- Email: [email protected]
An improved method for the gas chromatographic determination of methionine in proteins and crude plant materials
ANALYTICAL
BIOCHEMISTRY
118,
126- 130 ( 198 1)
An Improved Method for the Gas Chromatographic Determination Methionine in Proteins and Crude Plant Materials’ GERASIMOSAPOSTOLATOS~
ANDJOHAN
of
E. HOFF
Food Sciences Institute, Purdue University, West Lafayette, Indiana 47907 Received August 5, 1981 Procedures for the determination of methionine in crude plant materials by means of cyanogen bromide digestion and gas chromatographic analysis of the digest were modified to achieve complete reaction, enhanced precision, and shortened reaction times. Oxides of methionine and derivatives of S-methylcysteine did not add to the values obtained for free and peptide-bound methionine.
Since Inglis and Edman (5) described the reaction between peptide-bound methionine and cyanogen bromide and quantitated the released methylthiocyanate by gas-liquid chromatography, several methods based on this reaction have been proposed for the analysis of methionine in crude samples of animal and plant origin (2,3,6,7,9). Such methods employ rather lengthy incubation periods (up to 18 h) in an effort to allow the reaction to go to completion, but the analytical values obtained are still low, especially with crude materials. The lack of suitable column packings and of a convenient internal standard is also apparant. We are here addressing these problems and are proposing several modifications that jointly result in improved precision and a considerably shortened analysis time. EXPERIMENTAL
Reagents. Cyanogen bromide (CNBr,3 97%) methylthiocyanate (MeSCN, 99%), 5-nonanone (di-n-butyl ketone, 98%), and ’ Journal paper No. 8655 of the Purdue Agricultural Experiment Station. * Current address: Vegetable Research Institute, Gastouni, Bias, Greece. 3 Abbreviations used: CNBr, cyanogen bromide; MeSCN, methylthiocyanate. 0003-2697/81/170126-05$02.00/O Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
126
formic acid (97+%) were obtained from Aldrich Chemical Company. Formic acid was further purified by distillation using a reflux ratio of 4: 1. DL-Methionine, N-acetylDL-methionine, a-chymotrypsin (bovine, type II, 3X crystallized), fl-galactosidase, myoglobin (type I), papain (type III, 3~ crystallized), insulin (bovine, crystalline), Smethyl-L-cysteine, DL-methionine sulfoxide, and DL-methionine sulfone were obtained from Sigma Chemical Company. Legume seeds (10% moisture content) were ground to pass through an 80-mesh sieve. Procedure. Solution A: 1% CNBr (w/v) in 70% aqueous formic acid. Solution B: 1000 ppm 5-nonanone in 97% formic acid, Solution C: 2000 ppm MeSCN in 97% formic acid. The solutions were stable for at least 4 months when kept at 4°C. The reaction was performed in 1.5 ml Silli Vials (Applied Science Laboratories) stoppered by Teflon-laminated disks. To the dry samples, either 2030 mg finely ground seed meal or 2- 10 mg protein, were added 750 ~1 of solution A and 50 ~1 of solution B. A standard mixture was prepared each day from 700 ~1 of solution A and 50 ~1 of each of solutions B and C. Incubation at 95°C which continued for 60 min with seed meal and for 20 min with protein samples, was followed by centrifu-
IMPROVED
METHOD
FOR METHIONINE
127
DETERMINATION
There are two nonmethionine compounds in legume seed that could possibly release MeSCN and thereby contribute falsely to the apparent methionine values. S-Methylcysteine (2,10) was tested as the pure compound, while glutamyl-S-methylcysteine (10) was selectively extracted from Phase&s vulgaris seed with 70% aqueous acidic ethanol and the free amino acid fraction isolated (4). Both the ethanolic extract and the free amino acid fraction were analyzed by the proposed procedure. RESULTS AND DISCUSSION
012345 RETENTION
TIME
, min
FIG. 1. Typical chromatogram derived from the reaction of cyanogen bromide with Ph. vulgaris seed meal. I, MeSCN; 2, internal standard; 3, solvent peak.
