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[238] Diamine oxidase (pea seedling)
730
AMINE OXIDATION AND HYDROXYLATION
[238]
tains 12.2 millimicromoles of FAD per milligram of protein, s This value corresponds to 1 mole of FAD per mole of enzyme. Substrates. Cadaverine and spermidine are also oxidized, but at considerably lower rates. Other diamines, histamine, agmatine, and spermine are not oxidized. Spermidine is oxidatively degraded into y-aminobutyraldehyde and 1,3-diaminopropane stoichiometrically; no ammonia is liberated. pH Optimum and K,,. The pH optimum for enzyme activity is 8.0 for putrescine and 8.5 for spermidine. The K,, value is 2.3 × 10-4 M for both substrates. Inhibitors. The enzyme is inhibited by SH reagents such as p-chloromercurihenzoate. The inhibition is not immediate, but requires preincubation of the enzyme with the inhibitor. The enzyme is also inhibited by cyanide, hydroxylamine, and iproniazid, but not by phenylhydrazine, semicarbazide, isoniazid, and other metal-chelating agents. sv. Massey, G. Palmer, and R. Bennett, Biochim. Biophys. Acta 48, 1 (1961).
[238] Diamine Oxidase (Pea Seedling) 1 ByJ. M. D i a m i n e + H 2 0 + 02
HILL
) aldehyde + H202 + NH3
In the presence of catalase H202 is decomposed and the overall reaction then is Diamine + V202
) aldehyde + NH3
Assay Method Principle. The method is based on measuring the uptake of oxygen in the presence of catalase, with putrescine (1,4-diaminobutane) as the substrate. Cadaverine (1,5-diaminopentane) has been used in the assay of other amine oxidases, but cannot be used with crude plant extracts because inhibitors of the plant enzyme may be formed by secondary oxidations) a
1EC 1.4.3.6 diamine : oxygen oxidoreductase (deaminating); histaminase. For the preparation of various other amine oxidases, see this volume [229]-[239]. la]. M. Hill, Biochem.J. 104, 1048 (1967).
[238]
DIAMINE OXIDASE (PEA SEEDLING)
731
Reagents KH2PO4, 0.2 M, adjusted to pH 7.0 with KOH Catalase (a solution containing 250/zg/ml of the crystalline enzyme) Putrescine hydrochloride, 0.1 M KOH, 5 N Enzyme solution, dilute as necessary
Procedure. Manometers of the Warburg type are used with reaction flasks of about 25 ml volume. A reaction mixture containing 1 ml of the phosphate buffer, 0.1 ml catalase solution, enzyme solution, and glass-distilled water, to give a total volume of 2.9 ml, is placed in the main compartment; 0.1 ml of putrescine solution is put in the side arm and 0.2 ml of 5 N KOH plus a small piece of Whatman No. 40 filter paper in the center well. The putrescine solution is replaced by tile same volume of water in control experiments. The reaction mixtures are equilibrated by shaking at about 60 oscillations per minute f()r 15 minutes at 30 °. After initiation of the reaction by tipping the contents of the side arm into the reaction mixture, the oxygen uptake is measured at 5-minute intervals during the first 20 minutes. The greatest oxygen uptake over either the reaction period from 0-10 minutes, or 5-15 minutes is taken and is used to calculate the average oxygen uptake per minute. De/tuition 0/ Unit and Specific Activity'. The international unit (I.U.) is used and is defined as the amount of enzyme that will catalyze the oxidative deamination at pH 7 of 1 micromole of putrescine per minute at 30 ° . An oxygen uptake of 11.2 /xl is found for the oxidation of I micromole of putrescine in reaction mixtures containing catalase. Specific activity is defined as units per milligram of protein, the protein content being calculated from the nitrogen content, as found by tile Kjeldahl method, and multiplying this by the factor 6.25. P u r i f i c a t i o n P r o c e d u r e ",3
The method depends on removing much of the material ~¥om the crude extract by precipitation with a mixture of chloroform and ethanol (Tsuchihashi's reagent4), then precipitating the enzyme first with (NH4)2SO4 and secondly at pH 5 by the method of Tabor? The enzyme is finally purified by chromatography on hydroxyapatite and •zp..]. (;. Mann, Biochem.]. 5 9 , 6 0 9 (1955). :q'. J. (,. Mann, Bio~hem.]. 79, 623 ( 1961 ). ~M. Tstwhihashi, Biochem. Z. 140, 63 (1923). "H. "l'abov,[. Biol. (;hem. 188, 125 ( 1951 ); see also Vol. I I [54].
