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Desalting of proteins, peptides, and amino acids on polyacrylamide gel
ANALYTICAL
14, 321327
BIOCHEMISTRY
Desalting
of
Proteins, on
ALICE From
(1966)
Peptides,
Polyacrylamide
N. SCHWARTZ the Bio-Rad
and
AND
Laboratories,
Received
July
Amino
Acids
Gel BURTON Richmond,
A. ZABIN California
7, 1965
The technique of gel filtration was demonstrated by Porath and Flodin {l, 2) to be highly useful for the desalting of proteins. The protein molecules, being larger than the pore size of the gel, are excluded from the gel structure and are eluted in the first void volume from a column packed with a gel filtration material. The small salt molecules are able to penetrate the gel and are thus eluted later in a volume equal to the void volume plus the accessible volume in the gel matrix. Both cross-linked dextran and polyacrylamide gels are used for desalting of proteins (2,3). For the desalting of small molecules such as amino acids and peptides, a gel with small pore sizes is required. Such a material has not been available and various ion-exchange methods (4-7) are now used. A new highly cross-linked polyacrylamide gel was recently developed in this laboratory. Substances with molecular weights higher than about 2600 are excluded from this gel. Characterization of this gel by determining the Rf values for several peptides and amino acids gives an Rf versus log molecular weight curve with a lower limit of about 200 molecular weight. The possibility of extending gel filtration to the desalting of small molecules with such a material is apparent. This paper presents the results of experiments to determine the usefulness of a highly cross-linked polyacrylamide gel for desalting procedures and some of the parameters involved. EXPERIMENTAL
Materials. The highly cross-linked polyacrylamide gel used in this study was Bio-Gel P-2, lot 2834, 50-100 mesh and 100-200 mesh (from Bio-Rad Laboratories, Richmond, California). This gel had a hydrated bed volume of 3.8 ml/dry gram. Water regain values were 1.3 gm water/gm dry gel for the 50-100 mesh sample and 1.6 gm water/gm dry gel for the 100-200 mesh sample. The proteins, amino acids, and tris [tris(hydroxymethyl) aminomethane] buffer’ were obtained from Calbio321 8 1966 by Academic
Press
Inc.
322
SCHWARTZ
AND
ZARIN
them, Los Angeles, California. The ninhydrin paper used for detecting amino acids in the column effluent was prepared by immersing filter paper in a solution of ninhydrin in water and air drying. The chromatographic columns were of glass, 1.3 cm by approximately 40 cm over-all length, with the bottom drawn down to a small tip and plugged with quartz wool. All columns used in this laboratory for gel filtration are treated to reduce “wall effects” (4). The following column treatment is recommended where minimum band spreading is desired: A clean column is filled with a solution of lo/O dichlorodimethylsilane in benzene (available from Bio-Rad Laboratories) at 60°C. The solution is decanted after several minutes and the benzene allowed to evaporate in a drying oven. Two successive treatments give a column which can be used for several months. Methods. The dry Bio-Gel P-2, as supplied, was slowly poured with stirring into an excess of deionized water. Approximately lo-15 ml of water was used for every gram of gel to obtain a suspension suitable for pouring into the column. The gel was allowed to hydrate for l-2 hr. The gel suspension was then poured into the column through an attached funnel. The column and funnel were filled with a sufficient amount of the gel suspension, so that the total bed was packed with one pouring. Samples were introduced by draining the water level to the top of the bed, and carefully adding the sample using a pipet with a curved tip. The application was completed by allowing the sample to drain into the bed, followed by a rinse of about 0.5 ml of water. The column was then filled with deionized water and attached to a reservoir for gravity flow or to a Beckman Model 746 solution metering pump. The amino acids and bacitracin were detected in the effluent by spotting on ninhydrin paper and heating. Sodium chloride was detected on a spot plate with silver nitrate-nitric acid reagent. In the protein desalting experiments, hemoglobin, urea, and ammonium sulfate were detected by evaporating drops on a glass plate. Bovine serum albumin, urea, and tris buffer were monitored through a Vanguard model 1056 automatic ultraviolet analyzer. RESULTS The removal of ammonium sulfate, urea, and tris buffer from a protein solution by gel filtration through a short column of Bio-Gel P-2 is shown in Fig. 1. Percentage recovery was determined on bovine serum albumin and was 100%. Bacitracin, with a molecular weight of 1411, was chosen as a typical peptide which might be easily desalted on Bio-Gel P-2. A l-ml sample containing 10 mg of bacitracin and 10 mg of sodium chloride in water
DESALTING
ON
POLYACRYLAMIDE
IO
20 Effluent
323
GEL
40
30 Volume
(ml)
FIG. 1. Desalting of proteins by gel filtration through a short column of Bio-Gel P-2, 50-100 mesh. Column, 1.3 x 20 cm. Eluant, water at 0.5 ml/min. Samples, 1 ml, containing, respectively, 10 mg hemoglobin and 2 M (NHJSO,, 10 mg bovine serum albumin and 8M urea, 10 mg bovine serum albumin and 0.05 M tris-HCl buffer, pH 8.1.
