium data for this and other urea complexes of (NH₃)₅Co(III) are contrasted with the result for the analogous Rh(III)NH₂CONH₂ system K′_NO ≈ 1).     

Non-enzymatic hydrolysis of creatine ethyl ester

The rate of the non-enzymatic hydrolysis of creatine ethyl ester (CEE) was studied at 37 °C over the pH range of 1.6-7.0 using ¹H NMR. The ester can be present in solution in three forms: the unprotonated form (CEE), the monoprotonated form (HCEE⁺), and the diprotonated form (H₂CEE²⁺). The values of pK_a1 and pK_a2 of H₂CEE²⁺ were found to be 2.30 and 5.25, respectively. The rate law is found to be

Rate=-dC_CEE/dt=k₊₊[H₂CEE²⁺][OH^-]+k₊[HCEE⁺][OH^-]+k₀[CEE][OH^-]     

Sulphate transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

Synechococcus R-2 (PCC 1942) actively accumulates sulphate in the light and dark. Intracellular sulphate was 1.35 ± 0.23 mol m^?3 (light) and 0.894 ± 0.152 mol m^?3 (dark) under control conditions (BG-11 media: pH_o, 7.5; [SO₄^2?]_o, 0.304 mol m^?3). The sulphate transporter is different from that found in higher plants: it appears to be an ATP-driven pump transporting one SO₄^2?/ATP [ΔμSO₄^2?_i,o=+ 27.7 ± 0.24 kJ mol^?1 (light) and + 24 ± 0.34 kj mol^?1 (dark)]. The rate of metabolism of SO₄^2?at pH_o, 7.5 was 150 ± 28 pmol m^?2 s^?1 (n = 185) in the light but only 12.8 ± 3.6 pmol m^?2 s^?1 (n = 61) in the dark. Light-driven sulphate uptake is partially inhibited by DCMU and chloramphenicol. Sulphate uptake is not linked to potassium, proton, sodium or chloride transport. The alga has a constitutive over-capacity for sulphate uptake [light (n= 105): K_m= 0.3 ± 0.1 mmol m^?3, V_max, = 1.8 ± 0.6 nmol m^?2 s^?1; dark (n= 56): K_m= 1.4 ± 0.4 mmol m^?3, V_max= 41 ± 22 pmol m^?2 s^?1]. Sulphite (SO₃^2?) was a competitive inhibitor of sulphate uptake. Selenate (SeO₄^2?) was an uncompetitive inhibitor.     

Oxidation of Manganous and Ferrous Metalloporphyrins by Dioxygen in Aqueous Solutions

The stoichiometry and rate of oxidation with dioxygen of tetra-(p-sulfonatophenyl)-porphinatomanganese(II) and the bisimidazole tetra(p-sulfonatophenyl)porphinato-iron(II) were studied in aqueous solutions at neutral pH. The stoichiometry for both complexes was determined; two molecules of metalloporphyrin reacted with dioxygen to produce the +3 oxidation state of the metalloporphyrins and hydrogen peroxide. The rate law for the oxidation of Mn(II)-TPPS is rate = k′[Mn(II)-TPPS][O₂], with k′ at 26.5° of 2.6 × 10⁵ M^?1 sec^?1. The rate law for the oxidation of Fe(II)-TPPS in the presence of imidazole is

with k″ = 10,100 sec^?1. Some possible mechanisms consistent with these data are discussed.     

Disproportionation of pentaammineruthenium(III)–nucleoside complexes leads to two-electron oxidation of nucleosides without involving oxygen molecules

