Anionic Isoenzymes Research Articles

Purification and Biochemical Characterization of Two Anionic Peroxidase Isoenzymes from Raphanus sativus L. var niger Roots.

Peroxidase is a commonly used enzyme with a wide range of applications. Horseradish (Armoracia rusticana) is the most well-known source of peroxidase enzyme. Peroxidases extracted from other plant sources have also been proved as useful, sometimes even superior, comparing to traditional horseradish peroxidase (HRP). In the present study, two novel peroxidase isoenzymes were purified and characterized from Raphanus sativus L. var niger roots. Two anionic peroxidase isoenzymes were purified using diafiltration, ammonium sulfate precipitation, DEAE anion-exchange chromatography, and concanavalin A affinity chromatography. The heaviest anionic isoenzyme (isoenzyme A) has a MW of about 110 KD, and the other anionic isoenzyme (isoenzyme B) has a MW of 97 KD. Both isoenzymes have an optimum temperature of 40°C, but the activity of isoenzyme B is much more dependent on temperature with a Q10 of 3.5, while isoenzyme A has a Q10 of 1.7. These isoenzymes showed great thermal stability at 37°C and 4°C. Isoenzyme A showed the highest activity at pH 5 and it was found to be more stable at pH 6, whereas isoenzyme B showed the highest activity at pH 6 and is more stable at pH 7. Isoenzyme A has a Km value of 10.63mM and 0.043mM, and isoenzyme B has a Km of 15.38mM and 0.067mM for 4-aminoantipyrine and H2O2, respectively. The isoenzymes purified from Raphanus sativus L. var niger offer excellent chemical and thermal stability, which encourages further studies on their suitability for biotechnological applications.

Cadmium accumulation and alkaloid production of Narcissus tazetta plants grown under in vitro condition with cadmium stress

The capacity to accumulate cadmium (Cd) in Narcissus tazetta was investigated. Narcissus, an ornamental and medicinal plant can be used for the production of highly valuable alkaloids with anti-tumor, anti-viral and anti-cholinergic activities. For rapid propagation, in vitro micropropagation of this plant was done by three procedures: induction, proliferation and bulb production from twin scales as explants. After 4 months, micropropagated plants were treated by two concentrations of cadmium chloride (0.5, 1.0 mM) in the MS + 9% sucrose solidified media. After 3 weeks, growth, total proteins, peroxidase activity, Cd accumulation, quality and quantity of alkaloids were studied. Results showed that this plant accumulated 2778.13 μg g−1 DW, 801.87 μg g−1 DW and 162.8313 μg g−1 DW Cd in roots, bulbs and leaves respectively without any impact on growth. In order to study the ability of this plant in phytoremediation, tolerance index (TI), bioconcentration factors (BCF) and translocation factor (TF) were measured. Due to the TI > 0.6, BCF > 1 and TF < 1 concluded that Narcissus is suitable as phytostabilizer for remediation of Cd from contaminated media. Peroxidase activity increased under Cd stress. Also, isozyme pattern of peroxidase was changed and new anionic isoenzyme was appeared. Cd enhanced alkaloid content and altered its constituents as homolycorine was found instead of 9-O-demethylhomolycorine under Cd stress. This results presented in this study include the first report on the status of Narcissus alkaloids under Cd treatment.

Fungal penetration associated with recognition, signaling and defence-related genes and peroxidase activity during the resistance response of coffee to Colletotrichum kahawae

Resistant and susceptible coffee varieties were used aiming to unveil genes and proteins putatively involved in the resistance response to Colletotrichum kahawae. Gene expression analysis revealed a stronger induction of recognition, signaling and cell wall modification genes in the resistant variety during fungal penetration. It was also observed the increase of total peroxidase activity and the induction of lignin-forming anionic peroxidase-like gene which was further supported by the increase of an anionic isoenzyme. Peroxidases localized in the walls of host cells at infection sites can be related with cell wall lignification. Altogether, these defence responses contribute to restricting fungal growth.

