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A method for automated analyses of the activities of trypsin, chymotrypsin and their inhibitors
ANALYTICAL
BIOCHEMISTRY
A Method for of Trypsin,
51, 11-18 (1973)
Automated Chymotrypsin KENT
Protein
Nutrition
United
States
Analyses of the Activities and Their Inhibitors
K. STEWART
Laboratory, Nutrition Institute, Department of Agriculture,
Received November
Agricultural Beltsville,
Research Service, Maryland 10705
17, 1971; accepted August 25, 1972
Methods for the automated analyses of the activity of trypsin and chymotrypsin and their inhibitors using L-BAPA and L-GPNA, respectively, as substrates are described.
Today there is an increasing interest in trypsin and chymotrypsin inhibitors due to their possible role in altering the nutritive value of foodstuffs, their usefulness as models of protein-protein interactions and enzyme-substrate interact,ions, and their unique pharmacological potential for clinical applications (1,2). With this interest in these inhibitors there is an increased effort to isolate, purify, and characterize the large number of t.rypsin and chymotrypsin inhibitors found in nature. We have been engaged in the isolation, purification, and characterization of peanut trypsin and chymotrypsin inhibitors and have found it expedient to automate the assay of these inhibitors. In the process of automating the assay for the inhibitors we have also automated the assay of the enzymes. The chromogenic substrates N-benzoyl-m-arginine p-nitroanilide (DL-BAPA) and N-glutaryl-L-phenylalanine p-nitroanilide (L-GPNA) developed by Erlanger and his coworkers (3,4) have been quite useful in the manual assays for trypsin and chymotrypsin respectively due to the selectivity of the enzymes for the substrates and the low blank values obtained in the absence of enzyme. We have investigated the automation of these assays and report herein the details of an automated method for the assay of trypsin and trypsin inhibitor activities and of chymotrypsin and chymotrypsin inhibitor activities using L-BAPA and L-GPNA, respectively, as substrates. 11 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
12
KENT
METHODS
K.
AND
STEWART
MATERIALS
Substrates Ar-benzoyl-L-arginine p-nitroanilide hydrochloride (5) was purchased from the Protein Research Foundation, Osaka, Japan. The substrate solution was prepared fresh daily by dissolving L-BAPA in dimethyl sulfoxide (50 mg BAPA/ml) with gentle heating and then diluting the solution with 0.5 M Tris-HCl buffer (pH 8.2) which contained 0.02 M CaCl,. DL-BAPA was not used since it precipitated in the tubing and coils of the Autoanalyzer. N-glutaryl-L-phenylalanine p-nitroanilide was purchased from the Nutritional Biochemical Corporation. The substrate solution was prepared fresh daily by dissolving L-GPNA in methanol (20 mg GPNA/ml) with mild heating and then diluting the solution with 0.05 M Tris-HCl buffer (pH 7.6) containing 0.02 M CaCl,. Enzymes Trypsin (bovine pancreas, 2~ crystallized) was purchased from Sigma Chemical Company. Stock enzyme solution was prepared (2 mg/ ml in 1 mM HCI) and frozen in 2-ml ampoules. The enzyme assay solutions were prepared daily by diluting the stock solution with 1 mM HCl. The cy-chymotrypsin (bovine pancreas, 2 X crystallized) was purchased from Sigma Chemical Company. The chymotrypsin was weighed out directly prior to use and dissolved in 1 mM HCl. Inhibitor
Solutions
Soybean trypsin inhibitor (Type I S, lyophilized) (SBTI) and lima bean trypsin inhibitor (lyophilized) (LBTI) were purchased from Sigma Chemical Company and Worthington Biochemical Corporation, respectively. Peanut trypsin inhibitors (PTI) were prepared by a method which will be described at a later date. Aqueous solutions of the inhibitors were used. Equipment The schematic for the automated assay is shown in Fig. 1. The buffer for the trypsin assay was 0.05 M Tris-HCl (pH 8.2) with 0.02 M CaCl, and for the chymotrypsin assay was 0.05 M Tris-HCl (pH 7.6) with 0.02 M CaC12. Ten percent (v/v) acetic acid was used to stop the reaction. Standard Technicon Autoanalyzer parts were used except as noted below. The Technicon Sampler II was modified to have a dual pickup consisting of two sample probes, two sample probe assemblies, and two probe clamps mounted one on top of the other. The sample probe for the
AUTOMATED
TRYPSIN
AND
CHYMOTRYPSIN
13
ASSAYS
,p ., \ I’\\,
.\
\
5’ FLOW RATE ML/MIN
WASH BUFFER
FIG.
and
their
01
1. Flow diagram inhibitors.
for
/ 6
automated
determination
I
I 12 TIME
FIG. 2. Chymotrypsin to right the samples 800 &g/ml, respectively. was l/l.
had
of trypsin
I 24
or of chymotrypsin
I 30
dN8UTES)
assay using L-GPNA (0.5 mg/ml) as substrate. From left a chymotrypsin concentration of 160, 320, 480, 640, and The sampling rate was lO/hr and the wash sample ratio
14
KENT
K.