gation at 3000g for 5 min. An aliquot (l-2 ,ul) of the clear supernatant was injected into the gas chromatograph (Hewlett-Packard, Model 402 or 5730 A) equipped with flame ionization detector and automatic integrator. Glass columns (2 mm i.d. X 100 cm) were packed with 10% Carbowax-1000 and 2% Ucon LB 550X (Analabs, Inc.) on 80to loo-mesh Chromosorb W-AW (JohnsManville). The oven temperature was 105”C, while the injection port and flame ionization detectors were held at 165°C. Nitrogen carrier gas flow rate was 40 ml/min, while the flow rates of hydrogen and air were 30 and 300 ml/min, respectively. The methionine content of various seed meals was for comparison determined by ion-exchange chromatography as methionine sulfone after performic acid oxidation (1) followed by acid hydrolysis in refluxed constant boiling HCI for 22 h. The hydrolysates were analyzed by a Beckman Model 119 CL amino acid analyzer equipped with a Model 126 data system. Analyses of such samples by the amino acid analyzer were obtained by a 1Zmin short run employing a pH 3.45 citrate buffer at 52°C.
A typical gas chromatogram (Ph. vulgaris seed meal) of a cyanogen bromide digest is illustrated in Fig. 1. The methionine content in the sample was calculated as follows: Met, % (w/w) = kfIS/&)STD
x fhf/4SLnpIe Wsample
x l9 I6
* ,
where &, AM represent peak areas for 5nonanone and methylthiocyanate, respectively, W is the sample weight in milligrams, and 19. I6 is the product of four factors: the molecular ratio (1.794) of anhydromethionine to methylthiocyanate, the amount of methylthiocyanate in the standard mixture
TIME
, min
FIG. 2. The effect of reaction time on the conversion of methionine to methylthiocyanate; A, N-Acetyl-DLmethionine; B, Ph. vulgaris protein isolate; C, Ph. vulgo& seed meal.
128
APOSTOLATOS
(0.100 mg), the density of methylthiocyanate (1.068 mg/pl) at 25”C, and loo(%). The protein content of the sample must be known if the methionine content of the protein is to be reported. An incubation temperature of 95°C allowed the reaction to go to completion in 60 min with crude Ph. vulgaris seed meal while its protein isolate required only 15 min (Fig. 2). Incubation temperatures above the boiling point of formic acid (100.6”C) resulted in damage of septa with subsequent losses of reaction mixture. One percent (w/v) of CNBr was sufficient for complete reaction with bean seed meal. Lower concentrations, e.g. 0.5%, gave variable results, while concentrations in excess of 1% gave no additional recovery of MeSCN. The concentration of formic acid in the medium affected the conversion of methionine in protein samples (Fig. 3). Optimal conversions were obtained with formic acid concentrations above 70% (v/v). The internal standard, 5-nonanone, was stable under the conditions of the reaction and separated well from MeSCN. Other substances were evaluated as internal standards. 2-Octanone also emerged after the MeSCN, but tended to overlap with MeSCN when this occurred at high concentrations. 4-Heptanone emerged prior to MeSCN, but
FORMIC
ACID , X
FIG. 3. Yield of methylthiocyanate at different concentrations of formic acid: A, N-Acetyl-Dkmethionine; B, myoglobin; C, bovine serum albumin; D, Ph. vufg&s seed meal.
AND
HOFF TABLE
1
COMPARWNOFTHEORETICALANDEXPERIMENTAL ANALYTICAL VALUESOF METHIONINEIN COMPOUNDSOF KNOWN METHIONINE~ONTENT Methionine content (mg)
Source Methionine N-Acetyl-DLmethionine cr-Chymotrypsin /3-Galactosidase Pepsin Myoglobin Insulin Papain Methionine sulfoxide Methionine sulfone S-Methylcysteine
Sample size 6-w)
Experimental
Theoretical
0.050
0.050
0.050
0.050 6.0 3.5 8.1 8.8 5.0 8.0 0.080 0.050 0.050
0.039 0.07 1 0.046 0.101 0.154 0.0 0.0 0.0 0.0 0.0
0.039 0.071 0.046 0.103 0.154 0.0 0.0 0.071 0.040 0.054