Exploitation of neglected horseradish peroxidase izoenzymes for dye decolorization

Horseradish peroxidase (HRP) is enzyme first described more than 200 years ago and yet there are still some aspects of this potent enzyme to be tackled. Researchers were focused on most abundant isoenzyme HRP C1A while remaining, particularly anionic isoenzymes were discarded in purification process. This work describes exploitation of those isoenzymes for removal of recalcitrant pollutants such as reactive dyes. Results demonstrated that not only these enzymes can decolorize dyes but also in some cases anionic forms are more efficient than commercially produced cationic HRP form. Enzyme concentration of 0.14 U ml−1 was found to provide maximum dye removal at optimized reaction conditions with dye concentration of 30 mg l−1. Majority of dyes tested were successfully decolorized at pH 5 or 7 while some dyes like Orange 2 and Reactive black 5 are decolorized most efficiently at pH 9. Anionic isoenzymes act by disrupting chromophore of Reactive black 5 while cationic HRP oxidize dye but leaves chromophore present.

Cell wall bound anionic peroxidases from asparagus byproducts.

Asparagus byproducts are a good source of cationic soluble peroxidases (CAP) useful for the bioremediation of phenol-contaminated wastewaters. In this study, cell wall bound peroxidases (POD) from the same byproducts have been purified and characterized. The covalent forms of POD represent >90% of the total cell wall bound POD. Isoelectric focusing showed that whereas the covalent fraction is constituted primarily by anionic isoenzymes, the ionic fraction is a mixture of anionic, neutral, and cationic isoenzymes. Covalently bound peroxidases were purified by means of ion exchange chromatography and affinity chromatography. In vitro detoxification studies showed that although CAP are more effective for the removal of 4-CP and 2,4-DCP, anionic asparagus peroxidase (AAP) is a better option for the removal of hydroxytyrosol (HT), the main phenol present in olive mill wastewaters.

SOME PROPERTIES OF PEROXIDASE EXTRACTED FROM PULP OF STAR FRUIT (OXALIDACIA AVERRHOA)

The soluble and ionically bound peroxidase (EC. 1.11.1.7) was extracted from the star fruit in different stages of maturation. The extracts were prepared using solution 100 mM sodium phosphate buffer at pH 7.0 and the enzymatic activity of those extracts were measured. The maturation degree did not show an influence in the activity of soluble peroxidase; however the same change in the activity for the ionic bound peroxidase was observed. For the study of the thermal stability of the peroxidase, the crude extracts were submitted at temperatures of 60, 65, 70, 75, 80 and 85°C and the enzymatic activity was carried out in intervals of 0, 2, 4, 6, 8 and 10 minutes. It was observed that the enzyme lost activity with increased of the time and the temperature. In the purification step, the concentrated extract of the soluble peroxidase was dialyzed, using a solution of 10 mM of the sodium phosphate buffer pH 7.0, after the sample was applied in a chromatographic column (16 mm i.d. x 70 cm) packed with Sephacryl HS100 with flow rate of 30 ml/h. The results of electrophoresis show a presence of one pattern of the anionic isoenzyme.

Antioxidant defense system in leaves of Indian mustard (Brassica juncea) and rape (Brassica napus) under cadmium stress

Plant species capable of hyper-accumulating heavy metals are of considerable interest for phytoremediation, and differ in their ability to accumulate metals from environment. Using two brassica species (Brassica juncea and Brassica napus), nutrient solution experiments were conducted to study variation in tolerance to cadmium (Cd) toxicity based on (1) lipid peroxidation and (2) changes in antioxidative defense system in leaves of both plants (i.e., superoxide dismutase (SOD EC 1.15.1.1), catalase (CAT EC 1.11.1.6), ascorbate peroxidase (APX EC 1.11.1.11), guaiacol peroxidase (GPX EC 1.11.1.7), glutathione reductase (GR EC 1.6.4.2), levels of phytochelatins (PCs), non-protein thiols (NP-SH), and glutathione. Plants were grown in nutrient solution under controlled environmental conditions, and subjected to increasing concentrations of Cd (0, 10, 25 and 50 μM) for 15 days. Results showed marked differences between both species. Brassica napus under Cd stress exhibited increased level of lipid peroxidation, as was evidenced by the increased malondialdehyde (MDA) content in leaves. However, in Brassicajuncea treated plants, MDA content remained unchanged. In Brassica napus, with the exception of GPX, activity levels of some antioxidant enzymes involved in detoxification of reactive oxygen species (ROS), including SOD, CAT, GR, and APX, decreased drastically at high Cd concentrations. By contrast, in leaves of Brassica juncea treated plants, there was either only slight or no change in the activities of the antioxidative enzymes. Analysis of the profile of anionic isoenzymes of GPX revealed qualitative changes occurring during Cd exposure for both species. Moreover, levels of NP-SH and PCs, monitored as metal detoxifying responses, were much increased in leaves of Brassica juncea by increasing Cd supply, but did not change in Brassica napus. These results indicate that Brassica juncea plants possess the greater potential for Cd accumulation and tolerance than Brassica napus.