STEWART
enzyme pickup was a piece of 18 gauge Kel-F tubing. Preliminary studies indicated that the use of the standard stainless steel probes resulted in partial enzyme inactivation. The sample plate was modified to hold two 2-ml cups at each position. All the sample cups were soaked overnight in 0.1% EDTA solution, rinsed in distilled water, and air dried before use. Timing cams of 10 or 20 samples per hour at a l/l wash to sample ratio were used. The flow cell was the “N” tubular type which was modified to decrease the mixing volume at the debubbler and 420-nm filters were used in the calorimeter. It was always necessary to adjust the length of the inhibitor sample tubing and the enzyme sample tubing so that the two sample slugs arrived at. their mixing chamber at exactly the same time. RESULTS AND DISCUSSION
The results of a typical series of chymot,rypsin assays with L-GPNA as a substrate are shown in Fig. 2. As the sample peaks and their return to baseline meet the criteria of good peak shapes set by Roodyn (6) for this type of analysis, the absorbance values were calculated from the
0
-0
25
0
50
0
75
1.00
I25
I 50
L-BAPA mg/ml
FIG. 3. Effect of substrate concentration on trypsin activity at different enzyme concentrations. The assays were run at pH 8.2 with L-BAPA at the input concentrations shown. The number on each curve is the input concentration of trypsin in micrograms per milliliter.
AUTOMATED
TRTPSIN
ASD
CHYNOTR‘I-PSIS
15
ASSAYS
sample peak heights on the recorder tracing. The response of the trypsin assay system to increasing substrate concentration was a continuous increase in absorbance as shown in Fig. 3. The assay showed a linear response from 0 to 30 pg t,rypsin per ml at input L-BAPA concentrations of 0.50, 1.00 and 1.50 mg/ml; the latter concentration is close to the saturating concentration of L-BAPA in the buffer system. At higher enzyme concentrations (40-60 pg/ml) and the lower substrate concentration (0.125 mg/ml) the standard curves were not linear, although they were still reproducible. The response of the chymotrypsin assay system to increasing substrate concentration at different enzyme levels is shown in Fig. 4. Like the trypsin assay system, the plot of velocity versus substrate concentration does not level out, even at substrate concentrations close to t,he maximum solubility of GPNA. The chymotrypsin assay system showed a linear response in absorbance from 0 pg/ml to the highest enzyme concentrations tested (800 pg). Note that on a weight basis the trypsin assay was about 20 times more sensitive than the chymotrypsin assay which is similar to the differences in sensitivity observed with the manual assays of these two enzymes (4).
0
0.5-
04-
02
03 L-GPNA
1 mg/ml)
FIG. 4. Effect of substrate concentration on chymotrypsin activity at different enzyme concentrations. The assays were run at pH 7.6 using L-GPNA at the input concentration shown. The number on each curve is the input concentration of chymotrypsin in micrograms per milliliter.
16
KENT
K. STEWART
Standard curves for the assay of LBTI using the chymotrypsin assay (Fig. 5A) and of SBTI using the trypsin assay system (Fig. 5B) are shown in Fig. 5. Similar curves resulted when SBTI and PTI are assayed by the chymotrypsin assay and when LBTI and PTI are assayed by the trypsin assay. The difference in the shapes of the two inhibition curves is thought to be due to the differences in the binding constants of the enzymes for these inhibitors.
Oii
LBTI
(mg/ml)
FIG. 5. (A) Standard curve for lima bean trypsin inhibitor (LBTI) activity using the chymotrypsin assay. The reaction buffer was 0.05~ Tris-HCl (pH 7.6) with 0.02 M CaCh; chymotrypsin concentration was 500 pg/ml and the L-GPNA concentration was 1.0 mg/ml. The sampling rate was lO/hr with a l/l wash to sample ratio. (B) Standard curve for soybean trypsin (SBTI) inhibitor activity using the trypsin assay. The reaction buffer was 0.05 M Tris-HCl (pH 8.2) with 0.02 M CaCl,; trypsin concentration was 30 pg/ml and the L-BAPA concentration was 0.5 mg/ml. The sampling rate was lO/hr with a l/l wash to sample ratio.