was applied to a 1.3 X 36 cm column of Bio-Gel P-2, 50-100 mesh, in water. Elution was carried out with water at 0.5 ml/min flow rate. Figure 2 shows the complete separation of bacitracin from sodium chloride. The bacitracin fraction was collected and recovery was found to be 100%. Glycine was partially desalted on the same column under the same conditions (Fig. 3). NaCl Bacitracin
0) ., 2G I
, IO
,A 20 Effluent
FIU.
umn, water.
I\, 30 Volume
40
50
(ml)
2. Desalting of bacitracin by gel filtration on Bio-Gel P-2, 50-100 mesh. Col1.3 x 36 cm. Sample, 1 ml, containing 10 mg bacitracin and 10 mg NaCl in Eluted with water at a flow rate of 0.4 ml/min.
324
SCHWARTZ
AND
ZABIN
6
NaCl
.-6 L ‘r 8 s 0 aI 2 I z z (L
r\
Glycine
I IO
I 30
I 20 Effluent
Volume
I 50
40 (ml)
3. Desalting of glycine on Bio-Gel P-2, 50-160 mesh. Column, 1.3 X 36 cm. Sample, 1 ml, containing 10 mg glycine and 10 mg NaCl in water. Eluted with water at a flow rate of 0.5 ml/mm. FIQ.
Effect of Flow Rate and Particle Size on Desalting Figure 4 illustrates the effect of reducing the column flow rate and decreasing the particle size of the gel. With a small particle size of Bio-Gel P-2 and a slow flow rate, the elution curve was sharp and leucine was completely desalted. NaCl 0-e 2.0
-
Leucine .r.-0,
A.
s E s
0’
-,.-.-.-
I
OE 2 E $
I\
.’
l.O-
‘~.J */y-e-.-.
.’ I
‘1 _ ‘\-
e-0 2,0
-
(%
B.
/\
1.0-
i ,
I
O-
‘\.,
/*
,
1 t-9-
,. d
\
./“, 2.0
-
. .1
O-
I
I
25
30
. 2’
\ t
, 35
Effluent
Volume
I
0-o
C.
I.0 -
.
,.
/
I
40
I\
ii 45
(ml)
4. Effect of flow rate and gel particle size on desalting. Column, 1.3 X 36 cm. Sample, 10 mg leucine and 10 mg NaCl in 1 ml water. Eluant, water. (A) Bio-GeI P-2, M-100 mesh (dry). Eluant flow rate, 1.0 ml/min. (B) Bio-Gel P-2, 5&100 mesh (dry). Eluant flow rate, 0.5 ml/min. (C) Bio-Gel P-2, 100-206 mesh (dry). Eluant flow rate, 0.5 ml/min. FIO.
DESALTIKG
ON
POLYACRYLAMIDE
GEL
325
Eflect of Amino Acid Structure oni Desalting The differences in the elution curves of acidic, basic, and aromatic amino acids are shown in Figure 5. The relative Rf values calculated for
Phenylalanine
,/-‘t
1’ I
30
35
/
1’
,
40 Effluent
45 Volume
(ml)
FIG. 5. Desalting of acidic, basic, and aromatic amino acids. Column, 1.3 x 36 cm Bio-Gel P-2, 50-100 mesh. Sample, 1 ml, containing 10 mg amino acid and 10 mg NaCl. Eluted with water at a flow rate of approximately 0.5 ml/min.