A Novel Heme Peroxidase from Raphanus sativus Intrinsically Resistant to Hydrogen Peroxide

AbstractAll known heme peroxidases are susceptible to self‐inflicted oxidative damage. Substrate oxidation in peroxidases is a naturally imperfect process and damage emerges from the utilization of protein elements as electron sources through the catalytic intermediate Compound III. Given their wide application potential, the search for more stable heme peroxidases has been actively pursued in the past, albeit with limited success. Here, the identification and biochemical characterization of a novel H2O2‐resistant isoenzyme is reported which was isolated from daikon radish (Raphanus sativus L. cv. daikon) and named Zo peroxidase (ZoPrx). Partial protein sequencing demonstrated that ZoPrx is more related to anionic isoenzyme 53 from Arabidopsis thaliana and to anionic isoenzyme 2 from Armoracia rusticana (HRPA2) than to any other protein known to date. The stability of ZoPrx was demonstrated by rigorous evaluation of resistance to H2O2 in the absence or presence of exogenous reductants which was consistent with porphyrin integrity. ZoPrx systematically outperformed the reference HRPA2 isoenzyme, a heme peroxidase known for its relative oxidative stability. The catalytic cycle of ZoPrx is conventional in the sense that the usual intermediate Compounds I and II were identified although reaction rates are significantly different compared to HRPA2. The distinctive characteristic of ZoPrx is that when Compound III is formed in the presence of excess H2O2, it is particularly more stable and, instead of engaging into the regular deactivation decay pathways, it commits to the ground state restoration decay pathway. ZoPrx is the only known case of a naturally occurring stable heme peroxidase based on intrinsic properties. Deeper study of the structural determinants underlying this property may eventually allow us to graft it into other peroxidases with established commercial potential.

Transcriptional and translational analyses of poplar anionic peroxidase isoenzymes

Anionic peroxidases have been proposed to be a key enzyme for lignification in poplar. On the other hand, there are many genes encoding an anionic peroxidase in Populus trichocarpa genome, and their physiological functions are still unknown. Ampholine isoelectric focusing analysis revealed anionic peroxidases as dominant peroxidases in enzyme preparations from various organs. Using two-dimensional electrophoresis (2-DE) followed by peptide mass fingerprint (PMF) analysis, we surveyed the localization of anionic peroxidase isoenzymes in various organs of Populus alba L. Peroxidase isoenzymes were extracted from various organs and fractionated by a Concanavallin A Sepharose column. Each protein was separated by 2-DE gels and some anionic peroxidase isoenzymes in each organ were identified via PMF analysis. Transcript and protein of individual peroxidase indicate that the expression profile of each isoenzyme is quite different, for example, organspecific gene, stress-response gene, and multifunction gene, even though they are in the same cluster. These results suggest that individual anionic isoenzymes in this small cluster were differently regulated at transcription, translation, or posttranslation.

Abiotic Stress Generates ROS That Signal Expression of Anionic Glutamate Dehydrogenases to Form Glutamate for Proline Synthesis in Tobacco and Grapevine