AUTOMATED
TRTPSIN
A3D
CHYMOTRYPRIX
17
ASSAYS
The incubation time for an enzyme and its inhibitor might affect the observed inhibition. This was not seen with trypsin and SBTI. No difference in inhibition was noted when trypsin was mixed with SBTI for either 15, 30, 60, 300 or 900 set prior to the addition of the substrate. Slight variations in response to enzyme concentrations have been noted from day to day and between enzyme lots which necessitated running standard curves each day when quantitative assays were being performed. It was also necessary to “prerun” three to six enzyme samples before the system stabilized and gave a reproducible response to the enzyme concentration. The analytical system is sensitive to sample rates due to its mixing and washout characteristics. High sample rates (60 samples/hr ; l/l wash to sample ratio) result in decreased sensitivity due to nonattainment of steady state in the flow cell and increased error due to the cross cont’amination of the samples in the whole system. Sample rates of lO/hr are now routinely used for careful quantitative work while rates of 2O/hr are used for the analysis of chromatographic effluents. The automated enzyme assays reported herein are an important addition to the array of automated proteolytic enzyme assays available to the analyst, (7-14). The present system was designed to provide an automated assay system for selective specific enzyme activities, i.e., trypsin-like and chymotrypsin-like activity whereas previously reported automated enzyme methods were designed for the assay of a broad spectrum of proteolytic activity (7-10). In addition, the present system provides an automated method for the assay of specific inhibitors of trypsin and chymotrypsin. The automation of these assays provides an easy and reproducible assay system which could easily be adapted to other enzyme inhibitor combinations and can be routinely used for large number of samples. These assays have been useful in scanning chromatographic effluents for PTI inhibitor activity, for the quantitation of the inhibitor activity, and for the characterization of the isolated inhibitor(s). ACKNOWLEDGMENTS The author thanks Mr. F. Oldson for The technical assistance of Mr. Stephen is gratefully acknowledged.
making the M. Feltch
modifications and Mrs.
to Sandra
the sampler. H. Eldhart
REFERENCES I. E., AND KAKADE, M. L. (1969) in Toxic Constituents of Plant Food(Liener, I. E., ed.), p. 7, Academic Press, New York. 2. VOGEL, R., TRAUTSCHOLD. I., AND WERLE, E. (1968) Natural Proteinase Inhibitors, p. 1, Academic Press, New York. 3. ERLANQER, B. F., KOKOWSKY, N., AND COHEN, W. (1961) Arch. Biochem. Bioph?~s. 1. LIENER,
stuffs
95, 271.
18
KENT
K.
STEWART
B. F., EDEL: F., AND COOPER, A. G. (1966) Arch. Biochem. Biophys. 115, 296. NISHI, N., TOKURA, S., AND XOGUCHI, J. (1970) Bull. Chem. Sot. Jap. 43, 2900. ROODYN, D. B. (1970) Automated Enzyme Assays, p. 131, North-Holland/ American Elsevier, New York. HAZEN, C. G., HAUSE, J. A.! AND HUBICKI, J. A. (1965) Ann. N. Y. Acad. Sci. 130, 761. HEINICKE, R. M., LARSON, C.. LEVAND, O., AND MCCARTER, M. (1968) The 1967 Technicon Symposium, p. 207. TAPPEL, A. L. (1964) The 1964 Technicon International Symposium, p. 32. TAPPEL, A. L. (1968) Anal. Biochem. 23, 466. LEVINE, M., DORER, F. E., KAHN> J. R.. LENTZ, K. E., AND SKEGGS, L. T. (1970)
4. ERL.4NGER, 5.
6. 7. 8.
9. 10. 11.
Anal.
12. 13. 14.
Biochem.
34,
366.
M. L., CANAL, J., ASD JOUANNELLE, J. (1966) Ann. Biol. Clin. 24, 1191. Y., SUZUKI, K.? AND Moor, K. (1971) Anal. Biochem. 43, 15. VANDERMEERS, A., LELOTTE, H., .~ND CHRISTOPHE, J, (1971) Anal. Biochem. 42, 437.