Pentaammineruthenium(III) complexes of deoxyinosine (dIno) and xanthosine (Xao) ([Ru^III(NH₃)₅(L)], L?is?dIno, Xao) in basic solution were studied by UV?Cvis spectroscopy, liquid chromatography/electrospray ionization mass spectrometry, and high-performance liquid chromatography. Both Ru^III complexes disproportionate to Ru^II and Ru^IV. Disproportionation followed the rate law d[Ru^II]/dt?=?(k _o?+?k ₁[OH^?])[Ru^III]. k _o and k ₁ of disproportionation at 25?°C were 2.1 (±0.1)?×?10^?3?s^?1 and 21.4?±?3.2?M^?1 s^?1, respectively, for [Ru^III(NH₃)₅(dIno)], and 3.5 (±0.7)?×?10^?4?s^?1 and 59.7?±?3.6?M^?1?s^?1, respectively, for [Ru^III(NH₃)₅(Xao)]. The [Ru^III(NH₃)₅(Xao)] complex disproportionates at a faster rate than [Ru^III(NH₃)₅(dIno)] owing to the stronger electron-withdrawing effect of exocyclic oxygen in Xao. The activation parameters ??H ^? and ??S ^? for k ₁ of [Ru^III(NH₃)₅(dIno)] were 80.2?±?15.2?kJ?mol^?1 and 47.6?±?9.8?J?K^?1 mol^?1, respectively, indicating that the disproportionation of Ru^III to Ru^II and Ru^IV is favored owing to the positive entropy of activation. The final products of both complexes in basic solution under Ar were compared with those under O₂. Under both conditions [Ru(NH₃)₅(8-oxo-L)] was produced, but via different mechanisms. In both aerobic and anaerobic conditions, the deprotonation of highly positively polarized C8-H of Ru-L by OH^? initiates a two-electron redox reaction. For the next step, we propose a one-step two-electron redox reaction between L and Ru^IV under anaerobic conditions, which differentiates from Clarke??s mechanism of two consecutive one-electron redox reactions between L, Ru^III, and O₂.     

Dynamics of molecular organization in agarose sulphate

Nongelling solutions of structurally regular chain segments of agarose sulphate show disorder–order and order–disorder transitions (as monitored by the temperature dependence of optical rotation) that are closely similar to the conformational changes that accompany the sol–gel and gel–sol transitions of the unsegmented polymer. The transition midpoint temperature (T_m) for formation of the ordered structure on cooling is ～25 K lower than T_m for melting. Salt-induced conformational ordering, monitored by polarimetric stopped-flow, occurs on a millisecond time scale, and follows the dynamics expected for the process 2 coil ? helix. The equilibrium constant for helix growth (s) was calculated as a function of temperature from the calorimetric enthalpy change for helix formation (ΔH_cal = ?3.0 ± 0.3 kJ per mole of disaccharide pairs in the ordered state), measured by differential scanning calorimetry. The temperature dependence of the nucleation rate constant (k_nuc), calculated from the observed second-order rate constant (k_obs) by the relationship k_obs = k_nuc(1 ? 1/s) gave the following activation parameters for nucleation of the ordered structure of agarose sulphate (1 mg mL^?1; 0.5M Me₄NCl or KCl): ΔH* = 112 ± 5 kJ mol^?1; ΔS* = 262 ± 20 J mol^?1 K^?1; ΔG*₂₉₈ = 34 ± 6 kJ mol^?1; (k_nuc)₂₉₈ = (7.5 ± 0.5) × 10⁶ dm³ mol^?1 s^?1. The endpoint of the fast relaxation process corresponds to the metastable optical rotation values observed on cooling from the fully disordered form. Subsequent slow relaxation to the true equilibrium values (i.e., coincident with those observed on heating from the fully ordered state) was monitored by conventional optical rotation measurements over several weeks and follows second-order kinetics, with rate constants of (2.25 ± 0.07) × 10^?4 and (3.10 ± 0.10) × 10^?4 dm³ mol^?1 s^?1 at 293.7 and 296.2 K, respectively. This relaxation is attributed to the sequential aggregation processes helix + helix → dimer, helix + dimer → trimer, etc., with depletion of isolated helix driving the much faster coil–helix equilibrium to completion. Light-scattering measurements above and below the temperature range of the conformational transitions indicate an average aggregate size of 2–3 helices.     