732
AMINE OXIDATION AND HYDROXYLATION
[238]
DEAE-cellulose columns. If the chloroform-ethanol treatment is omitted, considerable difficulty may be experienced in the subsequent purification of the enzyme at the pH 5 precipitation and later stages. The diamine oxidase content of pea seedlings (Pisum sativum) is at a maximum over the period between 7 and 16 days after germination. We usually use them when 10 days old. Varieties differ slightly in their initial diamine oxidase activity, and in the ease with which the enzyme may be purified. In this laboratory we mainly use the cultivar "Fillbasket," but many other varieties have also been used successfully.
Step 1. Preparation of Crude Extract. About 1 kg of pea-seeds are soaked in r u n n i n g tap water for 18-24 hours and then sown thickly at a depth of 4-6 mm in coarse sand. The sand should previously have been well washed with tap water and fill, to a depth of 10-12 cm, the two plastic bowls, each measuring about 30 × 40 cm, in which the seeds are grown. The seeds are germinated and grown in a darkened cupboard; it is essential that the seedlings be grown in the dark as this makes the final stages in the purification of the enzyme easier than when plants have been grown in the light. About 10 days after sowing, when the etiolated shoots are 2-5 cm tall, the cotyledons and shoots (2-2.5 kg) are harvested, washed free from sand with cold tap water, drained, weighed, and put through a powered meat mincer. The mince is placed in cotton cloth and the juice is squeezed out in a screw press. The solid residue is mixed with 0.1 M potassium phosphate buffer pH 7 (1 ml/gram fresh material), and the liquid is squeezed out in the screw press. A further extraction of the solid residue is then made with 0.5 ml of the phosphate buffer for each gram of fresh material. The liquid extracts (2-3 liters) are combined and cooled to below 5°. Step 2. Precipitation of Inactive Material with An Ethanol-Chloroform Mixture. The volume of the crude extract is measured and a 2 : 1 v/v ethanol : chloroform mixture (30 ml for each 100 ml crude extract), which has been cooled to <--10 °, is added over a 30 minute period. Care must be taken to ensure that the temperature of the crude extract does not rise above +5 ° during this addition. The mixture is now allowed to stand for 1 hour at 0°-+5% after which the inactive precipitate is removed by centrifuging for 20 minutes at 3000-4000 g. The supernatant liquid is collected and saturated with (NH4)2804 (45 g/100 ml), and the temperature allowed to rise to about 10°. A solid separates and rises to the surface; the lower liquid is siphoned off and discarded, and the remaining slurry is centrifuged for 10-15 minutes at about 3000 g. When straight-sided centrifuge tubes are used, the hard curd that forms is easily removed with a flat spatula. This curd is homogenized with
[238]
DIAMINE OXIDASE (PEA SEEDLING)
733
0.02 M potassium phosphate buffer pH 7 (1 ml/2 g initial fresh pea seedlings), and allowed to stand overnight at about +2 ° . Step 3. Precipitation with (NH4)2S04. The 0.02 M phosphate buffer suspension is stirred for 1-2 hours at between 15-20 ° and the precipitate is removed by centrifuging for 20 minutes at 3000-4000 g. The supernatant liquid is treated with (NH4)2SO4 (45 g/100 ml) and, after standing at about 10° for 1 hour, the precipitate is collected by centrifuging at 3000 g for about 20 minutes. This precipitate is triturated with about 20 ml 0.2 M potassium phosphate buffer, pH 7, and then dialyzed, first against cold running tap water for 3-4 hours, and then against 3 changes, each of 2 liters of 0.005 M potassium phosphate buffer pH 7 over a period of 36 hours at 0-4 °. Step 4. Precipitation at pH 5. The dialyzed material is centrifuged at 2000-3000 g to remove inactive precipitate. The supernatant liquid, after cooling to about 5 °, is adjusted