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[85a] Iodination of tyrosine: Isolation of lactoperoxidase (bovine)
[85a]
IODINATION OF TYROSINE; LACTOPEROXIDASE
653
[85a] Iodination of Tyrosine: 1
Isolation of Lactoperoxidase (Bovine) By MARTIN
MORRISON
Assay Procedure Peroxidase activity can be measured by a number of techniques. T h e first two assay procedures to be described are commonly employed spectrophotometric methods. TM The third procedure is a recently developed assay for iodination reactions. 2
CH* OH
H
HsC~Hs
H~02 ~
Guaiacol
2.
3 I-
+
H~O~
H
2, 2'- Dihydroxy 3, 3'-dimethyldiphenyl
~
Is- +
2 OH-
X 3.
I- + H m O 2 + H O - - - ~ _ J / ~ - - - C H 2 C H C O O H k_____/ NH 2
Tyrosine
I
_
~-~H20+ OH- + H O ~ CH,CHCOOH k..__./ N H ~ Monoiodotyrosine
Procedure 1. The oxidation of the phenolic compound, guaiacol, can be followed spectrophotometrically and provides a convenient and very sensitive assay for the enzyme. The rate o f oxidation is followed on a recording spectrophotometer at 470 m~. T h e cuvette with a 1.0-cm light path contained 3 ml of a solution 33 rnM with respect to guaiacol in 0.1 M phosphate buffer, pH 7.4, 0.3 mM with respect to hydrogen ~EC 1.11.1.7; donor: hydrogen-peroxide oxidoreductase (peroxidase); and EC 1.11.1.8; iodide:hydrogen-peroxide oxidoreductase (iodinase). See article [85b] for the isolation of thyroid peroxidase from pig. See article [84] for the preparation ofchloroperoxidase, and a description of the iodination of tyrosine catalyzed by this enzyme. taA. C. Maehly and B. Chance, Methods Biochem. Anal. I, p. 357. Britton Chance and A. C. Maehly, Vol. II, p. 764. T. Hosoya and M. Morrison, J. Biol. Chem. 242, 2828 (1967). 2M. Morrison, Gunma Syrup. Endocrinol. 5,239 (1968).
654
AROMATIC AMINO ACIDS
[85a]
peroxide. The reaction is initiated with 0.010-0.100 ml of a solution containing the peroxidase. The assay was performed at room temperature (22-23°). One unit of enzyme is the amount of enzyme that gave an optical density change of 0.001 per second. For the calculations of the specific activities of the enzyme, a molar extinction coefficient of 5570 is employed for oxidized guaiacol. Procedure 2. The rate of oxidation of iodide was followed spectrophotometrically at 350 m g in a cuvette with a 1.0-cm light path. The assay mixture contained 3 ml of 33 mM phosphate buffer, pH 7.0, which was 5 mM with respect to potassium iodide, and 0.15 mM with respect to hydrogen peroxide. The reaction is initiated with 0.010 to 0.100 ml of a solution containing the peroxidase. Care was taken to ensure that the initial rate was as close as possible to zero order. One unit of enzyme is the amount of enzyme that gave an optical density change of 1.000 per second. For the calculations of the specific activities of the enzyme, a molar extinction coefficient of 26,000 is employed for the oxidized iodide, triiodide. Procedure 3. The rate of iodination of tyrosine or any one of a variety of phenolic compounds including proteins can be assessed by following the change of iodide concentration using an iodide sensor, a millivolt meter, and strip chart recorder. Commercial iodide-specific sensors.are available from Orion Research, Inc. or National Instrument Laboratories, Inc. For the assay procedure employed, the electrode should be standardized at iodide concentrations between 1 × 10 -4 M and 1 × 10 -e M, which is the range of iodide used for the assay. The conditions employed are optimal for the iodination of tyrosine catalyzed by lactoperoxidase at the physiological pH 7.4. ~a The jacketed reaction flask is maintained at a temperature of 25 ° and contains 5 ml of a solution with an initial concentration of 1.5 × 10 -4 M of KI, 1 × 10 -4 M H~O~, and 8 × 10 -4 M tyrosine in 0.05 M phosphate buffer pH 7.4. The reaction is initiated with 0.05-0.1 ml of a solution containing the peroxidase. The change in potential is recorded via a millivolt meter onto an appropriate recorder. The rate of change of iodide concentration is then calculated. Under the experimental conditions employed, this rate is proportional to the concentration of lactoperoxidase used to catalyze the reaction. Bovine lactoperoxidase will catalyze the iodination of 1 × 104 moles of tyrosine per minute per mole of lactoperoxidase. ~ A t neutral or higher pH values and at concentrations o f 1.5 × 10 -4 M KI, or lower, the oxidation o f 1- to I~ cannot be detected either spectrophotometrically or by the iodide electrode
