Pressure and temperature stability of 5-methyltetrahydrofolic acid: a kinetic study

Detailed kinetic studies of [6S] and [6RS] 5-methyltetrahydrofolic acid (5-CH3-H4folate) degradation during thermal (from 60 to 90 degrees C) and high pressure/thermal (from 30 to 45 degrees C; from 200 to 700 MPa) treatments were carried out. The results confirmed that the temperature and pressure induced degradation kinetics of [6S] 5-CH3-H4folate were identical (within 95% confidence interval) with those of [6RS] 5-CH3-H4folate. Under equal processing conditions, the estimated degradation rate constants (k), activation energy (E(a)), and activation volume (V(a)) values of [6S] and [6RS] 5-CH3-H4folate were the same (95% confidence interval). The modified thermodynamic model proposed by Nguyen and co-workers (J. Agric. Food Chem. 2003, 51, 3352-3357) to describe the pressure and temperature dependence of the rate constant for folate degradation was reevaluated.     

Comparative study on pressure and temperature stability of 5-methyltetrahydrofolic acid in model systems and in food products

A comparative study on the pressure and temperature stability of 5-methyltetrahydrofolic acid (5-CH(3)-H(4)folate) was performed in model/buffer systems and food products (i.e., orange juice, kiwi puree, carrot juice, and asparagus). Effects of pH and ascorbic acid (0.5 mg/g) on 5-CH(3)-H(4)folate stability in buffer systems were studied on a kinetic basis at different temperatures (from 65 to 160 degrees C) and different pressure/temperature combinations (from 100 to 700 MPa/from 20 to 65 degrees C). These studies showed that (i) the degradation of 5-CH(3)-H(4)folate in all model systems could be described by first-order reaction kinetics, (ii) the thermostability of 5-CH(3)-H(4)folate was enhanced by increasing pH up to 7, (iii) 5-CH(3)-H(4)folate was relatively pressure stable at temperatures lower than 40 degrees C, and (iv) ascorbic acid enhanced both the thermo- and barostabilities of 5-CH(3)-H(4)folate. In food products, temperature and pressure stabilities of 5-CH(3)-H(4)folate were studied at different temperatures (70-120 degrees C) and different pressure/temperature combinations (from 50 to 200 MPa/25 degrees C and 500 MPa/60 degrees C). 5-CH(3)-H(4)folate in orange juice and kiwi puree was relatively temperature (up to 120 degrees C) and pressure (up to 500 MPa/60 degrees C) stable in contrast to carrot juice and asparagus. Addition of ascorbic acid (0.5 mg/g) in carrot juice resulted in a remarkable protective effect on pressure (500 MPa/60 degrees C/40 min) and temperature degradation (120 degrees C/40 min) of 5-CH(3)-H(4)folate.     

Determination of 5-methyltetrahydrofolic acid and folic acid in citrus juices using stable isotope dilution-mass spectrometry

A stable isotope liquid chromatography-mass spectrometry (LC-MS) method was developed for the quantitative determination of 5-methyltetrahydrofolic acid (5-MTHFA) and folic acid in a variety of commercial citrus juices. Folates were extracted from juices, and the polyglutamyl side chain of 5-MTHFA was cleaved to the monoglutamate form using rat plasma conjugase. The folates were purified on a Bond-Elut column and analyzed by LC-MS with electrospray ionization. The analytes were quantified using the (13)C(5) analogues of 5-MTHFA and folic acid as internal standards. The relative standard error of the method was 3.35% based on replicate analyses (n = 4). This method was then applied to the determination of 5-MTHFA and folic acid in a variety of citrus juices obtained from local supermarkets. It was observed that although both "store" brands and "national" brands of fresh (nonfrozen) juices contained similar concentrations of 5-MTHFA, the "store" brands of fresh juices had on average >5-fold the amount of folic acid compared to the "national" brands. In addition, the "total" folate concentrations were generally below values listed on the food label.     