to pH 5 by the slow addition of 0.05 N acetic acid and allowed to stand for 1 hour at 2-4 °. The precipitate is collected by centrifuging, triturated with 10-15 ml water, and finally brought into solution by adjusting to pH 7 with 0.05 N KOH. This precipitation at pH 5 is repeated twice and the final solution made up to a suitable volume (1 ml for each 100-g pea seedlings) in 0.01 M potassium phosphate buffer pH 7. This enzyme solution can be stored for many months at--20 ° without appreciable loss of activity. Step 5. Fractionation on Hydroxyapatite. Hydroxyapatite is prepared by the method of Tiselius, Hjert6n, and Levin. 6 Five to eight of the enzyme preparations obtained at the end of the previous step are combined and centrifuged, and the supernatant solution (100-200 ml) is applied to a hydroxyapatite column (4 × 12 cm) equilibrated with 0.01 M potassium phosphate buffer pH 7. This and all subsequent fractionations are done at 0-5 °. The column is eluted first with 0.04 M potassium phosphate buffer, pH 7, and this is continued by stepwise increments (0.04 M) in the concentration of buffer up to a final concentration of 0.2 M. The volume of buffer at each concentration is 100 ml and the flow rate should be 50-80 ml/hour. The eluate is collected in 5-ml samples and the fractions containing the main amine oxidase peak showing the highest amine oxidase activity in relation to extinction at 280 m/~ (samples numbered approximately 100-140), are combined (approximately 150-250 ml), and dialyzed for 24 hours against 2 changes of 3 liters of distilled water. The ratio of amine oxidase units/E2s0 mu in the fractions combined should exceed 15. The amine oxidase fractions at this stage are usually pink. 6A. Tiselius, S. Hjert6n, and 0. Levin, Arch. Biochem. Biophy,~. 65, 132 (1956); 0. Levin, Vol. V [2].
see also
734
[238]
AMINE OXIDATION AND HYDROXYLATION
Step 6. Adsorption of Inert Material on Diethylaminoethyl Cellulose. Whatman DE 11 cellulose powder is suspended in water and adjusted to pH 7 with 1 M KH2PO4. A column (2 X 10 cm) is prepared and is equilibrated with 0.01 M phosphate buffer pH 7. The dialyzed amine oxidase preparation from the previous step is passed through this column and the column is then washed with 25 ml of 0.01 M phosphate buffer pH 7. Step 7. Concentration on Hydroxyapatite. The enzyme solution and washings (approximately 200-250 ml) are combined and applied to a column of hydroxyapatite (4 x 4 cm). The enzyme is eluted with 0.2 M potassium phosphate buffer pH 7 as a single pink band and is stored at < - 1 0 °. This enzyme can be stored at --20 ° for many months without loss of activity. SUMMARY OF THE PURIFICATION OF DIAMINE OXIDASE FROM PEA SEEDLINGS
Step 1. 3. 4. 4. 5. 7.
Fraction
Volume (ml)
Crude extract 2260 Dialyzed fraction after 31.5 (NH4)zSO4 precipitation After precipitation at pH 5 11.0 Combined fractions from 120 step 4 Eluate from hydroxyapatite 237 Eluate from DEAE after 20 concentration
Total units (I.U.)
Total protein (mg)
3320 1790
81,400 250
Specific activity (units/mg of protein) 0.041 7.16
1340 8820
44.6 329
30.0 26.8
5020 4560
114 62.4
44.1 73.1
Properties Specificity. This enzyme (specific activity greater than 60) catalyzes the oxidative deamination of a wide range of amines containing the ---CH2NHz grouping. The most active substrates are putrescine (1127) and cadaverine (112). 1,3-Diaminopropane is not attacked, but 1,6-diaminohexane (28) and 1,10-diaminodecane (21.5) are attacked. Other substrates are lysine (6.4), ornithine (0.7), histamine (6.0), spermine (0.4), spermidine (39), agmatine (39), tryptamine (7.6), benzylamine (4.9), tyramine (20), 2-phenylethylamine (32), n-propylamine (11), and some other aliphatic diaminesY pH Optimum. The optimum pH depends on the substrate and is about
rNumbers in parenthesis represent the relative activity observed with the different substrates.