[85a]
IODINATION OF TYROSINE" LACTOPEROXIDASE
655
Isolation from Raw Milk 3 A weak cation exchange resin such as IRC 50 or BioRad 70, 200-400 mesh, was washed free of fines. The resin was then buffered to pH 7 with phosphate buffer. The buffered resin was suspended in 0.01 M phosphate buffer pH 7 and was washed into a column 30 cm in diameter and 10 cm in height. Raw skim milk, adjusted to pH 6.9-7.0 with 6 N acetic acid or 6 N ammonium hydroxide, was then poured on to the resin bed at 4° . The milk was allowed to flow through this resin bed at the rate of about 4 liters per hour. After 40 liters of milk had passed through, the resin was washed with 5-10 liters of cold distilled water. When most of the excess milk had been removed, the resin was washed into large beakers with distilled water and packed into a column 10 cm in diameter by 30 cm in height. The column was washed with distilled water until free of 280 m/~ absorbing materials. Protein was then eluted from the resin with 0.5 M sodium acetate. T h e eluate was recovered on a fraction collector that was capable of collecting l-liter aliquots. A dark green lactoperoxidase band began migrating down the column as soon as the sodium acetate had entered the resin bed, and it increased in width as it flowed down the column. The lactoperoxidasecontaining fractions were collected in approximately 2-4 liters. These fractions were combined and 52 g of a m m o n i u m sulfate per 100 ml of solution was added with stirring at 4° . The solution was allowed to stand for at least 2 hours, after which the precipitate was collected by centrifugation for 30 minutes at 20,000 g. T h e precipitate was then suspended in a minimum volume of distilled water and dialyzed against at least three changes of 0.02 M sodium acetate adjusted to pH 7.35. This solution was centrifuged to remove any small amount of precipitate. The crude lactoperoxidase obtained in this way usually had a 412:280 absorbancy ratio o f 0.3-0.5.
Purification of the Crude Lactoperoxidase For the final purification of relatively large quantities of crude lactoperoxidase, a column, 2.5 by 40 cm, was packed with the ion-exchange resin equilibrated at pH 7.35. Crude lactoperoxidase, 250 rag, which had been dialyzed against the 7.35 buffer was allowed to enter the top of the resin, where it was adsorbed as a green zone. T h e column walls were rinsed with the dilute buffer, and the column was connected to a system for generating linear gradients. T h e mixing reservoir contained 450 ml o f the starting buffer, and the second reservoir contained 450 ml of 0.35 M sodium acetate. The elution volume was controlled by a Milton 3M. Morrison and D. E. Hultquist,J.Biol. Chem. 238, 2847 (1963).
656
AROMATIC AMINO ACIDS
[85a]
Roy Laboratory Pump adjusted to give approximately 60 ml per hour in 10-ml fractions. The buffers were changed after 5 hours to 480 ml of 0.35 M sodium acetate in the mixing reservoir and 0.5 M sodium acetate buffer in the other reservoir. After the lactoperoxidase had been completely eluted, 1 M sodium acetate buffer was run through the column to elute all proteins remaining on the column. After equilibration with the starting buffer, the column was then ready to be used again. Reproducible results were obtained in successive columns. The fractions of lactoperoxidase obtained as described above were combined and concentrated by ultrafiltration or by precipitation with 52 g of ammonium sulfate per 100 ml. The precipitate was redissolved and dialyzed again against any desired buffer. A final purification step employing gel filtration on Sephadex G-100, or Bio-Gel 100 was employed to remove traces of contaminating proteins. Five ml of a concentrated solution of purified lactoperoxidase was passed through a column 45 by 2.5 cm in diameter containing the Sephadex in equilibrium with 0.05 M phosphate buffer, pH 7.5. The central cut of the lactoperoxidase fraction was used. The enzyme obtained as indicated above had a 412:280 ratio of approximately 0.9 and is free of contaminating proteins as j u d g e d by immunochemical and electrophoretic assay.