overlapped with the early emerging peaks. Ethylthiocyanate, which was used by Varadi et al. (9) as an internal standard, was poorly resolved from MeSCN by the various column packings tested (Carbowax 1000, Carbowax 20M, DESG, EGA, OV-17, Porapak QS, EGA-Trimer acid). Carbowax 1000 and Ucon LB 550X when used in 5 to 1 combination gave good resolution of chromatographic components, provided short retention times, and possessed adequate stability. Performance was maintained after 1500 injections when 75% aqueous formic acid was the solvent. Lower formic acid concentrations, in addition to lowering methionine values, also decreased column stability and increased tailing. The standard error was 0.4% with N-acetyl-DL-methionine, 1.2% with ovalbumin, and 1.6% with Ph. vdgaris seed meal. The determinations were replicated 12 times in each case. Experimentally determined methionine values of various compounds of known methionine content are given in Table 1. For samples containing methionine the results were close to the theoretical values, while
IMPROVED
METHOD
FOR METHIONINE
samples containing no methionine did not release methylthiocyanate. Recrystallized methionine sulfone and methionine sulfoxide were unreactive to CNBr. There was a linear response between methionine recovered as MeSCN and amount of seed meal in the reaction mixture (Fig. 4). Determination in seed meals of total methionine as methionine sulfone after performic acid oxidation and subsequent amino acid analysis was in close agreement with the values obtained by the proposed method (Table 2). S-~ethylcysteine, a thioether homolog of methionine present in legumes (2,10), did not release MeSCN upon reaction with CNBr (Table 1). The related compound, the dipeptide glutamyl-S-methylcysteine, also occurs in legume seed (10). It could feasibly produce MeSCN when reacted with CNBr and thereby give rise to exaggerated methionine values. If present, it should be extracted by 70% ethanol and also be found in the acidic nonprotein fraction isolated by ion-exchange chromatography. The methionine content of the 70% aqueous ethanol extract accounted for about 5% of the total seed methionine (Table 3). The fraction isolated from an acidic ethanolic bean extract by ion-exchange chromatography, representing free amino acids and dipeptides,
129
DETERMINATION TABLE
2
COMPARISON OF Two METHODS FOR THE ANALYSIS OF METHIONINE IN SEED MEAL: PERFORMIC ACID OXIDATION AND AMINO ACID ANALYSIS (PAO) AND THE PRESENT METHOD (GLC)
Methionine
(g/ 100 g)
PA0
GLC
Soybean meal Meal 1 (defatted) Meal 2 (defatted)
0.64 0.69
0.62 0.66
Ph. vzdgaris seed meal Mea1 1 Meal 2 Meal 3
0.41 0.40 0.39
0.46 0.39 0.40
including glutamyl-S-methylcysteine, accounted for less than 1% of the total seed methionine. The possible contribution of glutamyl-S-methylcysteine to the analysis of methionine is therefore negligible. This method has been extensively used in our laboratory during the last 2 years. We find it to be rapid, economical, precise, and suitable for screening for methionine in plant breeding programs since more than 50 samples can be analyzed per man-day.
TABLE
3
APPARENT METHIONINE CONTENTS OF SEED MEAL, ETHANOLIC EXTRACTS AND FREE AMINO ACID FRACTIONS OF Ph. vulgaris CULTIVARS
Methionine (g/100 g seed)
SEED MEAL
, mg
FIG. 4. Recovered methionine as a function of sample size (Ph. vulgaris seed meal).
Cultivar
Seed meal
Crude ethanolic extract
Sanilac Lot 1 Lot 2 Star Cuva 168-N Rico-23
0.365 0.335 0.370 0.430 0.352
0.017 0.018 0.020 0.015 0.012
Free amino acid fraction
0.003 0.002 0.006 0.002 0.005
130
APOSTOLATOS
REFERENCES 1. Eastoe, J. E. (1966) in Glycoproteins (Gottschalk, A., ed.), Vol. 5, p. 112, Elsevier, New York. 2. Ellinger, G. M., and Duncan, A. (1976) Biochem. J. 155, 615-621. 3. Fintayson, A. J., and MacKenzie, S. L. (1976) Anal. Biochem. 70, 397-402. 4. Hoff, J. E. (1973) in Molecular Biology of Plants (Cherry, J. H., ed.), pp. 148-167, Columbia Univ. Press. New York.
AND
HOFF
5. Inglis, A. S., and Edman, P. (1970) Anal. Biochem. 37,73-80. 6. MacKenzie, S. L. ( 1977) J. Chrumatogr. 130,399402. 7. Paul, C. A. (1977) Pflanzenschutzberichte, 78,97112. 8. Schroeder, N. A., Shelton, J. B., and Shelton, J. R. (1970) Arch. ~~~hern. Biophys. 130, 551-556. 9. Varadi, A., Pongor, S., and Kaul, A. K. (1976) Acfa Biochem. Biophys. Acad. Sci. Hung. 11, 87-93. 10. Zacharius, R. M., Morris, C. J., and Thompson, J. F. (1959) Arch. Biochem. Biophys. 80, l99209.