[239]
DIAMINE OXIDASE (PIG KIDNEY)
735
7.0 for putrescine and 8.5 for /~-phenylethylamine in experiments carried out at 0.01 M and 0.002 M concentrations. Reaction with Substrate. The legume seedling diamine oxidases, unlike all other amine oxidases described so far, react with substrates under anaerobic conditions to give yellow complexes. The absorption spectra of these complexes are identical and have maxima at 350, 437, and 466 m/~.a'~ Inhibitors. This enzyme is strongly inhibited by carbonyl reagents, Cu z+ ions and some chelating agents including 8-hydroxyquinoline, 1,10-phenanthroline and sodium diethyldithiocarbamate;--SH group reagents do not significantly inhibit. Metal Requirements. Purified pea seedling diamine oxidase contains 0.08-0.09% copper. All the copper is in the divalent state and does not change valency during the enzymic reaction. Copper is firmly bound to the protein, but can be removed with sodium diethyldithiocarbamate: the inactive copper-free protein is reactivated specifically by Cu "+ ions. Color. The purified enzyme is pink with an absorption maximum at 505 m~ at pH 7; at the same pH the copper-free diamine oxidase absorbs maximally at 480 mtz. sj. M. Hill and P. J. G. Mann, Biochem..]. 91, 171 (1964).
[239] Diamine Oxidase (Pig Kidney) ~ By BRUNO MONDOVi, GIUSEPPE ROTILIO, MARIA TERESA COSTA, and ALESSANDRO FINAZZI AGR~) RCH2NH2 -+- 02 + H20 ~ R C H O + NH3 + H202
R indicates the residual chain of aliphatic diamines or of histamine. Although some authors have claimed that aliphatic diamines and histamine are oxidized by two different pig kidney enzymes, the identity between diamine oxidase and histaminase seems to be well established? a ~E.C. 1.4.3.6; diamine:oxygen oxidoreductase (deaminating); histaminase. A less purified preparation of this enzyme was described by H. Tabor in Vol. II [54]. Recently, homogeneous preparations of diamine oxidase from hog kidney have been obtained by somewhat different purification procedures by Goryachenkova, Shcherbatyuk, and Voronina [Biokhimiya 32, 398 (1967)] and by Yamada, Kumagai, Kawasaki, Matsui, and Ogata [Biochem. Biophys. Res. Commun. 29, 723 (1967)]. The latter preparation is crystalline, contains 2.17 g-moles of copper per mole of the enzyme, has a molecular weight of 185,000 and an absorption maximum at 470 naN. ~aB. Mondovl, (;. Rntilio, A. Finazzi Agr6, and A. Scioscia Santoro, Biochem..]. 91, 408 (1964).
AMINE OXIDATION AND HYDROXYLATION
[238]
tains 12.2 millimicromoles of FAD per milligram of protein, s This value corresponds to 1 mole of FAD per mole of enzyme. Substrates. Cadaverine and spermidine are also oxidized, but at considerably lower rates. Other diamines, histamine, agmatine, and spermine are not oxidized. Spermidine is oxidatively degraded into y-aminobutyraldehyde and 1,3-diaminopropane stoichiometrically; no ammonia is liberated. pH Optimum and K,,. The pH optimum for enzyme activity is 8.0 for putrescine and 8.5 for spermidine. The K,, value is 2.3 × 10-4 M for both substrates. Inhibitors. The enzyme is inhibited by SH reagents such as p-chloromercurihenzoate. The inhibition is not immediate, but requires preincubation of the enzyme with the inhibitor. The enzyme is also inhibited by cyanide, hydroxylamine, and iproniazid, but not by phenylhydrazine, semicarbazide, isoniazid, and other metal-chelating agents. sv. Massey, G. Palmer, and R. Bennett, Biochim. Biophys. Acta 48, 1 (1961).
[238] Diamine Oxidase (Pea Seedling) 1 ByJ. M. D i a m i n e + H 2 0 + 02
HILL
) aldehyde + H202 + NH3
In the presence of catalase H202 is decomposed and the overall reaction then is Diamine + V202
) aldehyde + NH3
Assay Method Principle. The method is based on measuring the uptake of oxygen in the presence of catalase, with putrescine (1,4-diaminobutane) as the substrate. Cadaverine (1,5-diaminopentane) has been used in the assay of other amine oxidases, but cannot be used with crude plant extracts because inhibitors of the plant enzyme may be formed by secondary oxidations) a
1EC 1.4.3.6 diamine : oxygen oxidoreductase (deaminating); histaminase. For the preparation of various other amine oxidases, see this volume [229]-[239]. la]. M. Hill, Biochem.J. 104, 1048 (1967).