the peak maxima in these curves are: glycine, 0.57; glutamic acid, 0.59; 0.52; tryptophan, 0.47. Thus, the basic arginine, 0.52; phenylalanine, and aromatic amino acids are slightly retarded on Bio-Gel P-2 during elution with water. Tryptophan showed the greatest amount of retardation. Elution of arginine in 0.1 M acetic acid from Bio-Gel P-2 equilibrated with acetic acid gave two ninhydrin-positive peaks (Fig. 6). Increasing the acetic acid concentration to 1 M resulted in an enhancement of the
first peak, with correspondingly less material in the second peak. A double peak was also obtained when glutamic acid was eluted with 0.1 M pyridine, the second peak being quite small. The retardation of arginine was increased in pyridine peak.
chloride
and the arginine
peak overlapped
the sodium
326
SCHWAFiTZ
Effluent
AND
Volume
ZABIN
(ml)
FIQ. 6. Elution of arginine and sodium chloride from B&Gel P-2 with 0.1 M acetic acid. Column, 1.3 X 36 cm of Bio-Gel P-2, 100-200 mesh, equilibrated with 0.1 M acetic acid. Sample, 10 mg arginine and 10 mg NaCl in 1 ml water. Eluant, 0.1 M acetic acid at 0.5 ml/min.
DISCUSSION Proteins and peptides with molecular weights as low as 2500 have an Rf of 1 when eluted through a column of Bio-Gel P-2. Such molecules should be easily desalted in the same manner as bovine serum albumin (Fig. 1). Peptides with molecular weights below 2500 penetrate the gel matrix to an extent determined, in part, by their molecular weight. Typical Rf values on Bio-Gel P-2 in 0.05&f phosphate buffer, pH 8, for small molecules in this range are: /3-MSH, M.W. 2200, Rf 0.97; bacitracin, M.W. 1400, Rt 0.86; glutathione (oxidized), M.W. 612, Rf 0.74; glutathione (reduced), M.W. 307, Rf 0.62; leucine, M.W. 131, Rf 0.55; sodium chloride, Rf 0.50. The volume (Av) between the peaks of the elution curves of two materials can be calculated from the void volume of the column (V,) and the Rf values of the two materials by the relationship:
Av = Vo(Rf, - Rd R&, From this formula and the Rf data above, the desalting of peptides from sodium chloride can be predicted. For example, a peptide of 1200 molecular weight would be expected to have an Rf of about 0.8 on Bio-Gel P-2. AV of such a peptide and sodium chloride would be equal to 3/4 V,. A peptide of 300 molecular weight would have an Rf of about 0.6 and AV would be 1/s V,. A peptide composed of a high percentage of basic, acidic, or aromatic amino acids may have a lower Rf value than a peptide of the same molecular
weight
containing
mostly
neutral
amino
acids because
of the retardation effects shown in Fig. 5. The AV would, therefore, be slightly lower than predicted from the above data. In order to obtain complete desalting of the small peptides and amino acids, it may be necessary to minimize the band spreading of the sample
DESALTING
ON
POLYACRYLAMIDE
GEL
327
which occurs as the sample is eluted through the column. Use of a small particle size of the gel and a slow flow rate serves to decrease the band spreading and sharpen the elution curves. For complete separation, the maximum sample load cannot exceed AV. Because of band spreading, the actual maximum load must be less than AV. It has been shown that sample dilution is decreased when sample loads are increased (2). Thus, in cases in which a minimum of sample dilution is desired, the column size should be chosen so that the sample load is as close to AV as possible. SUMMARY
The usefulness of a new polyacrylamide gel of small pore size for the desalting of proteins and peptides has been demonstrated. It has also been shown that neutral amino acids can be partially desalted. Maximum desalting of small molecules is obtained when a slow flow rate and a small particle size of gel are used. A method is discussed for predicting the desalting of peptides of molecular weight less than 2500. REFERENCES 1. PORATH, J., AND FJADIN, P., Nature 183,1657 (1959). 2. FLODIN, P., J. Chromatog. 5, 103 (1961). 3. HJER~~N, S., AND MOSBACH, R., Anal. Biochem. 3, 109 (1962). 4. DREZE, A., MOORE, S., AND BIGW~~D, E., Anal. Chim. Acta 11, 554 (1954). 5. HIRS, C. H. W., MOORE, S., AND STEIN, W. H., J. Biol. Chem. ‘219,623. 6. PIEZ, K. A., TOOPER, E. B., AND FOSDICK, L. S., J. Biol. Chem. 194, 669 (1952). 7. ROLLINS, C., JENSEN, L., AND SCHWARTZ, A. N., Anal. Chem. 34, ‘711 (1962).