Properties The enzyme lactoperoxidase has been isolated from salivary, lacrimal, and Harderian glands. 4-0 By all parameters tested, the bovine enzyme from the lacrimal or salivary gland is identical to the enzyme isolated from milk. T h e lactoperoxidase isolated from pig has a lower isoelectric point than the bovine enzyme and cannot be isolated by the procedure outlined. In other species, such as horse, goat, or sheep, the enzymes are immunochemically closely related, if not identical, to the bovine enzyme. Lactoperoxidase has a molecular weight of 78,000, a partial specific volume of 0.74, r and a diffusion coefficient a of 5.2 × 10 -r cm sec -1. This unique enzyme has the highest protein to heine ratio of any of the peroxidases yet isolated with a single heine per mole. The N-terminal amino acid residue is leucine, and the C terminal end may be asparagineY The 4M. Morrison and P. Z. Allen, Biochem. Biophys. Res. Comraun. 13, 490 (1963). 5M. Morrison, P. Z. Allen, J. Bright, and W. Jayasinghe, Arch. Biochem. Biophys. 111, 126 (1965). SM. Morrison and P. Z. Allen, Science 152, 1626 (1966). TW. A. Rombauts, W. A. Schroeder, and M. Morrison, Biochemistry6, 2965 (1967). sp. Z. Allen and M. Morrison, Arch. Biochem. Biophys. 102, 106 (1963).
[85a]
IODINATION o r TYROSINE: LACTOPEROXIDASE
657
enzyme has 20 moles of glucosamine, 20 moles of galactosamine, and 8 moles of neutral carbohydrate per mole of enzyme. The enzyme has an absorption maximum at 412 m/z in the Soret region with a millimolar extinction coefficient of 114. 9 The enzyme is stable in low ionic strength solutions up to 60 ° and from pH 3 to 10. It is not denatured by 8 M urea. The peroxidase is a hemoprotein and will interact with the usual hemoprotein ligands, such as cyanide, fluoride, azide. The ligands compete with peroxide and hence inhibit the enzyme activity) °- i3 Lactoperoxidase will catalyze the oxidation of a great variety of electron donors, such as phenolic compounds and aromatic amines. It will also catalyze the oxidation of anions, such as iodide and thiocyanate. Table I is a comparison of the specific activity of lactoperoxidase with a variety of other peroxidases in their ability to catalyze the oxidation of iodide and guaiacol. TM Table II compares the rate of iodination of various substrates catalyzed by lactoperoxidase. TABLE I COMPARISON OF SPECIFIC ACTIVITY OF A VARIETY OF PEROXIDASES a
Hemoprotein
Guaiacol Iodide Ratio (t~moles/min/mg protein) (tzmoles/min/mg protein) guaiacol:iodide
Horseradish peroxidase Lactoperoxidase Myeloperoxidase Thyroid peroxidase
215 175 101 48
0.6 103.0 5.0 7.4
344.0 1.7 20.2 6.6
aData calculated from Hosoya and Morrison? 4 TABLE II RATE OF IODINATION OF VARIOUS SUBSTRATES CATALYZED BY LACTOPEROXIDASE a
Substrates Tyrosine Monoiodotyrosine Thyronine Bovine serum albumin
AI-/min/mole enzyme 10 1.5 0.1 6.0
× × × ×
10 3 l0 s 10 3 l0 s
aData taken from M. Morrison, Gunma Syrup. Endocrinol. 5, 239 (1968). 9M. Morrison, H. B. Hamilton, and E. Stotz,J. Biol. Chem. 228, 767 (1957). ~°H. Theorell and A. Akeson,/lrkiv. Kemi, Mineral. Geol. 17B, 12 (1944). l iH. Theorell and K. G. Paul, Arkiv. Kemi, Mineral. Geol. 18A, 12 (1944). ~2B. D. Polis and H. W. Shmukler,J. Biol. Chem. 201,475 (1953). 13M. Morrison, in "Oxidases and Related Redox Systems" ( T. E. King, H. S. Mason, and M. Morrison, eds.), p. 659. Wiley, New York, 1965. ~4T. Hosoya and M. Morrison, Biochem. 6, 1021 (1967).