[238]
DIAMINE OXIDASE (PEA SEEDLING)
731
Reagents KH2PO4, 0.2 M, adjusted to pH 7.0 with KOH Catalase (a solution containing 250/zg/ml of the crystalline enzyme) Putrescine hydrochloride, 0.1 M KOH, 5 N Enzyme solution, dilute as necessary
Procedure. Manometers of the Warburg type are used with reaction flasks of about 25 ml volume. A reaction mixture containing 1 ml of the phosphate buffer, 0.1 ml catalase solution, enzyme solution, and glass-distilled water, to give a total volume of 2.9 ml, is placed in the main compartment; 0.1 ml of putrescine solution is put in the side arm and 0.2 ml of 5 N KOH plus a small piece of Whatman No. 40 filter paper in the center well. The putrescine solution is replaced by tile same volume of water in control experiments. The reaction mixtures are equilibrated by shaking at about 60 oscillations per minute f()r 15 minutes at 30 °. After initiation of the reaction by tipping the contents of the side arm into the reaction mixture, the oxygen uptake is measured at 5-minute intervals during the first 20 minutes. The greatest oxygen uptake over either the reaction period from 0-10 minutes, or 5-15 minutes is taken and is used to calculate the average oxygen uptake per minute. De/tuition 0/ Unit and Specific Activity'. The international unit (I.U.) is used and is defined as the amount of enzyme that will catalyze the oxidative deamination at pH 7 of 1 micromole of putrescine per minute at 30 ° . An oxygen uptake of 11.2 /xl is found for the oxidation of I micromole of putrescine in reaction mixtures containing catalase. Specific activity is defined as units per milligram of protein, the protein content being calculated from the nitrogen content, as found by tile Kjeldahl method, and multiplying this by the factor 6.25. P u r i f i c a t i o n P r o c e d u r e ",3
The method depends on removing much of the material ~¥om the crude extract by precipitation with a mixture of chloroform and ethanol (Tsuchihashi's reagent4), then precipitating the enzyme first with (NH4)2SO4 and secondly at pH 5 by the method of Tabor? The enzyme is finally purified by chromatography on hydroxyapatite and •zp..]. (;. Mann, Biochem.]. 5 9 , 6 0 9 (1955). :q'. J. (,. Mann, Bio~hem.]. 79, 623 ( 1961 ). ~M. Tstwhihashi, Biochem. Z. 140, 63 (1923). "H. "l'abov,[. Biol. (;hem. 188, 125 ( 1951 ); see also Vol. I I [54].
732
AMINE OXIDATION AND HYDROXYLATION
[238]
DEAE-cellulose columns. If the chloroform-ethanol treatment is omitted, considerable difficulty may be experienced in the subsequent purification of the enzyme at the pH 5 precipitation and later stages. The diamine oxidase content of pea seedlings (Pisum sativum) is at a maximum over the period between 7 and 16 days after germination. We usually use them when 10 days old. Varieties differ slightly in their initial diamine oxidase activity, and in the ease with which the enzyme may be purified. In this laboratory we mainly use the cultivar "Fillbasket," but many other varieties have also been used successfully.
Step 1. Preparation of Crude Extract. About 1 kg of pea-seeds are soaked in r u n n i n g tap water for 18-24 hours and then sown thickly at a depth of 4-6 mm in coarse sand. The sand should previously have been well washed with tap water and fill, to a depth of 10-12 cm, the two plastic bowls, each measuring about 30 × 40 cm, in which the seeds are grown. The seeds are germinated and grown in a darkened cupboard; it is essential that the seedlings be grown in the dark as this makes the final stages in the purification of the enzyme easier than when plants have been grown in the light. About 10 days after sowing, when the etiolated shoots are 2-5 cm tall, the cotyledons and shoots (2-2.5 kg) are harvested, washed free from sand with cold tap water, drained, weighed, and put through a powered meat mincer. The mince is placed in cotton cloth and the juice is squeezed out in a screw press. The solid residue is mixed with 0.1 M potassium phosphate buffer pH 7 (1 ml/gram fresh material), and the liquid is squeezed out in the screw press. A further extraction of the solid residue is then made with 0.5 ml of the phosphate buffer for each gram of fresh material. The liquid extracts (2-3 liters) are combined and cooled to below 5°. Step 2. Precipitation of Inactive Material with An Ethanol-Chloroform Mixture. The volume of the crude extract is measured and a 2 : 1 v/v ethanol : chloroform mixture (30 ml for each 100 ml crude extract), which has been cooled to <--10 °, is added over a 30 minute period. Care must be taken to ensure that the temperature of the crude extract does not rise above +5 ° during this addition. The mixture is now allowed to stand for 1 hour at 0°-+5% after which the inactive precipitate is removed by centrifuging for 20 minutes at 3000-4000 g. The supernatant liquid is collected and saturated with (NH4)2804 (45 g/100 ml), and the temperature allowed to rise to about 10°. A solid separates and rises to the surface; the lower liquid is siphoned off and discarded, and the remaining slurry is centrifuged for 10-15 minutes at about 3000 g. When straight-sided centrifuge tubes are used, the hard curd that forms is easily removed with a flat spatula. This curd is homogenized with
[238]
DIAMINE OXIDASE (PEA SEEDLING)
733
0.02 M potassium phosphate buffer pH 7 (1 ml/2 g initial fresh pea seedlings), and allowed to stand overnight at about +2 ° . Step 3. Precipitation with (NH4)2S04. The 0.02 M phosphate buffer suspension is stirred for 1-2 hours at between 15-20 ° and the precipitate is removed by centrifuging for 20 minutes at 3000-4000 g. The supernatant liquid is treated with (NH4)2SO4 (45 g/100 ml) and, after standing at about 10° for 1 hour, the precipitate is collected by centrifuging at 3000 g for about 20 minutes. This precipitate is triturated with about 20 ml 0.2 M potassium phosphate buffer, pH 7, and then dialyzed, first against cold running tap water for 3-4 hours, and then against 3 changes, each of 2 liters of 0.005 M potassium phosphate buffer pH 7 over a period of 36 hours at 0-4 °. Step 4. Precipitation at pH 5. The dialyzed material is centrifuged at 2000-3000 g to remove inactive precipitate. The supernatant liquid, after cooling to about 5 °, is adjusted to pH 5 by the slow addition of 0.05 N acetic acid and allowed to stand for 1 hour at 2-4 °. The precipitate is collected by centrifuging, triturated with 10-15 ml water, and finally brought into solution by adjusting to pH 7 with 0.05 N KOH. This precipitation at pH 5 is repeated twice and the final solution made up to a suitable volume (1 ml for each 100-g pea seedlings) in 0.01 M potassium phosphate buffer pH 7. This enzyme solution can be stored for many months at--20 ° without appreciable loss of activity. Step 5. Fractionation on Hydroxyapatite. Hydroxyapatite is prepared by the method of Tiselius, Hjert6n, and Levin. 6 Five to eight of the enzyme preparations obtained at the end of the previous step are combined and centrifuged, and the supernatant solution (100-200 ml) is applied to a hydroxyapatite column (4 × 12 cm) equilibrated with 0.01 M potassium phosphate buffer pH 7. This and all subsequent fractionations are done at 0-5 °. The column is eluted first with 0.04 M potassium phosphate buffer, pH 7, and this is continued by stepwise increments (0.04 M) in the concentration of buffer up to a final concentration of 0.2 M. The volume of buffer at each concentration is 100 ml and the flow rate should be 50-80 ml/hour. The eluate is collected in 5-ml samples and the fractions containing the main amine oxidase peak showing the highest amine oxidase activity in relation to extinction at 280 m/~ (samples numbered approximately 100-140), are combined (approximately 150-250 ml), and dialyzed for 24 hours against 2 changes of 3 liters of distilled water. The ratio of amine oxidase units/E2s0 mu in the fractions combined should exceed 15. The amine oxidase fractions at this stage are usually pink. 6A. Tiselius, S. Hjert6n, and 0. Levin, Arch. Biochem. Biophy,~. 65, 132 (1956); 0. Levin, Vol. V [2].
see also
734
[238]
AMINE OXIDATION AND HYDROXYLATION
Step 6. Adsorption of Inert Material on Diethylaminoethyl Cellulose. Whatman DE 11 cellulose powder is suspended in water and adjusted to pH 7 with 1 M KH2PO4. A column (2 X 10 cm) is prepared and is equilibrated with 0.01 M phosphate buffer pH 7. The dialyzed amine oxidase preparation from the previous step is passed through this column and the column is then washed with 25 ml of 0.01 M phosphate buffer pH 7. Step 7. Concentration on Hydroxyapatite. The enzyme solution and washings (approximately 200-250 ml) are combined and applied to a column of hydroxyapatite (4 x 4 cm). The enzyme is eluted with 0.2 M potassium phosphate buffer pH 7 as a single pink band and is stored at < - 1 0 °. This enzyme can be stored at --20 ° for many months without loss of activity. SUMMARY OF THE PURIFICATION OF DIAMINE OXIDASE FROM PEA SEEDLINGS
Step 1. 3. 4. 4. 5. 7.