BIOCHEMISTRY
118,
126- 130 ( 198 1)
An Improved Method for the Gas Chromatographic Determination Methionine in Proteins and Crude Plant Materials’ GERASIMOSAPOSTOLATOS~
ANDJOHAN
of
E. HOFF
Food Sciences Institute, Purdue University, West Lafayette, Indiana 47907 Received August 5, 1981 Procedures for the determination of methionine in crude plant materials by means of cyanogen bromide digestion and gas chromatographic analysis of the digest were modified to achieve complete reaction, enhanced precision, and shortened reaction times. Oxides of methionine and derivatives of S-methylcysteine did not add to the values obtained for free and peptide-bound methionine.
Since Inglis and Edman (5) described the reaction between peptide-bound methionine and cyanogen bromide and quantitated the released methylthiocyanate by gas-liquid chromatography, several methods based on this reaction have been proposed for the analysis of methionine in crude samples of animal and plant origin (2,3,6,7,9). Such methods employ rather lengthy incubation periods (up to 18 h) in an effort to allow the reaction to go to completion, but the analytical values obtained are still low, especially with crude materials. The lack of suitable column packings and of a convenient internal standard is also apparant. We are here addressing these problems and are proposing several modifications that jointly result in improved precision and a considerably shortened analysis time. EXPERIMENTAL
Reagents. Cyanogen bromide (CNBr,3 97%) methylthiocyanate (MeSCN, 99%), 5-nonanone (di-n-butyl ketone, 98%), and ’ Journal paper No. 8655 of the Purdue Agricultural Experiment Station. * Current address: Vegetable Research Institute, Gastouni, Bias, Greece. 3 Abbreviations used: CNBr, cyanogen bromide; MeSCN, methylthiocyanate. 0003-2697/81/170126-05$02.00/O Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.
126
formic acid (97+%) were obtained from Aldrich Chemical Company. Formic acid was further purified by distillation using a reflux ratio of 4: 1. DL-Methionine, N-acetylDL-methionine, a-chymotrypsin (bovine, type II, 3X crystallized), fl-galactosidase, myoglobin (type I), papain (type III, 3~ crystallized), insulin (bovine, crystalline), Smethyl-L-cysteine, DL-methionine sulfoxide, and DL-methionine sulfone were obtained from Sigma Chemical Company. Legume seeds (10% moisture content) were ground to pass through an 80-mesh sieve. Procedure. Solution A: 1% CNBr (w/v) in 70% aqueous formic acid. Solution B: 1000 ppm 5-nonanone in 97% formic acid, Solution C: 2000 ppm MeSCN in 97% formic acid. The solutions were stable for at least 4 months when kept at 4°C. The reaction was performed in 1.5 ml Silli Vials (Applied Science Laboratories) stoppered by Teflon-laminated disks. To the dry samples, either 2030 mg finely ground seed meal or 2- 10 mg protein, were added 750 ~1 of solution A and 50 ~1 of solution B. A standard mixture was prepared each day from 700 ~1 of solution A and 50 ~1 of each of solutions B and C. Incubation at 95°C which continued for 60 min with seed meal and for 20 min with protein samples, was followed by centrifu-
IMPROVED
METHOD
FOR METHIONINE
127
DETERMINATION
There are two nonmethionine compounds in legume seed that could possibly release MeSCN and thereby contribute falsely to the apparent methionine values. S-Methylcysteine (2,10) was tested as the pure compound, while glutamyl-S-methylcysteine (10) was selectively extracted from Phase&s vulgaris seed with 70% aqueous acidic ethanol and the free amino acid fraction isolated (4). Both the ethanolic extract and the free amino acid fraction were analyzed by the proposed procedure. RESULTS AND DISCUSSION
012345 RETENTION
TIME
, min
FIG. 1. Typical chromatogram derived from the reaction of cyanogen bromide with Ph. vulgaris seed meal. I, MeSCN; 2, internal standard; 3, solvent peak.