Glutamate dehydrogenase (GDH) may be a stress-responsive enzyme, as GDH exhibits considerable thermal stability, and de novo synthesis of the alpha-GDH subunit is induced by exogenous ammonium and senescence. NaCl treatment induces reactive oxygen species (ROS), intracellular ammonia, expression of tobacco (Nicotiana tabacum cv Xanthi) gdh-NAD;A1 encoding the alpha-subunit of GDH, increase in immunoreactive alpha-polypeptide, assembly of the anionic isoenzymes, and in vitro GDH aminating activity in tissues from hypergeous plant organs. In vivo aminating GDH activity was confirmed by gas chromatorgraphy-mass spectrometry monitoring of (15)N-Glu, (15)N-Gln, and (15)N-Pro in the presence of methionine sulfoximine and amino oxyacetic acid, inhibitors of Gln synthetase and transaminases, respectively. Along with upregulation of alpha-GDH by NaCl, isocitrate dehydrogenase genes, which provide 2-oxoglutarate, are also induced. Treatment with menadione also elicits a severalfold increase in ROS and immunoreactive alpha-polypeptide and GDH activity. This suggests that ROS participate in the signaling pathway for GDH expression and protease activation, which contribute to intracellular hyperammonia. Ammonium ions also mimic the effects of salinity in induction of gdh-NAD;A1 expression. These results, confirmed in tobacco and grape (Vitis vinifera cv Sultanina) tissues, support the hypothesis that the salinity-generated ROS signal induces alpha-GDH subunit expression, and the anionic iso-GDHs assimilate ammonia, acting as antistress enzymes in ammonia detoxification and production of Glu for Pro synthesis.

Avaliação da atividade da peroxidase em carambola (Oxalidacia averrhoa) em diferentes estádios de maturação

The soluble and ionically bound peroxidase (EC. 1.11.1.7) was extracted from the star fruit in different stages of maturation. The extracts were prepared using solution 100 mM sodium phosphate buffer at pH 7.0 and the enzymatic activity of those extracts was measured. The maturation degree did not influence the soluble peroxidase activity; however, some changes were observed in the activity for the ionic bound peroxidase. For the study of peroxidase thermal stability, the crude extracts were submitted to temperatures of 60, 65, 70, 75, 80 and 85°C and the enzymatic activity was carried out at intervals of 0, 2, 4, 6, 8 and 10 minutes. It was observed that the enzyme lost activity with increasing time and temperature. For purification, the concentrated extract of the soluble peroxidase was dialysed, using a solution of 10mM of the sodium phosphate buffer pH 7.0, after the sample was applied in a chromatographyc column (16 mm i.d x 70 cm) packed with Sephacryl HS100 with flow rate of 30 mL/h. The electrophoresis results showed the presence of an anionic isoenzyme with 64kDa MW.

Isolation and thermostability of peroxidase isoenzymes from apple cultivars Gala and Fuji

Extracts of soluble peroxidase and ionically-bound peroxidase were obtained from the peel and pulp of apples ( Mallus comunis), cultivars Gala and Fuji, using 100 mM sodium phosphate buffer and pH 6.0 and 7.0 for pulp and peel of Gala cultivar, respectively and pH 5.5 for the pulp and the peel of cultivar Fuji. Then the enzymatic activity of peroxidase (POD) was determined in both extracts, soluble fractions and ionically-bound. Electrophoresis results shows similar compositions for the anionic and cationic isoenzymes in both cultivars. The molecular weights and p I values of the isolated isoenzymes from the Fuji cultivar were: two anionic, A 1 and A 2 (38, 28 kDa and p I 4.4; 5.0, respectively) and three cationic C 1, C 2 and C 3 (40, 34, 26 kDa and p I 8.0, 8.3, 9.0, respectively). From the Gala cultivar, also, two anionic isoenzymes were isolated A 2 and A 4 (28, 26 kDa and p I 5.0 and 5.7, respectively) and three cationic isoenzymes C 1, C 2 and C 3 (40, 34, 26 kDa and p I 8.0, 8.3 and 9.0, respectively). The cationic isoenzymes were more heat-stable under the heat treatments at 65, 70, 75 and 80 °C in both cultivars.

Efeito do extrato aquoso de leucena sobre o desenvolvimento, índice mitótico e atividade da peroxidase em plântulas de milho