Trans-pyridine tetraammine complexes of RuII and RuIII with N7-coordninated purine nucleosides

 The synthesis, spectroscopic, and electrochemical properties of trans-[L(Pyr)(NH₃)₄Ru^II/III] (Pyr=py, 3-phpy, 4-phpy, 3-bnpy, or 4-bnpy; L=H₂O, Guo, dGuo, 1MeGuo, Gua, Ino, or G⁷-DNA) are reported. As expected, the Pyr ligand slows DNA binding by trans-[(H₂O)(Pyr)(NH₃)₄Ru^II]²⁺ relative to [(H₂O)(NH₃)₅Ru^II]²⁺ and favors reduction of Ru^III by about 150 mV. The pyridine ligand also promotes the disproportionation of Ru^III to afford the corresponding complexes of Ru^II and, presumably, Ru^IV. For L=Ino, disproportionation follows the rate law: d[Ru^II]/dt=k ₀[Ru^III]+k ₁[OH^–][Ru^III], k ₀=(2.7±0.7)×10^–4s^–1 and k ₁=70±1 M^–1s^–1. Received: 11 May 1998 / Accepted: 3 March 1999     

Dimeric chlorite dismutase from the nitrogen‐fixing cyanobacterium Cyanothece sp. PCC7425

It is demonstrated that cyanobacteria (both azotrophic and non‐azotrophic) contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite ‘dismutase’, Cld). Beside the water‐splitting manganese complex of photosystem II, this metalloenzyme is the second known enzyme that catalyses the formation of a covalent oxygen–oxygen bond. All cyanobacterial Clds have a truncated N‐terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in Escherichia coli and shown to efficiently degrade chlorite with an activity optimum at pH 5.0 [k_cat 1144 ± 23.8 s^?1, K_M 162 ± 10.0 μM, catalytic efficiency (7.1 ± 0.6) × 10⁶ M^?1 s^?1]. The resting ferric high‐spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of ?126 ± 1.9 mV at pH 7.0. Cyanide mediates the formation of a low‐spin complex with k_on = (1.6 ± 0.1) × 10⁵ M^?1 s^?1 and k_off = 1.4 ± 2.9 s^?1 (K_D ～ 8.6 μM). Both, thermal and chemical unfolding follows a non‐two‐state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure–function relationships of Clds. We ask for the physiological substrate and putative function of these O₂‐producing proteins in (nitrogen‐fixing) cyanobacteria.     

Aryl acylamidase activity of human serum albumin with o-nitrotrifluoroacetanilide as the substrate

Albumin is generally regarded as an inert protein with no enzyme activity. However, albumin has esterase activity as well as aryl acylamidase activity. A new acetanilide substrate, o-nitrotrifluoroacetanilide (o-NTFNAC), which is more reactive than the classical o-nitroacetanilide, made it possible to determine the catalytic parameters for hydrolysis by fatty-acid free human serum albumin. Owing to the low enzymatic activity of albumin, kinetic studies were performed at high albumin concentration (0.075 mM). The albumin behavior with this substrate was Michaelis-Menten like. Kinetic analysis was performed according to the formalism used for catalysis at high enzyme concentration. This approach provided values for the turnover and dissociation constant of the albumin-substrate complex: k_cat = 0.13 ± 0.02 min ? ¹ and K_s = 0.67 ± 0.04 mM. MALDI-TOF experiments showed that unlike the ester substrate p-nitrophenyl acetate, o-NTFNAC does not form a stable adduct (acetylated enzyme). Kinetic analysis and MALDI-TOF experiments demonstrated that hydrolysis of o-NTFNAC by albumin is fully rate-limited by the acylation step (k_cat = k₂). Though the aryl acylamidase activity of albumin is low (k_cat/K_s = 195 M^{? 1}min^{? 1}), because of its high concentration in human plasma (0.6–1 mM), albumin may participate in hydrolysis of aryl acylamides through second-order kinetics. This suggests that albumin may have a role in the metabolism of endogenous and exogenous aromatic amides, including drugs and xenobiotics.     

The cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942 has a sodium-dependent chloride transporter

Synechococcus R-2 (PCC 7942) actively accumulated Cl^? in the light and dark, under control conditions (BG-11 media: pH_o, 7·5; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 molm^?3). In BG-11 medium [Cl^?], was 17·2±0·848 mol m^?3 (light), electrochemical potential of Cl^? (ΔμCl^?_i,o) =+211±2mV; [Cl^?]_i= 1·24±0·11 mol m^?3(dark), ΔμCl^?_i,o=+133±4mV. Cl^? fluxes, but not permeabilities, were much higher in the light: ?Cl^?_i,o= 4·01±5·4 nmol m^?2 s^?1, ^PCl^?_i,o= 47±5pm s^?1 (light); ?Cl^?_i,o= 0·395±0·071 nmol m^?2 s^?1, _PCl^?_i,o= 69±14 pm s^?1 (dark). Chloride fluxes are inhibited by acid pH_o (pH_o 5; ?Cl^?_i,o= 0·14±0·04 nmol m^?2 s^?1); optimal at pH_o 7·5 and not strongly inhibited by alkaline pH_o (pH_o 10; ?Cl^?1_i,o= 1·7±0·14 nmol m^?2 s^?1). A Cl^?_in/2H⁺_in coporter could not account for the accumulation of Cl^? alkaline pH_o. Permeability of Cl^? is very low, below 100pm s^?1 under all conditions used, and appears to be maximal at pH_o 7·5 (50–70 pm s^?1) and minimal in acid pH_o (20pm s^?1). DCCD (dicyclohexyl-carbodiimide) inhibited ?Cl^?_i,o in the light about 75% and [Cl^?]_i fell to 2·2±0·26 (4) mol m^?3. Valinomycin had no effect but monensin severely inhibited Cl^? uptake ([Cl^?]_i= 1·02±0·32 mol m^?3; ?Cl^?_i,o= 0·20±0·1 nmol m^?2 s^?1). Vanadate (200 mmol m^?3) accelerated the Cl^? flux (?Cl^?_i,o= 5·28±0·64 nmol m^?2 s^?1) but slightly decreased accumulation of Cl^? ([Cl^?], = 13·9±1·3 mol m^?3) in BG-11 medium but had no significant effect in Na⁺-free media. DCMU (dichlorophenyldimethylurea) did not reduce [Cl^?], or ?Cl^?_i,o to that found in the dark ([Cl^?]_i= 8·41±0·76 mol m^?3; ?Cl^?_i,o= 2·06±0·36 nmol m^?2 s^?1). Synechococcus also actively accumulated Cl^? in Na⁺-free media, [Cl^?]_i was lower but ΔΨ_i,o hyperpolarized in Na⁺-free media and so the ΔμCl^?_i,o was little changed ([Cl^?]_i= 7·98±0·698 mol m^?3; ΔμCl^?_i,o=+203±3 mV). Net Cl^? uptake was stimulated by Na⁺; Li⁺ acted as a partial analogue for Na⁺. Synechococcus has a Na⁺ activated Cl^? transporter which is probably a primary 2Cl^?/ATP pump. The Cl^? pump is voltage sensitive. ΔμCl^?_i,o is directly proportional to ΔΨ_i,o(P»0·01%): ΔμCl^?_i,o= -1·487 (±0·102) ×ΔΨ_i,o, r= -0·983, n= 31. The ΔμCl^?_i,o increased (more positive) as the Δμ_i,o became more negative. The ΔμCl^?_i,o has no known function, but might provide a driving force for the uptake of micronutrients.     

Astaxanthin diferulate as a bifunctional antioxidant

Abstract

Astaxanthin when esterified with ferulic acid is better singlet oxygen quencher with k₂ = (1.58 ± 0.1) 10¹⁰ L mol^{? 1}s^{? 1} in ethanol at 25°C compared with astaxanthin with k₂ = (1.12 ± 0.01) 10⁹ L mol^{? 1}s^{? 1}. The ferulate moiety in the astaxanthin diester is a better radical scavenger than free ferulic acid as seen from the rate constant of scavenging of 1-hydroxyethyl radicals in ethanol at 25°C with a second-order rate constant of (1.68 ± 0.1) 10⁸ L mol^{? 1}s^{? 1} compared with (1.60 ± 0.03) 10⁷ L mol^{? 1}s^{? 1} for the astaxanthin:ferulic acid mixture, 1:2 equivalents. The mutual enhancement of antioxidant activity for the newly synthetized astaxanthin diferulate becoming a bifunctional antioxidant is rationalized according to a two-dimensional classification plot for electron donation and electron acceptance capability.     