gation at 3000g for 5 min. An aliquot (l-2 ,ul) of the clear supernatant was injected into the gas chromatograph (Hewlett-Packard, Model 402 or 5730 A) equipped with flame ionization detector and automatic integrator. Glass columns (2 mm i.d. X 100 cm) were packed with 10% Carbowax-1000 and 2% Ucon LB 550X (Analabs, Inc.) on 80to loo-mesh Chromosorb W-AW (JohnsManville). The oven temperature was 105”C, while the injection port and flame ionization detectors were held at 165°C. Nitrogen carrier gas flow rate was 40 ml/min, while the flow rates of hydrogen and air were 30 and 300 ml/min, respectively. The methionine content of various seed meals was for comparison determined by ion-exchange chromatography as methionine sulfone after performic acid oxidation (1) followed by acid hydrolysis in refluxed constant boiling HCI for 22 h. The hydrolysates were analyzed by a Beckman Model 119 CL amino acid analyzer equipped with a Model 126 data system. Analyses of such samples by the amino acid analyzer were obtained by a 1Zmin short run employing a pH 3.45 citrate buffer at 52°C.
A typical gas chromatogram (Ph. vulgaris seed meal) of a cyanogen bromide digest is illustrated in Fig. 1. The methionine content in the sample was calculated as follows: Met, % (w/w) = kfIS/&)STD
x fhf/4SLnpIe Wsample
x l9 I6
* ,
where &, AM represent peak areas for 5nonanone and methylthiocyanate, respectively, W is the sample weight in milligrams, and 19. I6 is the product of four factors: the molecular ratio (1.794) of anhydromethionine to methylthiocyanate, the amount of methylthiocyanate in the standard mixture
TIME
, min
FIG. 2. The effect of reaction time on the conversion of methionine to methylthiocyanate; A, N-Acetyl-DLmethionine; B, Ph. vulgaris protein isolate; C, Ph. vulgo& seed meal.
128
APOSTOLATOS
(0.100 mg), the density of methylthiocyanate (1.068 mg/pl) at 25”C, and loo(%). The protein content of the sample must be known if the methionine content of the protein is to be reported. An incubation temperature of 95°C allowed the reaction to go to completion in 60 min with crude Ph. vulgaris seed meal while its protein isolate required only 15 min (Fig. 2). Incubation temperatures above the boiling point of formic acid (100.6”C) resulted in damage of septa with subsequent losses of reaction mixture. One percent (w/v) of CNBr was sufficient for complete reaction with bean seed meal. Lower concentrations, e.g. 0.5%, gave variable results, while concentrations in excess of 1% gave no additional recovery of MeSCN. The concentration of formic acid in the medium affected the conversion of methionine in protein samples (Fig. 3). Optimal conversions were obtained with formic acid concentrations above 70% (v/v). The internal standard, 5-nonanone, was stable under the conditions of the reaction and separated well from MeSCN. Other substances were evaluated as internal standards. 2-Octanone also emerged after the MeSCN, but tended to overlap with MeSCN when this occurred at high concentrations. 4-Heptanone emerged prior to MeSCN, but
FORMIC
ACID , X
FIG. 3. Yield of methylthiocyanate at different concentrations of formic acid: A, N-Acetyl-Dkmethionine; B, myoglobin; C, bovine serum albumin; D, Ph. vufg&s seed meal.
AND
HOFF TABLE
1
COMPARWNOFTHEORETICALANDEXPERIMENTAL ANALYTICAL VALUESOF METHIONINEIN COMPOUNDSOF KNOWN METHIONINE~ONTENT Methionine content (mg)
Source Methionine N-Acetyl-DLmethionine cr-Chymotrypsin /3-Galactosidase Pepsin Myoglobin Insulin Papain Methionine sulfoxide Methionine sulfone S-Methylcysteine
Sample size 6-w)
Experimental
Theoretical
0.050
0.050
0.050
0.050 6.0 3.5 8.1 8.8 5.0 8.0 0.080 0.050 0.050
0.039 0.07 1 0.046 0.101 0.154 0.0 0.0 0.0 0.0 0.0
0.039 0.071 0.046 0.103 0.154 0.0 0.0 0.071 0.040 0.054