Kinetics of the stability of broccoli (Brassica oleracea Cv. Italica) myrosinase and isothiocyanates in broccoli juice during pressure/temperature treatments

The Brassicaceae plant family contains high concentrations of glucosinolates, which can be hydrolyzed by myrosinase yielding products having an anticarcinogenic activity. The pressure and temperature stabilities of endogenous broccoli myrosinase, as well as of the synthetic isothiocyanates sulforaphane and phenylethyl isothiocyanate, were studied in broccoli juice on a kinetic basis. At atmospheric pressure, kinetics of thermal (45-60 degrees C) myrosinase inactivation could be described by a consecutive step model. In contrast, only one phase of myrosinase inactivation was observed at elevated pressure (100-600 MPa) combined with temperatures from 10 up to 60 degrees C, indicating inactivation according to first-order kinetics. An antagonistic effect of pressure (up to 200 MPa) on thermal inactivation (50 degrees C and above) of myrosinase was observed indicating that pressure retarded the thermal inactivation. The kinetic parameters of myrosinase inactivation were described as inactivation rate constants (k values), activation energy (Ea values), and activation volume (Va values). On the basis of the kinetic data, a mathematical model describing the pressure and temperature dependence of myrosinase inactivation rate constants was constructed. The stability of isothiocyanates was studied at atmospheric pressure in the temperature range from 60 to 90 degrees C and at elevated pressures in the combined pressure-temperature range from 600 to 800 MPa and from 30 to 60 degrees C. It was found that isothiocyanates were relatively thermolabile and pressure stable. The kinetics of HP/T isothiocyanate degradation could be adequately described by a first-order kinetic model. The obtained kinetic information can be used for process evaluation and optimization to increase the health effect of Brassicaceae.     

Impact of high-pressure carbon dioxide combined with thermal treatment on degradation of red beet (Beta vulgaris L.) pigments

A combined high-pressure carbon dioxide (HP-CO 2) and thermal degradation reaction of betanin and isobetanin in aqueous solution was investigated and can be described by a first-order decay. At 45 degrees C, the degradation rate constant ( k) for each pigment component significantly increased (the half-life ( t 1/2) decreased, p < 0.05) with elevated pressure. Furthermore, HP-CO 2 treatment led to lower k values (higher t 1/2 values) than thermal treatment. However, k and t 1/2 values approached those of thermal treatment when the pressure was >30 MPa combined with temperatures exceeding 55 degrees C. Moreover, betanin was more stable than isobetanin under HP-CO 2. E a values ranged from 94.01 kJ/mol for betanin and 97.16 kJ/mol for isobetanin at atmospheric pressure to 170.83 and 142.69 kJ/mol at 50 MPa, respectively. A higher pressure and temperature as well as longer exposure time resulted in higher values of L*, b*, C*, and h degrees . HP-CO 2 induced more degradation products from betanin and isobetanin than thermal treatment with an identical temperature and exposure time.     

Reaction of folic acid with reducing sugars and sugar degradation products

The reaction of folic acid with reducing sugars (nonenzymatic glycation) under conditions that can occur during food processing and preparation was studied by high-performance liquid chromatography with diode array detection. N-(p-Aminobenzoyl)-L-glutamic acid, a well-established oxidation product, was detected in the reaction mixtures. Furthermore, a new product was isolated and identified as N2-[1-(carboxyethyl)]folic acid (CEF). CEF was the main product that was formed by the nonenzymatic glycation of folic acid. For preparation, N2-[1-(carboxyethyl)]folic acid was obtained in high yields when folic acid and dihydroxyacetone (DHA), a sugar degradation product, were heated at 100 degrees C in phosphate buffer. Mixtures of folic acid and different sugars or DHA were heated under variation of reaction time and temperature, and CEF was quantified. Up to 50% of the vitamin was converted to CEF, with highest yields formed from maltose (49%) and lactose (43%).     

Changes in sulfhydryl content of egg white proteins due to heat and pressure treatment

The sulfhydryl (SH) content of egg white proteins (10% v/v or 9.64 mg of protein/mL) after heat (50-85 degrees C) and combined heat- and high-pressure treatments (100-700 MPa, 10-60 degrees C) was determined using 5',5-dithiobis (2-nitrobenzoic acid) (DTNB), both for the soluble fraction and the total protein fraction. Only irreversible changes were taken into account. Both physical treatments were performed at two pH levels: pH 7.6, corresponding to the pH of fresh egg white, and pH 8.8, corresponding to that of aged egg white. Both heat and combined heat- and high-pressure treatment resulted in an exposure of buried SH groups. These exposed SH groups were involved in the formation of disulfide bond stabilized protein aggregates, as shown by gel electrophoresis. Under severe processing conditions (above 70 degrees C at atmospheric pressure or above 500-600 MPa, depending on the temperature applied), a decrease in total SH content could be observed, probably due to the formation of disulfide bonds by oxidation, especially at alkaline pH when the thiolate anion was more reactive. The high degree of exposure of sulfhydryl groups, and subsequent oxidation and sulfhydryl-disulfide bond exchange reactions resulting in soluble aggregates, can explain why pressure-induced egg white gels are softer and more elastic than heat-induced ones. When pressure treatment was performed at low temperatures (e.g., 10 degrees C), a lower pressure was required to induce similar changes in the sulfhydryl content, as compared to higher temperatures (e.g., 25 degrees C), indicating an antagonistic effect between pressure and temperature in the domain studied (10-60 degrees C, 100-700 MPa). Treatment conditions resulting in extensive protein insolubilization were accompanied by a transfer of free sulfhydryl groups from the soluble to the insoluble protein fraction. These SH groups were mainly accessible to DTNB.     