Effects of salinity,light intensity and sediment on growth,pigments, agar production and reproduction in Gracilaria tenuistipitata from Songkhla Lagoon in Thailand

The effects of salinity, light intensity and sediment on Gracilaria tenuistipitata C.F. Chang & B.M. Xia on growth, pigments, agar production, and net photosynthesis rate were examined in the laboratory under varying conditions of salinity (0, 25 and 33 psu), light intensity (150, 400, 700 and 1000 µmol photons m^?2 s^?1) and sediment (0, 0.67 and 2.28 mg L^?1). These conditions simulated field conditions, to gain some understanding of the best conditions for cultivation of G. tenuistipitata. The highest growth rate was at 25 psu, 700 µmol photons m^?2 s^?1 with no sediments, that provided a 6.7% increase in weight gain. The highest agar production (24.8 ± 3.0 %DW) was at 25 psu, 150–400 µmol photons m^?2 s^?1 and no sediment. The highest pigment contents were phycoerythrin (0.8 ± 0.5 mg g^?1FW) and phycocyanin (0.34 ± 0.05 mg g^?1 FW) produced in low light conditions, at 150 µmol photons m^?2 s^?1. The highest photosynthesis rate was 161.3 ± 32.7 mg O₂ g^?1 DW h^?1 in 25 psu, 400 µmol photons m^?2 s^?1 without sediment in the short period of cultivation, (3 days) and 60.3 ± 6.7 mg O₂ g^?1 DW h^?1 in 25 psu, 700 µmol photons m^?2 s^?1 without sediment in the long period of cultivation (20 days). The results indicated that salinity was the most crucial factor affecting G. tenuistipitata growth and production. This would help to promote the cultivation of Gracilaria cultivation back into the lagoon using these now determined baseline conditions. Extrapolation of the results from the laboratory study to field conditions indicated that it was possible to obtain two crops of Gracilaria a year in the lagoon, with good yields of agar, from mid‐January to the end of April (dry season), and from mid‐July to the end of September (first rainy season) when provided sediment was restricted.     

Kinetics of chloride transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

The kinetics of the light-driven Cl^? uptake pump of Synechococcus R-2 (PCC 7942) were investigated. The kinetics of Cl^? uptake were measured in BG-11 medium (pH_o, 7·5; [K⁺]_o, 0·35 mol m^?3; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 mol m^?3) or modified media based on the above. Net³⁶Cl^? fluxes (?Cl^?_o,i) followed Michaelis-Menten kinetics and were stimulated by Na⁺ [18 mol m^?3 Na⁺ BG-11 ?Cl^?_max= 3·29±0·60 (49) nmol m^?2 s^?1 versus Na⁺-free BG-11 ?Cl^?_max= 1·02±0·13 (54) nmol m^?2 s^?1] but the Km was not significantly different in the presence or absence of Na⁺ at pH_o 10; the Km was lower, but not affected by the presence or absence of Na⁺ [Km = 22·3±3·54 (20) mmol m^?3]. Na⁺ is a non-competitive activator of net ?Cl^?_o,i. High [K⁺]_o (18 mol m^?3) did not stimulate net ?Cl^?_o,i or change the Km in Na⁺-free medium. High [K⁺]_o (18 mol m^?3) added to Na⁺ BG-11 medium decreased net ?Cl^?_o,i [18 mol m^?3K⁺ BG-11; ?Cl^?_max= 2·50±0·32 (20) nmol m^?2 s^?1 versus BG-11 medium; ?Cl^?_max= 3·35±0·56 (20) nmol m^?2 s^?1] but did not affect the Km 55·8±8·100 (40) mmol m^?3]. Na⁺-stimulation of net ?Cl^?_o,i followed Michaelis-Menten kinetics up to 2–5 mol m^?3 [Na⁺]_o but higher concentrations were inhibitory. The Km for Na⁺-stimulation of net ?Cl^?_o,i [K_1/2(Na⁺)] was different at 47 mmol m^?3 [Cl^?]_o (K_1/2[Na⁺] = 123±27 (37) mmol m^?3]. Li⁺ was only about one-third as effective as Na⁺ in stimulating Cl^? uptake but the activation constant was similar [K_1/2(Li⁺) = 88±46 (16) mmol m^?3]. Br^? was a competitive inhibitor of Cl^? uptake. The inhibition constant (Ki) was not significantly different in the presence and absence of Na⁺. The overall Ki was 297±23 (45) mmol m^?3. The discrimination ratio of Cl^? over Br^? (δCl^?/δBr^?) was 6·38±0·92 (df = 147). Synechococcus has a single Na⁺-stimulated Cl^? pump because the Km of the Cl^? transporter and its discrimination between Cl^? and Br^? are not significantly different in the presence and absence of Na⁺. The Cl^? pump is probably driven by ATP.     