Fraction
Volume (ml)
Crude extract 2260 Dialyzed fraction after 31.5 (NH4)zSO4 precipitation After precipitation at pH 5 11.0 Combined fractions from 120 step 4 Eluate from hydroxyapatite 237 Eluate from DEAE after 20 concentration
Total units (I.U.)
Total protein (mg)
3320 1790
81,400 250
Specific activity (units/mg of protein) 0.041 7.16
1340 8820
44.6 329
30.0 26.8
5020 4560
114 62.4
44.1 73.1
Properties Specificity. This enzyme (specific activity greater than 60) catalyzes the oxidative deamination of a wide range of amines containing the ---CH2NHz grouping. The most active substrates are putrescine (1127) and cadaverine (112). 1,3-Diaminopropane is not attacked, but 1,6-diaminohexane (28) and 1,10-diaminodecane (21.5) are attacked. Other substrates are lysine (6.4), ornithine (0.7), histamine (6.0), spermine (0.4), spermidine (39), agmatine (39), tryptamine (7.6), benzylamine (4.9), tyramine (20), 2-phenylethylamine (32), n-propylamine (11), and some other aliphatic diaminesY pH Optimum. The optimum pH depends on the substrate and is about
rNumbers in parenthesis represent the relative activity observed with the different substrates.
[239]
DIAMINE OXIDASE (PIG KIDNEY)
735
7.0 for putrescine and 8.5 for /~-phenylethylamine in experiments carried out at 0.01 M and 0.002 M concentrations. Reaction with Substrate. The legume seedling diamine oxidases, unlike all other amine oxidases described so far, react with substrates under anaerobic conditions to give yellow complexes. The absorption spectra of these complexes are identical and have maxima at 350, 437, and 466 m/~.a'~ Inhibitors. This enzyme is strongly inhibited by carbonyl reagents, Cu z+ ions and some chelating agents including 8-hydroxyquinoline, 1,10-phenanthroline and sodium diethyldithiocarbamate;--SH group reagents do not significantly inhibit. Metal Requirements. Purified pea seedling diamine oxidase contains 0.08-0.09% copper. All the copper is in the divalent state and does not change valency during the enzymic reaction. Copper is firmly bound to the protein, but can be removed with sodium diethyldithiocarbamate: the inactive copper-free protein is reactivated specifically by Cu "+ ions. Color. The purified enzyme is pink with an absorption maximum at 505 m~ at pH 7; at the same pH the copper-free diamine oxidase absorbs maximally at 480 mtz. sj. M. Hill and P. J. G. Mann, Biochem..]. 91, 171 (1964).
[239] Diamine Oxidase (Pig Kidney) ~ By BRUNO MONDOVi, GIUSEPPE ROTILIO, MARIA TERESA COSTA, and ALESSANDRO FINAZZI AGR~) RCH2NH2 -+- 02 + H20 ~ R C H O + NH3 + H202
R indicates the residual chain of aliphatic diamines or of histamine. Although some authors have claimed that aliphatic diamines and histamine are oxidized by two different pig kidney enzymes, the identity between diamine oxidase and histaminase seems to be well established? a ~E.C. 1.4.3.6; diamine:oxygen oxidoreductase (deaminating); histaminase. A less purified preparation of this enzyme was described by H. Tabor in Vol. II [54]. Recently, homogeneous preparations of diamine oxidase from hog kidney have been obtained by somewhat different purification procedures by Goryachenkova, Shcherbatyuk, and Voronina [Biokhimiya 32, 398 (1967)] and by Yamada, Kumagai, Kawasaki, Matsui, and Ogata [Biochem. Biophys. Res. Commun. 29, 723 (1967)]. The latter preparation is crystalline, contains 2.17 g-moles of copper per mole of the enzyme, has a molecular weight of 185,000 and an absorption maximum at 470 naN. ~aB. Mondovl, (;. Rntilio, A. Finazzi Agr6, and A. Scioscia Santoro, Biochem..]. 91, 408 (1964).