Inactivation of orange pectinesterase by combined high-pressure and -temperature treatments: a kinetic study

Pressure and/or temperature inactivation of orange pectinesterase (PE) was investigated. Thermal inactivation showed a biphasic behavior, indicating the presence of labile and stable fractions of the enzyme. In a first part, the inactivation of the labile fraction was studied in detail. The combined pressure-temperature inactivation of the labile fraction was studied in the pressure range 0.1-900 MPa combined with temperatures from 15 to 65 degrees C. Inactivation in the pressure-temperature domain specified could be accurately described by a first-order fractional conversion model, estimating the inactivation rate constant of the labile fraction and the remaining activity of the stable fraction. Pressure and temperature dependence of the inactivation rate constants of the labile fraction was quantified using the Eyring and Arrhenius relations, respectively. By replacing in the latter equation the pressure-dependent parameters (E(a), k(ref)(T)()) by mathematical expressions, a global model was formulated. This mathematical model could accurately predict the inactivation rate constant of the labile fraction of orange PE as a function of pressure and temperature. In a second part, the stable fraction was studied in more detail. The stable fraction inactivated at temperatures exceeding 75 degrees C. Acidification (pH 3.7) enhanced thermal inactivation of the stable fraction, whereas addition of Ca(2+) ions (1 M) suppressed inactivation. At elevated pressure (up to 900 MPa), an antagonistic effect of pressure and temperature on the inactivation of the stable fraction was observed. The antagonistic effect was more pronounced in the presence of a 1 M CaCl(2) solution as compared to the inactivation in water, whereas it was less pronounced for the inactivation in acid medium.     

Tomato (Lycopersicon esculentum) pectin methylesterase and polygalacturonase behaviors regarding heat- and pressure-induced inactivation.

The combined high pressure/thermal (HP/T) inactivation of tomato pectin methyl esterase (PME) and polygalacturonase (PG) was investigated as a possible alternative to thermal processing classically used for enzyme inactivation. The temperature and pressure ranges tested were from 60 degrees C to 105 degrees C, and from 0.1 to 800 MPa, respectively. PME, a heat-labile enzyme at ambient pressure, is dramatically stabilized against thermal denaturation at pressures above atmospheric and up to 500-600 MPa. PG, however, is very resistant to thermal denaturation at 0.1 MPa, but quickly and easily inactivated by combinations of moderate temperatures and pressures. Selective inactivation of either PME or PG was achieved by choosing proper combinations of P and T. The inactivation kinetics of these enzymes was measured and described mathematically over the investigated portion of the P/T plane. Whereas medium composition and salinity had little influence on the inactivation rates, PME was found less sensitive to both heat and pressure when pH was raised above its physiological value. PG, on the other hand, became more labile at higher pH values. The results are discussed in terms of isoenzymes and other physicochemical features of PME and PG.     

Yeast survival during thermal and osmotic shocks is related to membrane phase change

The aim of this work was to study the survival of yeast cells exposed to a combination of thermal and osmotic treatments, as occurs during drying processes, and assess associated changes in fluidity of the plasma membrane, which have been studied previously in this laboratory. Cells that were maintained at a nonlethal dehydration temperature (5 or 30 degrees C) were rapidly dehydrated up to 120 MPa, and then thermal stress was suddenly applied between 5 and 30 degrees C. Cell viability was measured after a return to initial conditions (i.e., 1.38 MPa and 25 degrees C). Results showed that the viability of yeast cells exposed to identical combined thermal and osmotic treatments was dependent on the chronology of the stress application. Finally, the temperature at which the dehydration was conducted, up to 120 MPa, appeared to be the main factor involved in cell survival and could be related to fluidity variations of the plasma membrane.     