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.     

Presteady-state kinetic study of the elementary processes in the chymotrypsin-catalyzed hydrolysis of specific ester substrate. Rate-limiting association process due to the secondary binding.

Presteady-state kinetic studies of α-chymotrypsin-catalyzed hydrolysis of a specific chromophoric substrate, N-(2-furyl)acryloyl-l-tryptophan methyl ester, were performed by using a stopped-flow apparatus both under [E]₀ ? [S]₀ and [S]₀ ? [E]₀ conditions in the pH range of 5–9, at 25 °C. The results were accounted for in terms of the three-step mechanism involving enzyme-substrate complex (E · S) and acylated enzyme (ES′); no other intermediate was observed. This substrate was shown to react very efficiently, i.e., the maximum of the second-order acylation rate constant ($\text{k}{}_2\text{K}{}_{\mathrm{s}}\text{′}\text{)}{}_{\mathrm{<mtext>max</mtext>}}\text{= 4.2 × 10}{}^7\text{M}{}^{\mathrm{?1}}\text{s}{}^{\mathrm{?1}}$. The limiting values of K_s′ (dissociation constant of E · S), K₂ (acylation rate) and k₃ (deacylation rate) were obtained from the pH profiles of these parameters to be 0.6 ± 0.2 × 10^?5 m, 360 ± 15 s^?1 and 29.3 ± 0.8 s^?1, respectively. Likewise small values were observed for K_i of N-(2-furyl)-acryloyl-l-tryptophan and N-(2-furyl)acryloyl-d-tryptophan methyl ester and K_m of N-(2-furyl)acryloyl-l-tryptophan amide. The strong affinities observed may be due to intense interaction of β-(2-furyl)acryloyl group with a secondary binding site of the enzyme. This interaction led to a $\text{k}{}_{\mathrm{?1}}\text{k}{}_2$ value lower than unity, i.e., the rate-limiting process of the acylation was the association, even with the relatively low k₂ value of this methyl ester substrate, compared to those proposed for labile p-nitrophenyl esters.     

Kinetics of CO substitution in Co4(CO)12 and Rh4(CO)12

The kinetics of rapid CO substitution by PPh₃ in Co₄(CO)₁₂ and Rh₄(CO)₁₂ have been examined by stopped-flow and low temperature FT-IR methods. In Co₄(CO)₁₂ rapid (k_obs ∼ 1.8 s⁻¹) substitution of CO occurs after a 1–15 s induction period at 28 °C in C₆H₅Cl solvent by a catalytic process. Addition of PPh₃ to Rh₄(CO)₁₂ yields Rh₄(CO)₁₁(PPh₃) according to a predominantly second order rate law k₁[Rh₄- (CO)₁₂] + k₂[Rh₄(CO)₁₂][PPh₃] with k₁ = 25 ± 11 s⁻¹ and k₂ = 2.97 ± 0.27 X 10⁴ M⁻¹ s⁻¹ at 28 °C. Substitution of a second CO ligand also occurs rapidly with k₁ = 0.15 ± 0.09 s⁻¹ and k₂ = 6.54 ± 0.07 X 10² M⁻¹ s⁻¹ at 28 °C. The reactivity of Rh₄(CO)₁₂ toward associative substitution is 10⁴– 10¹¹ faster than for the Co and Ir analogues, In Rh₄(CO)₁₁(PPh₃) the increase in CO substitution rates over Co and Rh analogues is 10²–10⁷. The ordering of associative substitution rates Co << Rh >>> Ir in these clusters exaggerates the trend seen in mononuclear metal complexes.     