overlapped with the early emerging peaks. Ethylthiocyanate, which was used by Varadi et al. (9) as an internal standard, was poorly resolved from MeSCN by the various column packings tested (Carbowax 1000, Carbowax 20M, DESG, EGA, OV-17, Porapak QS, EGA-Trimer acid). Carbowax 1000 and Ucon LB 550X when used in 5 to 1 combination gave good resolution of chromatographic components, provided short retention times, and possessed adequate stability. Performance was maintained after 1500 injections when 75% aqueous formic acid was the solvent. Lower formic acid concentrations, in addition to lowering methionine values, also decreased column stability and increased tailing. The standard error was 0.4% with N-acetyl-DL-methionine, 1.2% with ovalbumin, and 1.6% with Ph. vdgaris seed meal. The determinations were replicated 12 times in each case. Experimentally determined methionine values of various compounds of known methionine content are given in Table 1. For samples containing methionine the results were close to the theoretical values, while
IMPROVED
METHOD
FOR METHIONINE
samples containing no methionine did not release methylthiocyanate. Recrystallized methionine sulfone and methionine sulfoxide were unreactive to CNBr. There was a linear response between methionine recovered as MeSCN and amount of seed meal in the reaction mixture (Fig. 4). Determination in seed meals of total methionine as methionine sulfone after performic acid oxidation and subsequent amino acid analysis was in close agreement with the values obtained by the proposed method (Table 2). S-~ethylcysteine, a thioether homolog of methionine present in legumes (2,10), did not release MeSCN upon reaction with CNBr (Table 1). The related compound, the dipeptide glutamyl-S-methylcysteine, also occurs in legume seed (10). It could feasibly produce MeSCN when reacted with CNBr and thereby give rise to exaggerated methionine values. If present, it should be extracted by 70% ethanol and also be found in the acidic nonprotein fraction isolated by ion-exchange chromatography. The methionine content of the 70% aqueous ethanol extract accounted for about 5% of the total seed methionine (Table 3). The fraction isolated from an acidic ethanolic bean extract by ion-exchange chromatography, representing free amino acids and dipeptides,
129
DETERMINATION TABLE
2
COMPARISON OF Two METHODS FOR THE ANALYSIS OF METHIONINE IN SEED MEAL: PERFORMIC ACID OXIDATION AND AMINO ACID ANALYSIS (PAO) AND THE PRESENT METHOD (GLC)
Methionine
(g/ 100 g)
PA0
GLC
Soybean meal Meal 1 (defatted) Meal 2 (defatted)
0.64 0.69
0.62 0.66
Ph. vzdgaris seed meal Mea1 1 Meal 2 Meal 3
0.41 0.40 0.39
0.46 0.39 0.40
including glutamyl-S-methylcysteine, accounted for less than 1% of the total seed methionine. The possible contribution of glutamyl-S-methylcysteine to the analysis of methionine is therefore negligible. This method has been extensively used in our laboratory during the last 2 years. We find it to be rapid, economical, precise, and suitable for screening for methionine in plant breeding programs since more than 50 samples can be analyzed per man-day.
TABLE
3
APPARENT METHIONINE CONTENTS OF SEED MEAL, ETHANOLIC EXTRACTS AND FREE AMINO ACID FRACTIONS OF Ph. vulgaris CULTIVARS
Methionine (g/100 g seed)
SEED MEAL
, mg
FIG. 4. Recovered methionine as a function of sample size (Ph. vulgaris seed meal).
Cultivar
Seed meal
Crude ethanolic extract
Sanilac Lot 1 Lot 2 Star Cuva 168-N Rico-23
0.365 0.335 0.370 0.430 0.352
0.017 0.018 0.020 0.015 0.012
Free amino acid fraction
0.003 0.002 0.006 0.002 0.005
130
APOSTOLATOS
REFERENCES 1. Eastoe, J. E. (1966) in Glycoproteins (Gottschalk, A., ed.), Vol. 5, p. 112, Elsevier, New York. 2. Ellinger, G. M., and Duncan, A. (1976) Biochem. J. 155, 615-621. 3. Fintayson, A. J., and MacKenzie, S. L. (1976) Anal. Biochem. 70, 397-402. 4. Hoff, J. E. (1973) in Molecular Biology of Plants (Cherry, J. H., ed.), pp. 148-167, Columbia Univ. Press. New York.
AND
HOFF
5. Inglis, A. S., and Edman, P. (1970) Anal. Biochem. 37,73-80. 6. MacKenzie, S. L. ( 1977) J. Chrumatogr. 130,399402. 7. Paul, C. A. (1977) Pflanzenschutzberichte, 78,97112. 8. Schroeder, N. A., Shelton, J. B., and Shelton, J. R. (1970) Arch. ~~~hern. Biophys. 130, 551-556. 9. Varadi, A., Pongor, S., and Kaul, A. K. (1976) Acfa Biochem. Biophys. Acad. Sci. Hung. 11, 87-93. 10. Zacharius, R. M., Morris, C. J., and Thompson, J. F. (1959) Arch. Biochem. Biophys. 80, l99209.