Stability of copigmented anthocyanins and ascorbic acid in a grape juice model system

The stability of red grape anthocyanins (Vitis vinifera) was evaluated in a model juice system during normal (25 degrees C) and accelerated storage (35 degrees C) in the presence of ascorbic acid. Rosemary polyphenolic cofactors (0, 0.2, and 0.4% v/v) were evaluated as anthocyanin stabilizing agents. Cofactor addition resulted in concentration-dependent hyperchromic (up to 178%) and bathochromic (up to 23 nm) shifts, indicating a more intense red coloration of the models. Anthocyanin and ascorbic acid degradation followed first-order kinetics during storage. Results showed that copigmented treatments underwent a lower conversion of L-ascorbic acid into dehydroascorbic acid during storage when compared to the control, favorably impacting the vitamin retention of these models. Copigmentation did not affect anthocyanin degradation in the absence of ascorbic acid but in its presence aided to retain a higher anthocyanin content than the control. This study indicated that the addition of anthocyanin cofactors could be used to reduce the pigment and vitamin degradation while masking detrimental color changes in anthocyanin containing products.     

Effect of temperature and/or pressure on tomato pectinesterase activity

The activity of tomato pectinesterase (PE) was studied as a function of pressure (0.1-900 MPa) and temperature (20-75 degrees C). Tomato PE was rather heat labile at atmospheric pressure (inactivation in the temperature domain 57-65 degrees C), but it was very pressure resistant. Even at 900 MPa and 60 degrees C the inactivation was slower as compared to the same treatment at atmospheric pressure. At atmospheric pressure, optimal catalytic activity of PE was found at neutral pH and a temperature of 55 degrees C. Increasing pressure up to 300 MPa increased the enzyme activity as compared to atmospheric pressure. A maximal enzyme activity was found at 100-200 MPa combined with a temperature of 60-65 degrees C. The presence of Ca(2+) ions (60 mM) decreased the enzyme activity at atmospheric pressure in the temperature range 45-60 degrees C but increased enzyme activity at elevated pressure (up to 300 MPa). Maximal enzyme activity in the presence of Ca(2+) ions was noted at 200-300 MPa in combination with a temperature of 65-70 degrees C.     

Effect of combined heat and high-pressure treatments on the texture of chicken breast muscle (pectoralis fundus)

Commercially supplied chicken breast muscle was subjected to simultaneous heat and pressure treatments. Treatment conditions ranged from ambient temperature to 70 degrees C and from 0.1 to 800 MPa, respectively, in various combinations. Texture profile analysis (TPA) of the treated samples was performed to determine changes in muscle hardness. At treatment temperatures up to and including 50 degrees C, heat and pressure acted synergistically to increase muscle hardness. However, at 60 and 70 degrees C, hardness decreased following treatments in excess of 200 MPa. TPA was performed on extracted myofibrillar protein gels that after treatment under similar conditions revealed similar effects of heat and pressure. Differential scanning calorimetry analysis of whole muscle samples revealed that at ambient pressure the unfolding of myosin was completed at 60 degrees C, unlike actin, which completely denatured only above 70 degrees C. With simultaneous pressure treatment at >200 MPa, myosin and actin unfolded at 20 degrees C. Unfolding of myosin and actin could be induced in extracted myofibrillar protein with simultaneous treatment at 200 MPa and 40 degrees C. Electrophoretic analysis indicated high pressure/temperature regimens induced disulfide bonding between myosin chains.     

Thermal stability of ascorbic acid and ascorbic acid oxidase in african cowpea leaves ( Vigna unguiculata ) of different maturities

Cowpea, an African leafy vegetable ( Vigna unguiculata ), contains a high level of vitamin C. The leaves harvested at 4-9 weeks are highly prone to vitamin C losses during handling and processing. Therefore, the purpose of this research was to study the effect of thermal treatment on the stability of ascorbic acid oxidase (AAO), total vitamin C content (l-ascorbic acid, l-AA), and dehydroascorbic acid (DHAA) and l-AA/DHAA ratio in cowpea leaves harvested at different maturities (4, 6, and 8 weeks old). The results showed that AAO activity, total vitamin C content, and l-AA/DHAA ratio in cowpea leaves increased with increasing maturity (up to 8 weeks). Eight-week-old leaves were the best source of total vitamin C and showed a high ratio of l-AA/DHAA (4:1). Thermal inactivation of AAO followed first-order reaction kinetics. Heating at temperatures above 90 °C for short times resulted in a complete AAO inactivation, resulting in a protective effect of l-AA toward enzyme-catalyzed oxidation. Total vitamin C in young leaves (harvested at 4 and 6 weeks) was predominantly in the form of DHAA, and therefore temperature treatment at 30-90 °C for 10 min decreased the total vitamin C content, whereas total vitamin C in 8-week-old cowpea leaves was more than 80% in the form of l-AA, so that a high retention of the total vitamin C can be obtained even after heating and/or reheating (30-90 °C for 10 min) before consumption. The results indicated that the stability of total vitamin C in situ was strongly dependent on the plant maturity stage and the processing conditions applied.     