IODINATION OF TYROSINE; LACTOPEROXIDASE
653
[85a] Iodination of Tyrosine: 1
Isolation of Lactoperoxidase (Bovine) By MARTIN
MORRISON
Assay Procedure Peroxidase activity can be measured by a number of techniques. T h e first two assay procedures to be described are commonly employed spectrophotometric methods. TM The third procedure is a recently developed assay for iodination reactions. 2
CH* OH
H
HsC~Hs
H~02 ~
Guaiacol
2.
3 I-
+
H~O~
H
2, 2'- Dihydroxy 3, 3'-dimethyldiphenyl
~
Is- +
2 OH-
X 3.
I- + H m O 2 + H O - - - ~ _ J / ~ - - - C H 2 C H C O O H k_____/ NH 2
Tyrosine
I
_
~-~H20+ OH- + H O ~ CH,CHCOOH k..__./ N H ~ Monoiodotyrosine
Procedure 1. The oxidation of the phenolic compound, guaiacol, can be followed spectrophotometrically and provides a convenient and very sensitive assay for the enzyme. The rate o f oxidation is followed on a recording spectrophotometer at 470 m~. T h e cuvette with a 1.0-cm light path contained 3 ml of a solution 33 rnM with respect to guaiacol in 0.1 M phosphate buffer, pH 7.4, 0.3 mM with respect to hydrogen ~EC 1.11.1.7; donor: hydrogen-peroxide oxidoreductase (peroxidase); and EC 1.11.1.8; iodide:hydrogen-peroxide oxidoreductase (iodinase). See article [85b] for the isolation of thyroid peroxidase from pig. See article [84] for the preparation ofchloroperoxidase, and a description of the iodination of tyrosine catalyzed by this enzyme. taA. C. Maehly and B. Chance, Methods Biochem. Anal. I, p. 357. Britton Chance and A. C. Maehly, Vol. II, p. 764. T. Hosoya and M. Morrison, J. Biol. Chem. 242, 2828 (1967). 2M. Morrison, Gunma Syrup. Endocrinol. 5,239 (1968).
654
AROMATIC AMINO ACIDS
[85a]
peroxide. The reaction is initiated with 0.010-0.100 ml of a solution containing the peroxidase. The assay was performed at room temperature (22-23°). One unit of enzyme is the amount of enzyme that gave an optical density change of 0.001 per second. For the calculations of the specific activities of the enzyme, a molar extinction coefficient of 5570 is employed for oxidized guaiacol. Procedure 2. The rate of oxidation of iodide was followed spectrophotometrically at 350 m g in a cuvette with a 1.0-cm light path. The assay mixture contained 3 ml of 33 mM phosphate buffer, pH 7.0, which was 5 mM with respect to potassium iodide, and 0.15 mM with respect to hydrogen peroxide. The reaction is initiated with 0.010 to 0.100 ml of a solution containing the peroxidase. Care was taken to ensure that the initial rate was as close as possible to zero order. One unit of enzyme is the amount of enzyme that gave an optical density change of 1.000 per second. For the calculations of the specific activities of the enzyme, a molar extinction coefficient of 26,000 is employed for the oxidized iodide, triiodide. Procedure 3. The rate of iodination of tyrosine or any one of a variety of phenolic compounds including proteins can be assessed by following the change of iodide concentration using an iodide sensor, a millivolt meter, and strip chart recorder. Commercial iodide-specific sensors.are available from Orion Research, Inc. or National Instrument Laboratories, Inc. For the assay procedure employed, the electrode should be standardized at iodide concentrations between 1 × 10 -4 M and 1 × 10 -e M, which is the range of iodide used for the assay. The conditions employed are optimal for the iodination of tyrosine catalyzed by lactoperoxidase at the physiological pH 7.4. ~a The jacketed reaction flask is maintained at a temperature of 25 ° and contains 5 ml of a solution with an initial concentration of 1.5 × 10 -4 M of KI, 1 × 10 -4 M H~O~, and 8 × 10 -4 M tyrosine in 0.05 M phosphate buffer pH 7.4. The reaction is initiated with 0.05-0.1 ml of a solution containing the peroxidase. The change in potential is recorded via a millivolt meter onto an appropriate recorder. The rate of change of iodide concentration is then calculated. Under the experimental conditions employed, this rate is proportional to the concentration of lactoperoxidase used to catalyze the reaction. Bovine lactoperoxidase will catalyze the iodination of 1 × 104 moles of tyrosine per minute per mole of lactoperoxidase. ~ A t neutral or higher pH values and at concentrations o f 1.5 × 10 -4 M KI, or lower, the oxidation o f 1- to I~ cannot be detected either spectrophotometrically or by the iodide electrode