GIRARD, OZAWA,
BIOCHEMISTRY
A Method for of Trypsin,
51, 11-18 (1973)
Automated Chymotrypsin KENT
Protein
Nutrition
United
States
Analyses of the Activities and Their Inhibitors
K. STEWART
Laboratory, Nutrition Institute, Department of Agriculture,
Received November
Agricultural Beltsville,
Research Service, Maryland 10705
17, 1971; accepted August 25, 1972
Methods for the automated analyses of the activity of trypsin and chymotrypsin and their inhibitors using L-BAPA and L-GPNA, respectively, as substrates are described.
Today there is an increasing interest in trypsin and chymotrypsin inhibitors due to their possible role in altering the nutritive value of foodstuffs, their usefulness as models of protein-protein interactions and enzyme-substrate interact,ions, and their unique pharmacological potential for clinical applications (1,2). With this interest in these inhibitors there is an increased effort to isolate, purify, and characterize the large number of t.rypsin and chymotrypsin inhibitors found in nature. We have been engaged in the isolation, purification, and characterization of peanut trypsin and chymotrypsin inhibitors and have found it expedient to automate the assay of these inhibitors. In the process of automating the assay for the inhibitors we have also automated the assay of the enzymes. The chromogenic substrates N-benzoyl-m-arginine p-nitroanilide (DL-BAPA) and N-glutaryl-L-phenylalanine p-nitroanilide (L-GPNA) developed by Erlanger and his coworkers (3,4) have been quite useful in the manual assays for trypsin and chymotrypsin respectively due to the selectivity of the enzymes for the substrates and the low blank values obtained in the absence of enzyme. We have investigated the automation of these assays and report herein the details of an automated method for the assay of trypsin and trypsin inhibitor activities and of chymotrypsin and chymotrypsin inhibitor activities using L-BAPA and L-GPNA, respectively, as substrates. 11 Copyright @ 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
12
KENT
METHODS
K.
AND
STEWART
MATERIALS
Substrates Ar-benzoyl-L-arginine p-nitroanilide hydrochloride (5) was purchased from the Protein Research Foundation, Osaka, Japan. The substrate solution was prepared fresh daily by dissolving L-BAPA in dimethyl sulfoxide (50 mg BAPA/ml) with gentle heating and then diluting the solution with 0.5 M Tris-HCl buffer (pH 8.2) which contained 0.02 M CaCl,. DL-BAPA was not used since it precipitated in the tubing and coils of the Autoanalyzer. N-glutaryl-L-phenylalanine p-nitroanilide was purchased from the Nutritional Biochemical Corporation. The substrate solution was prepared fresh daily by dissolving L-GPNA in methanol (20 mg GPNA/ml) with mild heating and then diluting the solution with 0.05 M Tris-HCl buffer (pH 7.6) containing 0.02 M CaCl,. Enzymes Trypsin (bovine pancreas, 2~ crystallized) was purchased from Sigma Chemical Company. Stock enzyme solution was prepared (2 mg/ ml in 1 mM HCI) and frozen in 2-ml ampoules. The enzyme assay solutions were prepared daily by diluting the stock solution with 1 mM HCl. The cy-chymotrypsin (bovine pancreas, 2 X crystallized) was purchased from Sigma Chemical Company. The chymotrypsin was weighed out directly prior to use and dissolved in 1 mM HCl. Inhibitor
Solutions
Soybean trypsin inhibitor (Type I S, lyophilized) (SBTI) and lima bean trypsin inhibitor (lyophilized) (LBTI) were purchased from Sigma Chemical Company and Worthington Biochemical Corporation, respectively. Peanut trypsin inhibitors (PTI) were prepared by a method which will be described at a later date. Aqueous solutions of the inhibitors were used. Equipment The schematic for the automated assay is shown in Fig. 1. The buffer for the trypsin assay was 0.05 M Tris-HCl (pH 8.2) with 0.02 M CaCl, and for the chymotrypsin assay was 0.05 M Tris-HCl (pH 7.6) with 0.02 M CaC12. Ten percent (v/v) acetic acid was used to stop the reaction. Standard Technicon Autoanalyzer parts were used except as noted below. The Technicon Sampler II was modified to have a dual pickup consisting of two sample probes, two sample probe assemblies, and two probe clamps mounted one on top of the other. The sample probe for the
AUTOMATED
TRYPSIN
AND
CHYMOTRYPSIN
13
ASSAYS
,p ., \ I’\\,
.\
\
5’ FLOW RATE ML/MIN
WASH BUFFER
FIG.
and
their
01
1. Flow diagram inhibitors.
for
/ 6
automated
determination
I
I 12 TIME
FIG. 2. Chymotrypsin to right the samples 800 &g/ml, respectively. was l/l.
had
of trypsin
I 24
or of chymotrypsin
I 30
dN8UTES)
assay using L-GPNA (0.5 mg/ml) as substrate. From left a chymotrypsin concentration of 160, 320, 480, 640, and The sampling rate was lO/hr and the wash sample ratio
14
KENT
K.