14, 321327
BIOCHEMISTRY
Desalting
of
Proteins, on
ALICE From
(1966)
Peptides,
Polyacrylamide
N. SCHWARTZ the Bio-Rad
and
AND
Laboratories,
Received
July
Amino
Acids
Gel BURTON Richmond,
A. ZABIN California
7, 1965
The technique of gel filtration was demonstrated by Porath and Flodin {l, 2) to be highly useful for the desalting of proteins. The protein molecules, being larger than the pore size of the gel, are excluded from the gel structure and are eluted in the first void volume from a column packed with a gel filtration material. The small salt molecules are able to penetrate the gel and are thus eluted later in a volume equal to the void volume plus the accessible volume in the gel matrix. Both cross-linked dextran and polyacrylamide gels are used for desalting of proteins (2,3). For the desalting of small molecules such as amino acids and peptides, a gel with small pore sizes is required. Such a material has not been available and various ion-exchange methods (4-7) are now used. A new highly cross-linked polyacrylamide gel was recently developed in this laboratory. Substances with molecular weights higher than about 2600 are excluded from this gel. Characterization of this gel by determining the Rf values for several peptides and amino acids gives an Rf versus log molecular weight curve with a lower limit of about 200 molecular weight. The possibility of extending gel filtration to the desalting of small molecules with such a material is apparent. This paper presents the results of experiments to determine the usefulness of a highly cross-linked polyacrylamide gel for desalting procedures and some of the parameters involved. EXPERIMENTAL
Materials. The highly cross-linked polyacrylamide gel used in this study was Bio-Gel P-2, lot 2834, 50-100 mesh and 100-200 mesh (from Bio-Rad Laboratories, Richmond, California). This gel had a hydrated bed volume of 3.8 ml/dry gram. Water regain values were 1.3 gm water/gm dry gel for the 50-100 mesh sample and 1.6 gm water/gm dry gel for the 100-200 mesh sample. The proteins, amino acids, and tris [tris(hydroxymethyl) aminomethane] buffer’ were obtained from Calbio321 8 1966 by Academic
Press
Inc.
322
SCHWARTZ
AND
ZARIN
them, Los Angeles, California. The ninhydrin paper used for detecting amino acids in the column effluent was prepared by immersing filter paper in a solution of ninhydrin in water and air drying. The chromatographic columns were of glass, 1.3 cm by approximately 40 cm over-all length, with the bottom drawn down to a small tip and plugged with quartz wool. All columns used in this laboratory for gel filtration are treated to reduce “wall effects” (4). The following column treatment is recommended where minimum band spreading is desired: A clean column is filled with a solution of lo/O dichlorodimethylsilane in benzene (available from Bio-Rad Laboratories) at 60°C. The solution is decanted after several minutes and the benzene allowed to evaporate in a drying oven. Two successive treatments give a column which can be used for several months. Methods. The dry Bio-Gel P-2, as supplied, was slowly poured with stirring into an excess of deionized water. Approximately lo-15 ml of water was used for every gram of gel to obtain a suspension suitable for pouring into the column. The gel was allowed to hydrate for l-2 hr. The gel suspension was then poured into the column through an attached funnel. The column and funnel were filled with a sufficient amount of the gel suspension, so that the total bed was packed with one pouring. Samples were introduced by draining the water level to the top of the bed, and carefully adding the sample using a pipet with a curved tip. The application was completed by allowing the sample to drain into the bed, followed by a rinse of about 0.5 ml of water. The column was then filled with deionized water and attached to a reservoir for gravity flow or to a Beckman Model 746 solution metering pump. The amino acids and bacitracin were detected in the effluent by spotting on ninhydrin paper and heating. Sodium chloride was detected on a spot plate with silver nitrate-nitric acid reagent. In the protein desalting experiments, hemoglobin, urea, and ammonium sulfate were detected by evaporating drops on a glass plate. Bovine serum albumin, urea, and tris buffer were monitored through a Vanguard model 1056 automatic ultraviolet analyzer. RESULTS The removal of ammonium sulfate, urea, and tris buffer from a protein solution by gel filtration through a short column of Bio-Gel P-2 is shown in Fig. 1. Percentage recovery was determined on bovine serum albumin and was 100%. Bacitracin, with a molecular weight of 1411, was chosen as a typical peptide which might be easily desalted on Bio-Gel P-2. A l-ml sample containing 10 mg of bacitracin and 10 mg of sodium chloride in water
DESALTING
ON
POLYACRYLAMIDE
IO
20 Effluent
323
GEL
40
30 Volume
(ml)
FIG. 1. Desalting of proteins by gel filtration through a short column of Bio-Gel P-2, 50-100 mesh. Column, 1.3 x 20 cm. Eluant, water at 0.5 ml/min. Samples, 1 ml, containing, respectively, 10 mg hemoglobin and 2 M (NHJSO,, 10 mg bovine serum albumin and 8M urea, 10 mg bovine serum albumin and 0.05 M tris-HCl buffer, pH 8.1.