[85a]
IODINATION OF TYROSINE" LACTOPEROXIDASE
655
Isolation from Raw Milk 3 A weak cation exchange resin such as IRC 50 or BioRad 70, 200-400 mesh, was washed free of fines. The resin was then buffered to pH 7 with phosphate buffer. The buffered resin was suspended in 0.01 M phosphate buffer pH 7 and was washed into a column 30 cm in diameter and 10 cm in height. Raw skim milk, adjusted to pH 6.9-7.0 with 6 N acetic acid or 6 N ammonium hydroxide, was then poured on to the resin bed at 4° . The milk was allowed to flow through this resin bed at the rate of about 4 liters per hour. After 40 liters of milk had passed through, the resin was washed with 5-10 liters of cold distilled water. When most of the excess milk had been removed, the resin was washed into large beakers with distilled water and packed into a column 10 cm in diameter by 30 cm in height. The column was washed with distilled water until free of 280 m/~ absorbing materials. Protein was then eluted from the resin with 0.5 M sodium acetate. T h e eluate was recovered on a fraction collector that was capable of collecting l-liter aliquots. A dark green lactoperoxidase band began migrating down the column as soon as the sodium acetate had entered the resin bed, and it increased in width as it flowed down the column. The lactoperoxidasecontaining fractions were collected in approximately 2-4 liters. These fractions were combined and 52 g of a m m o n i u m sulfate per 100 ml of solution was added with stirring at 4° . The solution was allowed to stand for at least 2 hours, after which the precipitate was collected by centrifugation for 30 minutes at 20,000 g. T h e precipitate was then suspended in a minimum volume of distilled water and dialyzed against at least three changes of 0.02 M sodium acetate adjusted to pH 7.35. This solution was centrifuged to remove any small amount of precipitate. The crude lactoperoxidase obtained in this way usually had a 412:280 absorbancy ratio o f 0.3-0.5.
Purification of the Crude Lactoperoxidase For the final purification of relatively large quantities of crude lactoperoxidase, a column, 2.5 by 40 cm, was packed with the ion-exchange resin equilibrated at pH 7.35. Crude lactoperoxidase, 250 rag, which had been dialyzed against the 7.35 buffer was allowed to enter the top of the resin, where it was adsorbed as a green zone. T h e column walls were rinsed with the dilute buffer, and the column was connected to a system for generating linear gradients. T h e mixing reservoir contained 450 ml o f the starting buffer, and the second reservoir contained 450 ml of 0.35 M sodium acetate. The elution volume was controlled by a Milton 3M. Morrison and D. E. Hultquist,J.Biol. Chem. 238, 2847 (1963).
656
AROMATIC AMINO ACIDS
[85a]
Roy Laboratory Pump adjusted to give approximately 60 ml per hour in 10-ml fractions. The buffers were changed after 5 hours to 480 ml of 0.35 M sodium acetate in the mixing reservoir and 0.5 M sodium acetate buffer in the other reservoir. After the lactoperoxidase had been completely eluted, 1 M sodium acetate buffer was run through the column to elute all proteins remaining on the column. After equilibration with the starting buffer, the column was then ready to be used again. Reproducible results were obtained in successive columns. The fractions of lactoperoxidase obtained as described above were combined and concentrated by ultrafiltration or by precipitation with 52 g of ammonium sulfate per 100 ml. The precipitate was redissolved and dialyzed again against any desired buffer. A final purification step employing gel filtration on Sephadex G-100, or Bio-Gel 100 was employed to remove traces of contaminating proteins. Five ml of a concentrated solution of purified lactoperoxidase was passed through a column 45 by 2.5 cm in diameter containing the Sephadex in equilibrium with 0.05 M phosphate buffer, pH 7.5. The central cut of the lactoperoxidase fraction was used. The enzyme obtained as indicated above had a 412:280 ratio of approximately 0.9 and is free of contaminating proteins as j u d g e d by immunochemical and electrophoretic assay.