A leucena (Leucaena leucocephala (Lam.) de Wit), quando usada como cobertura no solo, apresenta a propriedades de controlar plantas daninhas, sendo esse efeito resultante da presença de aleloquímicos, principalmente mimosina, encontrados na parte aérea da planta. A maioria dos estudos em alelopatia refere-se apenas ao efeito do aleloquímico sobre a germinação e o crescimento da planta-teste, sem considerar os eventos celulares relacionados às mudanças fisiológicas. Com este trabalho objetivou-se avaliar o efeito do extrato aquoso da leucena sobre o desenvolvimento, índice mitótico radicular e atividade da peroxidase e suas isoformas na parte aérea e raízes de plântulas de milho. Os bioensaios foram conduzidos em casa-de-vegetação utilizando-se sementes de milho híbrido desenvolvidas nas concentrações 0; 0,4; 0,8; 1,6; 3,2 e 6,4 % do extrato aquoso de leucena. Verificaram-se inibição do crescimento de raízes e redução do índice mitótico proporcional ao incremento na dose do extrato, não sendo observada divisão celular a partir da concentração de 1,6 %. A atividade da peroxidase, tanto em raízes como em folhas, cresceu com o aumento da concentração do extrato, sem, contudo, apresentar diferença nos zimogramas de suas isoformas. A maior atividade dessa enzima, nas raízes, foi correlacionada com a presença das isoformas aniônicas, pI 4,99 e 4,86, o que possivelmente esteja contribuindo para maior espessamento das raízes, o que foi verificado nas doses elevadas do extrato. As análises por HPLC revelaram teor de mimosina crescente com aumentos na concentração do extrato, sugerindo o envolvimento desse aleloquímico no desenvolvimento das plântulas.

The syringaldazine-oxidizing peroxidase PXP 3-4 from poplar xylem: cDNA isolation, characterization and expression.

The cell wall polymer lignin is believed to be condensed by specific cell wall-localized oxidoreductases. In many plants species, including poplar, the peroxidase-directed oxidation of the lignin analogue syringaldazine (SYR) has been localized to cells that undergo secondary wall formation, a process that includes lignification. As a first step to analyse the corresponding peroxidases. we have isolated previously two anionic isoenzymes (PXP 3-4 and PXP 5) from poplar xylem (Populus trichocarpa), which use SYR as a substrate. Here, we demonstrate that these enzymes are responsible for the visualized SYR oxidation in the developing xylem. The cDNA that corresponds to PXP 3-4 was isolated and the deduced protein was found closely related to the other SYR-oxidizing peroxidase PXP 5 (ca. 98% of identity). PXP 3-4 was expressed in a baculovirus expression system yielding high levels of active peroxidase (3 mg/l medium). The heterologously produced protein showed characteristics similar to those of the corresponding protein from poplar xylem (enzymatic properties, isoelectric point, and migration in a native gel). PXP 3-4 was expressed in the stem and in the root xylem. The data demonstrate that PXP 3-4 (and/or PXP 5) are present in differentiating xylem. supporting a function in secondary cell wall formation.

Purification and kinetic characterization of an anionic peroxidase from melon (Cucumis melo L.) cultivated under different salinity conditions.

The partial characterization of an anionic peroxidase in melon fruit is described. Four melon peroxidase (MPX) isoenzymes were detected in crude extracts after isoelectric focusing. The major MPX isoenzyme (pI = 3.7) was partially purified by including hydrophobic and anion-exchange chromatography in the purification scheme. The sample obtained was used to characterize MPX. This peroxidase did not show activity on ascorbic acid but oxidized guaiacol at a high rate, showing an optimum pH of 5.5 when acting on this last reducing substrate. Melon fruits grown under highly saline conditions showed slightly increased levels of this anionic isoenzyme. Kinetic studies using 2,2'-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) as reducing substrate showed that increased salinity in the growth medium did not modify the kinetic parameters of melon peroxidase on both hydrogen peroxide and reducing substrate.

A study of peroxidase and amylase isoenzymes in different radish cultivars during winter storage

The changes in activities of soluble peroxidase (EC 1.11.1.7; POD) and amylase (EC 3.2.1.1) and in levels of soluble protein and reducing sugar are investigated in three cultivars of radishes (Raphanus Sativus L) in winter storage for 150 days. It has been shown that the activity of peroxidase increases at first and then decreases during the storage. The highest POD activity appears on the 90th day. The levels of reducing sugars change similarly. Peroxidase isoenzymes (types and activities) in these radish cultivars are obviously different during the storage. The highest activities of peroxidase isoenzymes are observed in Xinlimei (XLM) and the lowest in Dihuangying (DHY). The difference between the radish cultivars is also observed in the peroxidase isoenzyme types. Both cationic and anionic peroxidase isoenzymes appear in the radish during storage. The activities and PAGE bands of the amylase isoenzymes decrease with increasing storage time. The activities of amylase are the lowest in the XLM but highest in the DHY. A similar change is found for the levels of soluble protein during the storage: the levels are highest in XLM and lowest in DHY. These results suggest a possible correlation between the activity of peroxidase and the level of reducing sugar during storage. © 2000 Society of Chemical Industry

PURIFICATION AND PROPERTIES OF TWO ACID PEROXIDASES FROM BRUSSELS SPROUTS (Brassica oleraceae L.)