was applied to a 1.3 X 36 cm column of Bio-Gel P-2, 50-100 mesh, in water. Elution was carried out with water at 0.5 ml/min flow rate. Figure 2 shows the complete separation of bacitracin from sodium chloride. The bacitracin fraction was collected and recovery was found to be 100%. Glycine was partially desalted on the same column under the same conditions (Fig. 3). NaCl Bacitracin
0) ., 2G I
, IO
,A 20 Effluent
FIU.
umn, water.
I\, 30 Volume
40
50
(ml)
2. Desalting of bacitracin by gel filtration on Bio-Gel P-2, 50-100 mesh. Col1.3 x 36 cm. Sample, 1 ml, containing 10 mg bacitracin and 10 mg NaCl in Eluted with water at a flow rate of 0.4 ml/min.
324
SCHWARTZ
AND
ZABIN
6
NaCl
.-6 L ‘r 8 s 0 aI 2 I z z (L
r\
Glycine
I IO
I 30
I 20 Effluent
Volume
I 50
40 (ml)
3. Desalting of glycine on Bio-Gel P-2, 50-160 mesh. Column, 1.3 X 36 cm. Sample, 1 ml, containing 10 mg glycine and 10 mg NaCl in water. Eluted with water at a flow rate of 0.5 ml/mm. FIQ.
Effect of Flow Rate and Particle Size on Desalting Figure 4 illustrates the effect of reducing the column flow rate and decreasing the particle size of the gel. With a small particle size of Bio-Gel P-2 and a slow flow rate, the elution curve was sharp and leucine was completely desalted. NaCl 0-e 2.0
-
Leucine .r.-0,
A.
s E s
0’
-,.-.-.-
I
OE 2 E $
I\
.’
l.O-
‘~.J */y-e-.-.
.’ I
‘1 _ ‘\-
e-0 2,0
-
(%
B.
/\
1.0-
i ,
I
O-
‘\.,
/*
,
1 t-9-
,. d
\
./“, 2.0
-
. .1
O-
I
I
25
30
. 2’
\ t
, 35
Effluent
Volume
I
0-o
C.
I.0 -
.
,.
/
I
40
I\
ii 45
(ml)
4. Effect of flow rate and gel particle size on desalting. Column, 1.3 X 36 cm. Sample, 10 mg leucine and 10 mg NaCl in 1 ml water. Eluant, water. (A) Bio-GeI P-2, M-100 mesh (dry). Eluant flow rate, 1.0 ml/min. (B) Bio-Gel P-2, 5&100 mesh (dry). Eluant flow rate, 0.5 ml/min. (C) Bio-Gel P-2, 100-206 mesh (dry). Eluant flow rate, 0.5 ml/min. FIO.
DESALTIKG
ON
POLYACRYLAMIDE
GEL
325
Eflect of Amino Acid Structure oni Desalting The differences in the elution curves of acidic, basic, and aromatic amino acids are shown in Figure 5. The relative Rf values calculated for
Phenylalanine
,/-‘t
1’ I
30
35
/
1’
,
40 Effluent
45 Volume
(ml)
FIG. 5. Desalting of acidic, basic, and aromatic amino acids. Column, 1.3 x 36 cm Bio-Gel P-2, 50-100 mesh. Sample, 1 ml, containing 10 mg amino acid and 10 mg NaCl. Eluted with water at a flow rate of approximately 0.5 ml/min.