Properties The enzyme lactoperoxidase has been isolated from salivary, lacrimal, and Harderian glands. 4-0 By all parameters tested, the bovine enzyme from the lacrimal or salivary gland is identical to the enzyme isolated from milk. T h e lactoperoxidase isolated from pig has a lower isoelectric point than the bovine enzyme and cannot be isolated by the procedure outlined. In other species, such as horse, goat, or sheep, the enzymes are immunochemically closely related, if not identical, to the bovine enzyme. Lactoperoxidase has a molecular weight of 78,000, a partial specific volume of 0.74, r and a diffusion coefficient a of 5.2 × 10 -r cm sec -1. This unique enzyme has the highest protein to heine ratio of any of the peroxidases yet isolated with a single heine per mole. The N-terminal amino acid residue is leucine, and the C terminal end may be asparagineY The 4M. Morrison and P. Z. Allen, Biochem. Biophys. Res. Comraun. 13, 490 (1963). 5M. Morrison, P. Z. Allen, J. Bright, and W. Jayasinghe, Arch. Biochem. Biophys. 111, 126 (1965). SM. Morrison and P. Z. Allen, Science 152, 1626 (1966). TW. A. Rombauts, W. A. Schroeder, and M. Morrison, Biochemistry6, 2965 (1967). sp. Z. Allen and M. Morrison, Arch. Biochem. Biophys. 102, 106 (1963).
[85a]
IODINATION o r TYROSINE: LACTOPEROXIDASE
657
enzyme has 20 moles of glucosamine, 20 moles of galactosamine, and 8 moles of neutral carbohydrate per mole of enzyme. The enzyme has an absorption maximum at 412 m/z in the Soret region with a millimolar extinction coefficient of 114. 9 The enzyme is stable in low ionic strength solutions up to 60 ° and from pH 3 to 10. It is not denatured by 8 M urea. The peroxidase is a hemoprotein and will interact with the usual hemoprotein ligands, such as cyanide, fluoride, azide. The ligands compete with peroxide and hence inhibit the enzyme activity) °- i3 Lactoperoxidase will catalyze the oxidation of a great variety of electron donors, such as phenolic compounds and aromatic amines. It will also catalyze the oxidation of anions, such as iodide and thiocyanate. Table I is a comparison of the specific activity of lactoperoxidase with a variety of other peroxidases in their ability to catalyze the oxidation of iodide and guaiacol. TM Table II compares the rate of iodination of various substrates catalyzed by lactoperoxidase. TABLE I COMPARISON OF SPECIFIC ACTIVITY OF A VARIETY OF PEROXIDASES a
Hemoprotein
Guaiacol Iodide Ratio (t~moles/min/mg protein) (tzmoles/min/mg protein) guaiacol:iodide
Horseradish peroxidase Lactoperoxidase Myeloperoxidase Thyroid peroxidase
215 175 101 48
0.6 103.0 5.0 7.4
344.0 1.7 20.2 6.6
aData calculated from Hosoya and Morrison? 4 TABLE II RATE OF IODINATION OF VARIOUS SUBSTRATES CATALYZED BY LACTOPEROXIDASE a
Substrates Tyrosine Monoiodotyrosine Thyronine Bovine serum albumin
AI-/min/mole enzyme 10 1.5 0.1 6.0
× × × ×
10 3 l0 s 10 3 l0 s
aData taken from M. Morrison, Gunma Syrup. Endocrinol. 5, 239 (1968). 9M. Morrison, H. B. Hamilton, and E. Stotz,J. Biol. Chem. 228, 767 (1957). ~°H. Theorell and A. Akeson,/lrkiv. Kemi, Mineral. Geol. 17B, 12 (1944). l iH. Theorell and K. G. Paul, Arkiv. Kemi, Mineral. Geol. 18A, 12 (1944). ~2B. D. Polis and H. W. Shmukler,J. Biol. Chem. 201,475 (1953). 13M. Morrison, in "Oxidases and Related Redox Systems" ( T. E. King, H. S. Mason, and M. Morrison, eds.), p. 659. Wiley, New York, 1965. ~4T. Hosoya and M. Morrison, Biochem. 6, 1021 (1967).