Thermal oxidation of 9'-cis-neoxanthin in a model system containing peroxyacetic acid leads to the potent odorant beta-damascenone

The potent odorant beta-damascenone was formed directly from 9'-cis-neoxanthin in a model system by peroxyacetic acid oxidation and two-phase thermal degradation without the involvement of enzymatic activity. Beta-damascenone formation was heavily dependent on pH (optimum at 5.0) and temperature, occurring over the two sequential phases. The first was incubation with peroxyacetic acid at 60 degrees C for 90 min, and the second was at above 90 degrees C for 20 min. Only traces of beta-damascenone were formed on application of only one of the two phases. Formate and citrate solutions produced a much better environment for beta-damascenone formation than acetate and phosphate. About 7 microg/L beta-damascenone was formed from 5.8 mg/L 9'-cis-neoxanthin under optimal experimental condition. The detailed pathway by which beta-damascenone is formed remains to be elucidated.     

Capillary electrophoresis and high-performance liquid chromatography determination of polyglutamyl 5-methyltetrahydrofolate forms in citrus products

The 5-methyltetrahydrofolate (5mTHF) polyglutamates in citrus products were analyzed by capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). Folate species were purified from citrus products and concentrated from 2- to 100-fold using combined folate-affinity chromatography and C18 extraction. Seven polyglutamyl 5mTHFs were found in most not-from-concentrate (NFC) orange juices (OJ) in total amounts of approximately 1 nmol/mL, with varying distributions of individual polyglutamates. Folate amounts and distributions were also measured in orange fractions, single-strength OJ from concentrate, NFC grapefruit juice, and citrus peel molasses. Models containing ascorbic acid had folate thermal degradation rates one-seventh that of models without ascorbic acid. Pasteurization studies demonstrated that folate loss was <2% for commercial OJ pasteurization conditions (i.e., 93 degrees C for 5 s, 88 degrees C for 15 s, and 82 degrees C for 30 s). Both methods were precise, reproducible, and potentially faster than traditional analytical procedures requiring enzymatic deconjugation and microbial assays.     

Combined pressure-temperature effects on carotenoid retention and bioaccessibility in tomato juice

This study highlights the changes in lycopene and β-carotene retention in tomato juice subjected to combined pressure-temperature (P-T) treatments ((high-pressure processing (HPP; 500-700 MPa, 30 °C), pressure-assisted thermal processing (PATP; 500-700 MPa, 100 °C), and thermal processing (TP; 0.1 MPa, 100 °C)) for up to 10 min. Processing treatments utilized raw (untreated) and hot break (～93 °C, 60 s) tomato juice as controls. Changes in bioaccessibility of these carotenoids as a result of processing were also studied. Microscopy was applied to better understand processing-induced microscopic changes. TP did not alter the lycopene content of the tomato juice. HPP and PATP treatments resulted in up to 12% increases in lycopene extractability. all-trans-β-Carotene showed significant degradation (p < 0.05) as a function of pressure, temperature, and time. Its retention in processed samples varied between 60 and 95% of levels originally present in the control. Regardless of the processing conditions used, <0.5% lycopene appeared in the form of micelles (<0.5% bioaccessibility). Electron microscopy images showed more prominent lycopene crystals in HPP and PATP processed juice than in thermally processed juice. However, lycopene crystals did appear to be enveloped regardless of the processing conditions used. The processed juice (HPP, PATP, TP) showed significantly higher (p < 0.05) all-trans-β-carotene micellarization as compared to the raw unprocessed juice (control). Interestingly, hot break juice subjected to combined P-T treatments showed 15-30% more all-trans-β-carotene micellarization than the raw juice subjected to combined P-T treatments. This study demonstrates that combined pressure-heat treatments increase lycopene extractability. However, the in vitro bioaccessibility of carotenoids was not significantly different among the treatments (TP, PATP, HPP) investigated.     