the peak maxima in these curves are: glycine, 0.57; glutamic acid, 0.59; 0.52; tryptophan, 0.47. Thus, the basic arginine, 0.52; phenylalanine, and aromatic amino acids are slightly retarded on Bio-Gel P-2 during elution with water. Tryptophan showed the greatest amount of retardation. Elution of arginine in 0.1 M acetic acid from Bio-Gel P-2 equilibrated with acetic acid gave two ninhydrin-positive peaks (Fig. 6). Increasing the acetic acid concentration to 1 M resulted in an enhancement of the
first peak, with correspondingly less material in the second peak. A double peak was also obtained when glutamic acid was eluted with 0.1 M pyridine, the second peak being quite small. The retardation of arginine was increased in pyridine peak.
chloride
and the arginine
peak overlapped
the sodium
326
SCHWAFiTZ
Effluent
AND
Volume
ZABIN
(ml)
FIQ. 6. Elution of arginine and sodium chloride from B&Gel P-2 with 0.1 M acetic acid. Column, 1.3 X 36 cm of Bio-Gel P-2, 100-200 mesh, equilibrated with 0.1 M acetic acid. Sample, 10 mg arginine and 10 mg NaCl in 1 ml water. Eluant, 0.1 M acetic acid at 0.5 ml/min.
DISCUSSION Proteins and peptides with molecular weights as low as 2500 have an Rf of 1 when eluted through a column of Bio-Gel P-2. Such molecules should be easily desalted in the same manner as bovine serum albumin (Fig. 1). Peptides with molecular weights below 2500 penetrate the gel matrix to an extent determined, in part, by their molecular weight. Typical Rf values on Bio-Gel P-2 in 0.05&f phosphate buffer, pH 8, for small molecules in this range are: /3-MSH, M.W. 2200, Rf 0.97; bacitracin, M.W. 1400, Rt 0.86; glutathione (oxidized), M.W. 612, Rf 0.74; glutathione (reduced), M.W. 307, Rf 0.62; leucine, M.W. 131, Rf 0.55; sodium chloride, Rf 0.50. The volume (Av) between the peaks of the elution curves of two materials can be calculated from the void volume of the column (V,) and the Rf values of the two materials by the relationship:
Av = Vo(Rf, - Rd R&, From this formula and the Rf data above, the desalting of peptides from sodium chloride can be predicted. For example, a peptide of 1200 molecular weight would be expected to have an Rf of about 0.8 on Bio-Gel P-2. AV of such a peptide and sodium chloride would be equal to 3/4 V,. A peptide of 300 molecular weight would have an Rf of about 0.6 and AV would be 1/s V,. A peptide composed of a high percentage of basic, acidic, or aromatic amino acids may have a lower Rf value than a peptide of the same molecular
weight
containing
mostly
neutral
amino
acids because
of the retardation effects shown in Fig. 5. The AV would, therefore, be slightly lower than predicted from the above data. In order to obtain complete desalting of the small peptides and amino acids, it may be necessary to minimize the band spreading of the sample
DESALTING
ON
POLYACRYLAMIDE
GEL
327
which occurs as the sample is eluted through the column. Use of a small particle size of the gel and a slow flow rate serves to decrease the band spreading and sharpen the elution curves. For complete separation, the maximum sample load cannot exceed AV. Because of band spreading, the actual maximum load must be less than AV. It has been shown that sample dilution is decreased when sample loads are increased (2). Thus, in cases in which a minimum of sample dilution is desired, the column size should be chosen so that the sample load is as close to AV as possible. SUMMARY
The usefulness of a new polyacrylamide gel of small pore size for the desalting of proteins and peptides has been demonstrated. It has also been shown that neutral amino acids can be partially desalted. Maximum desalting of small molecules is obtained when a slow flow rate and a small particle size of gel are used. A method is discussed for predicting the desalting of peptides of molecular weight less than 2500. REFERENCES 1. PORATH, J., AND FJADIN, P., Nature 183,1657 (1959). 2. FLODIN, P., J. Chromatog. 5, 103 (1961). 3. HJER~~N, S., AND MOSBACH, R., Anal. Biochem. 3, 109 (1962). 4. DREZE, A., MOORE, S., AND BIGW~~D, E., Anal. Chim. Acta 11, 554 (1954). 5. HIRS, C. H. W., MOORE, S., AND STEIN, W. H., J. Biol. Chem. ‘219,623. 6. PIEZ, K. A., TOOPER, E. B., AND FOSDICK, L. S., J. Biol. Chem. 194, 669 (1952). 7. ROLLINS, C., JENSEN, L., AND SCHWARTZ, A. N., Anal. Chem. 34, ‘711 (1962).