STEWART
enzyme pickup was a piece of 18 gauge Kel-F tubing. Preliminary studies indicated that the use of the standard stainless steel probes resulted in partial enzyme inactivation. The sample plate was modified to hold two 2-ml cups at each position. All the sample cups were soaked overnight in 0.1% EDTA solution, rinsed in distilled water, and air dried before use. Timing cams of 10 or 20 samples per hour at a l/l wash to sample ratio were used. The flow cell was the “N” tubular type which was modified to decrease the mixing volume at the debubbler and 420-nm filters were used in the calorimeter. It was always necessary to adjust the length of the inhibitor sample tubing and the enzyme sample tubing so that the two sample slugs arrived at. their mixing chamber at exactly the same time. RESULTS AND DISCUSSION
The results of a typical series of chymot,rypsin assays with L-GPNA as a substrate are shown in Fig. 2. As the sample peaks and their return to baseline meet the criteria of good peak shapes set by Roodyn (6) for this type of analysis, the absorbance values were calculated from the
0
-0
25
0
50
0
75
1.00
I25
I 50
L-BAPA mg/ml
FIG. 3. Effect of substrate concentration on trypsin activity at different enzyme concentrations. The assays were run at pH 8.2 with L-BAPA at the input concentrations shown. The number on each curve is the input concentration of trypsin in micrograms per milliliter.
AUTOMATED
TRTPSIN
ASD
CHYNOTR‘I-PSIS
15
ASSAYS
sample peak heights on the recorder tracing. The response of the trypsin assay system to increasing substrate concentration was a continuous increase in absorbance as shown in Fig. 3. The assay showed a linear response from 0 to 30 pg t,rypsin per ml at input L-BAPA concentrations of 0.50, 1.00 and 1.50 mg/ml; the latter concentration is close to the saturating concentration of L-BAPA in the buffer system. At higher enzyme concentrations (40-60 pg/ml) and the lower substrate concentration (0.125 mg/ml) the standard curves were not linear, although they were still reproducible. The response of the chymotrypsin assay system to increasing substrate concentration at different enzyme levels is shown in Fig. 4. Like the trypsin assay system, the plot of velocity versus substrate concentration does not level out, even at substrate concentrations close to t,he maximum solubility of GPNA. The chymotrypsin assay system showed a linear response in absorbance from 0 pg/ml to the highest enzyme concentrations tested (800 pg). Note that on a weight basis the trypsin assay was about 20 times more sensitive than the chymotrypsin assay which is similar to the differences in sensitivity observed with the manual assays of these two enzymes (4).
0
0.5-
04-
02
03 L-GPNA
1 mg/ml)
FIG. 4. Effect of substrate concentration on chymotrypsin activity at different enzyme concentrations. The assays were run at pH 7.6 using L-GPNA at the input concentration shown. The number on each curve is the input concentration of chymotrypsin in micrograms per milliliter.
16
KENT
K. STEWART
Standard curves for the assay of LBTI using the chymotrypsin assay (Fig. 5A) and of SBTI using the trypsin assay system (Fig. 5B) are shown in Fig. 5. Similar curves resulted when SBTI and PTI are assayed by the chymotrypsin assay and when LBTI and PTI are assayed by the trypsin assay. The difference in the shapes of the two inhibition curves is thought to be due to the differences in the binding constants of the enzymes for these inhibitors.
Oii
LBTI
(mg/ml)
FIG. 5. (A) Standard curve for lima bean trypsin inhibitor (LBTI) activity using the chymotrypsin assay. The reaction buffer was 0.05~ Tris-HCl (pH 7.6) with 0.02 M CaCh; chymotrypsin concentration was 500 pg/ml and the L-GPNA concentration was 1.0 mg/ml. The sampling rate was lO/hr with a l/l wash to sample ratio. (B) Standard curve for soybean trypsin (SBTI) inhibitor activity using the trypsin assay. The reaction buffer was 0.05 M Tris-HCl (pH 8.2) with 0.02 M CaCl,; trypsin concentration was 30 pg/ml and the L-BAPA concentration was 0.5 mg/ml. The sampling rate was lO/hr with a l/l wash to sample ratio.