COMPARISON OF DEVELOPMENT AND PHOTOSYNTHETIC GROWTH FOR FILAMENT CLUMPS AND REGENERATED MICROPLANTLET CULTURES OF AGARDHIELLA SUBULATA (RHODOPHYTA, GIGARTINALES)

Two axenic, in vitro liquid suspension cultures were established for Agardhiella subulata (C. Agardh) Kraft et Wynne, and their growth characteristics were compared. This study illustrated how reliable routes for the development of suspension cultures of macrophytic red algae of terete thallus morphology can be achieved for biotechnology applications. Undifferentiated filament clumps of 2–8 mm diameter were established by induction of callus-like tissue from thallus explants, and lightly branched microplantlets of 2–10 mm length were established by regeneration of filament clumps. The filament clumps were susceptible to regeneration. Adventitious shoot formation was reliably induced from 40% to 70% of the filament clumps by gentle mixing at 100 rev min^?1 on an orbital shaker. The specific growth rate of the microplantlets was higher than the filament clumps in nonagitated well plate culture (4%–6% per day for microplantlets vs. 2%–3% per day for filament clumps) at 24° C and 8–36 μmol photons·m^?2·s^?1 irradiance (10:14 h LD cycle) when grown on ASP12 artificial seawater medium at pH 8.6–8.9 with 20%–25% per day medium replacement. Oxygen evolution rate vs. irradiance measurements showed that relative to the filament clumps, microplantlets had a higher maximum specific oxygen evolution rate (P_o,max= 0.181 ± 0.035 vs. 0.130 ± 0.023 mmol O₂·g^?1 dry cell mass·h^?1), but comparable respiration rate (Q_o= 0.040 ± 0.013 vs. 0.033 ± 0.017 mmol O₂·g^?1 dry cell mass·h^?1), compensation point (I_c= 3.8 ± 2.4 vs. 5.7 ± 1.2 μmol photons·m^?2·s^?1), and light intensity at 63.2% of saturation (I_k= 17.5 ± 3.9 vs. 14.9 ± 2.6 μmol photons·m^?2·s^?1). The microplantlet culture was more suitable for suspension culture development than the filament clump culture because it was morphologically stable and exhibited higher growth rates.     

Reaction Kinetics of the Invertase from Yeast (S. Cerevisiae)

Invertase converts sucrose to glucose and fructose. The reaction mechanism for the formation of glucose and fructose was studied by stopped flow spectrophotometer and circular dichroism. The reaction mechanism follows biphasic mode with rate constants of k₁0.0053 s^?1?±?0.001 s^?1 and k₂ 0.030 s^?1?±?0.01 s^?1 for 25 mM concentration of sucrose. Far UV circular dichroic spectrum of invertase in presence of sucrose shows 18 % increase in β conformation as a function of time. Taken together, the invertase hydrolysis follows biphasic mode where it undergoes conformational changes followed by hydrolysis of the sucrose.     

Kinetics of some electron-transfer reactions in biological photosystems. I. Pulse radiolysis study of spinach ferredoxin reduction by the hydrated electron and CO−2 radical

The reduction of spinach ferredoxin by the CO^?₂ radical and the hydrated electron (e^?_aq) has been studied by pulse radiolysis in the pH range between 5.05 and 9.67. The reduction of oxidized spinach ferredoxin by both CO^?₂ and e^?_aq was found to be essentially quantitative. The CO^?₂ radical reduces spinach ferredoxin by a single second-order process at a rate k₅ = (6.2 ± 0.6) · 10⁷ M^?1 · s^?1. Reduction by e^?_aq follows a biphasic pathway. The first phase obeys second-order kinetics for the reduction of the cluster, k_app = (9.4 ± 0.3) · 10⁹ M^?1 · s^?1. The second phase follows an intramolecular first-order reaction k_B = (8.3 ± 1.7) · 10² s^?1 which is observed as a further reduction of the active site. Spectral changes accompanying the reduction of oxidized spinach ferredoxin in the ultraviolet and visible range are discussed.