An acid peroxidase isoenzyme (A1) from Brussels sprouts (Brassica oleraceae L.) has been purified while another isoenzyme (A2) has been partially purified. Studies of their properties leading to a potential application in immunoassays as an alternative to horseradish peroxidase were conducted. Isoenzyme Al was purified 503 fold, through ammonium sulfate and acetone fractionation, and successive chromatography on DEAE-cellulose, Sephadex G-100 and Mono-S (FPLC system) columns. Isoenzyme A1 had a pI of 4.0, a molecular weight of 90 kDa, and contained two identical molecular weight subunits. Preliminary studies indicated a pI of 4.7 for isoenzyme A2. ABTS [2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid)] K m values (0.2 mM) for both isoenzymes are 20 times lower than those reported for commercial horseradish peroxidase anionic isoenzymes. Optimum pH for activity of isoenzymes A1 and A2 were 4.3 and 4.5, respectively. Optimum temperature for isoenzyme A1 was 57C, with an activation energy for inactivation of 148.8 kJ/mol.

The thermostability of purified isoperoxidases from Brassica oleracea VAR. gemmifera

The thermostabilities of four previously purified isoperoxidases from Brussels sprouts ( Brassica oleracea VAR. gemmifera.)have been determined. The heating time periods selected (10 s – 0.5 min intervals) are comparable to those used during commercial blanching. Non-linear regression (NLR) equation fitting, using common goodness of fit criteria (low chi-squared value, high regression coefficient and low residuals) points to a mechanism of peroxidase heat inactivation involving two consecutive reactions during the initial periods of heating. In the consecutive model, native peroxidase ( E 0) is converted into a partially active from ( E 1) and then into the inactivated enzyme ( E 2) during short periods of heating. The order of calculated decimal reduction times for the two anionic (A1 and A2) and two cationic (C1 and C2) Brussels sprouts isoperoxidases was A1⩾ C1> A2> C2. Calculated concentration changes for E 0, E 1 and E 2 during heat inactivation were quite different for the four isoperoxidase preparations and indicated the generation of more stable forms of partially denatured peroxidases. The anionic isoenzymes showed greater regeneration of enzymatic activity after heat treatment and this could have been due to their greater ability to regain previously liberated haem. ©

Nickel-induced oxidative damage and antioxidant responses in Zea mays shoots

Zea mays (cv. LG 23/01) seedlings precultured in hydroponic culture for seven days were treated with 250 μM NiCl 2 for five days. Several metabolic parameters representative of oxidative damage and antioxidant activity in shoots were regularly studied after the metal treatment. Two days after Ni-application, membrane lipid peroxidation intensified. A stimulation of guaiacol peroxidase (EC 1.11.1.7) activity was recorded. Analysis of the profile of anionic isoenzymes of guaiacol peroxidase revealed quantitative changes occurring during Ni-exposure. Superoxide dismutase (EC 1.15.1.1) activity was stimulated early and transiently. Later, the activities of ascorbate peroxidase (EC 1.11.1.11), catalase (EC 1.11.1.6) and glutathione reductase (EC 1.6.4.2) increased. The results are discussed with regard to nickel-induced oxidative stress and potential antioxidant reactions.

Estudo da termoestabilidade de peroxidases extraídas da polpa e casca de mexerica (Citrus deliciosa)

Extracts of the soluble and ionic-bound peroxidases were prepared from tangerine (Citrus deliciosa) pulp and peel and submitted to heat treatments at 60ºC, 65ºC and 70ºC during a period of 0 to 10 minutes for the study of the peroxidase enzymatic activity. Ion exchange chromatography (DEAE-Sepharose) was carried out in the soluble fraction of the peel to isolate cationic and anionic iso-enzymes. Separation was confirmed by electrophoresis. Two anionic and two cationic iso-enzymes were found in the soluble fraction of peel. Enzyme activity showed itself to be non-linear with increase of temperature.