AUTOMATED
TRTPSIN
A3D
CHYMOTRYPRIX
17
ASSAYS
The incubation time for an enzyme and its inhibitor might affect the observed inhibition. This was not seen with trypsin and SBTI. No difference in inhibition was noted when trypsin was mixed with SBTI for either 15, 30, 60, 300 or 900 set prior to the addition of the substrate. Slight variations in response to enzyme concentrations have been noted from day to day and between enzyme lots which necessitated running standard curves each day when quantitative assays were being performed. It was also necessary to “prerun” three to six enzyme samples before the system stabilized and gave a reproducible response to the enzyme concentration. The analytical system is sensitive to sample rates due to its mixing and washout characteristics. High sample rates (60 samples/hr ; l/l wash to sample ratio) result in decreased sensitivity due to nonattainment of steady state in the flow cell and increased error due to the cross cont’amination of the samples in the whole system. Sample rates of lO/hr are now routinely used for careful quantitative work while rates of 2O/hr are used for the analysis of chromatographic effluents. The automated enzyme assays reported herein are an important addition to the array of automated proteolytic enzyme assays available to the analyst, (7-14). The present system was designed to provide an automated assay system for selective specific enzyme activities, i.e., trypsin-like and chymotrypsin-like activity whereas previously reported automated enzyme methods were designed for the assay of a broad spectrum of proteolytic activity (7-10). In addition, the present system provides an automated method for the assay of specific inhibitors of trypsin and chymotrypsin. The automation of these assays provides an easy and reproducible assay system which could easily be adapted to other enzyme inhibitor combinations and can be routinely used for large number of samples. These assays have been useful in scanning chromatographic effluents for PTI inhibitor activity, for the quantitation of the inhibitor activity, and for the characterization of the isolated inhibitor(s). ACKNOWLEDGMENTS The author thanks Mr. F. Oldson for The technical assistance of Mr. Stephen is gratefully acknowledged.
making the M. Feltch
modifications and Mrs.
to Sandra
the sampler. H. Eldhart
REFERENCES I. E., AND KAKADE, M. L. (1969) in Toxic Constituents of Plant Food(Liener, I. E., ed.), p. 7, Academic Press, New York. 2. VOGEL, R., TRAUTSCHOLD. I., AND WERLE, E. (1968) Natural Proteinase Inhibitors, p. 1, Academic Press, New York. 3. ERLANQER, B. F., KOKOWSKY, N., AND COHEN, W. (1961) Arch. Biochem. Bioph?~s. 1. LIENER,
stuffs
95, 271.
18
KENT
K.
STEWART
B. F., EDEL: F., AND COOPER, A. G. (1966) Arch. Biochem. Biophys. 115, 296. NISHI, N., TOKURA, S., AND XOGUCHI, J. (1970) Bull. Chem. Sot. Jap. 43, 2900. ROODYN, D. B. (1970) Automated Enzyme Assays, p. 131, North-Holland/ American Elsevier, New York. HAZEN, C. G., HAUSE, J. A.! AND HUBICKI, J. A. (1965) Ann. N. Y. Acad. Sci. 130, 761. HEINICKE, R. M., LARSON, C.. LEVAND, O., AND MCCARTER, M. (1968) The 1967 Technicon Symposium, p. 207. TAPPEL, A. L. (1964) The 1964 Technicon International Symposium, p. 32. TAPPEL, A. L. (1968) Anal. Biochem. 23, 466. LEVINE, M., DORER, F. E., KAHN> J. R.. LENTZ, K. E., AND SKEGGS, L. T. (1970)
4. ERL.4NGER, 5.
6. 7. 8.
9. 10. 11.
Anal.
12. 13. 14.
Biochem.
34,
366.
M. L., CANAL, J., ASD JOUANNELLE, J. (1966) Ann. Biol. Clin. 24, 1191. Y., SUZUKI, K.? AND Moor, K. (1971) Anal. Biochem. 43, 15. VANDERMEERS, A., LELOTTE, H., .~ND CHRISTOPHE, J, (1971) Anal. Biochem. 42, 437.
GIRARD, OZAWA,