Effect of different dosage and administration schedules of folic acid on blood folate levels in a population of Honduran women of reproductive age

BACKGROUND: Observational studies and clinical trials have shown conclusive evidence that periconceptional folic acid supplementation prevents up to 70 % of neural tube defects (NTD). The Honduran government wanted to implement a supplementation programme of folic acid but needed to assess the relative effects of two dosages of folic acid. OBJECTIVE: To determine the effect of two dosages of folic acid on blood folate levels in Honduran female factory workers aged 18 to 49 years. DESIGN: This was a randomized, double-blind control supplementation trial conducted in Choloma, Honduras. A total of 140 eligible women were randomly assigned to two dosage groups and followed up for 12 weeks. One group received a daily dosage of 1 mg folic acid and the other a once weekly dosage of 5 mg. Serum folate and red blood cell folate levels were determined by radioassay at baseline, 6 weeks and 12 weeks. RESULTS: Serum folate levels increased from 6.3 (se 0.2) to 14.9 (se 0.6) ng/ml (P < 0.0001) in women assigned to the 1 mg/d group and from 6.9 (se 0.3) to 10.1 (se 0.4) ng/ml (P < 0.0001) in those assigned to the 5 mg/week group. Red blood cell folate concentrations also increased significantly in both groups, albeit more slowly. Educational level, age and BMI were not associated with the changes in serum and red blood cell folate levels during the supplementation period. However, a differential effect on serum folate levels by dosage group and time was observed. CONCLUSIONS: Although both folate supplementation regimens increased serum and red blood cell folate levels significantly among the women studied, blood folate levels that are considered to be protective of NTD were reached faster with the daily dosage of 1 mg folic acid.     

Lipoxygenase inactivation in green beans (Phaseolus vulgaris L.) due to high pressure treatment at subzero and elevated temperatures

The kinetics of lipoxygenase (LOX) inactivation in green beans due to high-pressure treatment were studied in the pressure-temperature area of 0.1 up to 650 MPa and -10 up to 70 degrees C for systems with different levels of food complexity, i.e., in green bean juice and intact green beans (in situ study). For both systems, LOX was irreversibly inactivated by high-pressure treatment combined with subzero and elevated temperatures and the inactivation could be described as a first-order reaction. At ambient pressure, in situ LOX was less thermostable than in the juice at temperatures below 68 degrees C whereas the stability ranking was reverse at temperatures above 68 degrees C. At temperatures below 63 degrees C, sensitivity of the inactivation rate constants to temperature changes was on the same order of magnitude in the juice and in situ, while at higher temperature it was lower in situ. The pressure needed to obtain the same rate of LOX inactivation at a given temperature was lower in situ than in the juice. Application of high-pressure treatment at low/subzero temperature resulted in an antagonistic effect on LOX inactivation for both systems, whereas no such effect was found above room temperature. The pressure-temperature dependence of the LOX inactivation rate constants in green beans was successfully modeled.     

Biochemical characterization of a cambialistic superoxide dismutase isozyme from diatom Thallassiosira weissflogii: cloning, expression, and enzyme stability

A cDNA clone of 1081 bp encoding a second putative superoxide dismutase (SOD) from diatom Thallassiosira weissflogii was cloned by the polymerase chain reaction technique. The cDNA encodes a protein of 286 amino acid residues. Alignment of the truncated SOD sequence containing 217 amino acid residues with Mn-SODs from Vibrio mimicus and Escherichia coli, as well as two Fe-SODs from E. coli and Photobacterium leiognathi, this SOD showed greater homology to Mn-SOD. The residues required to coordinate the manganese ion were conserved in all reported Mn-SOD. The recombinant SOD has a half life of deactivation of 14.7 min at 65 degrees C. Its thermal inactivation rate constant Kd was 3.21 x 10(-2) min(-1). The enzyme was stable in a broad pH range from 4 to 12. The presence of imidazole (up to 0.8 M) and sodium dodecylsulfate (up to 4%) had little effect on the enzyme's activity. The atomic absorption spectrometric assay showed the presence of 0.3 atom of iron/manganese (2:1) in each SOD subunit. Reconstituted activity suggested that diatom SOD was cambialistic Fe/Mn-SOD.