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Separation of proline and hydroxyproline using thin-layer chromatography
\NALYTICAL
BIOCHEMISTRY
Separation
6,
of
193-198
Proline
Thimn-Layer DEREK Prom
the Depart,ments
of
and
Hydroxyproline
Using
Chromatography’
MYHILL IWedical
(1963)
DAVID
AND
S. JACKSON
Surgery and Biochemistry, School, Portland, Oregon
Received
November
17nizjetxity
of OregorL
26, 1962
INTRODUCTION
Hydroxyproline is unique to collagen and is derived biosynthetically from proline (1). Thus, there are many problems of collagen and hydroxyproline biosynthesis that require a simple rapid method of isolating pure samples of proline and hydroxyproline, particularly where radioactive isotopes are involved. Several methods of achieving this objective have been described that use filter paper chromatography but these involve preliminary desalting and can handle only small amounts of material; they are, hence, time consuming and unsuitable for multiple samples. The most successful method is that of Prockop et al. (2) in which the pyrrole formed by oxidation of hydroxyproline is extracted into toluene and determined calorimetrically in this form and is also immediately available for radioactivity measurement by scintillation counting. However, not only is 335% of the tritium label lost from hydroxyproline during oxidation but proline cannot be separated simultaneously by this met,hod, a feature often to be desired.? Thin-layer chromatography is a rapid adsorption chromatographic method originally applied to partitioning of oils and lipids and recently extended to many organic compounds including amino acids (3). Simplicity of method and apparatus, speed of operation, and quantities handled are the main advantages of applying Stahl’s thin-layer technique to imino acid separation. ‘This investigation was supported by a PHS Research Grant RG-6483 from the Division of General Medical Sciences. ‘Since preparation of this manuscript, Peterkofsky and Prockop have modified this method to allow simultaneous measurement of the radioactivity of proline-C” hydroxyproline-Cl4 in biological materials [Anal. Biochem. 4, 400 (1962)l. 193
194
MYHILL
.4ND
JACKSON
EXPERIMENTAL
Apparatus
Desaga-Brinkmann apparatus was used for thin-layer chromatography. The applicator was nonadjustable, giving a standard layer of 250 p thickness. The glass plates used were 8” X 8#‘. Reagents
Nitrous acid was prepared by slowly adding 3 ml of 40% aqueous sodium nitrite to 10 ml of concentrated hydrochloric acid, in an ice bath. Proline and hydroxyproline standards were purified by chromatography on Dowex 50 X 8 (H+) resin with an elution gradient from 1.5 to 4 N HCl. Scintillator solution: reagent-grade toluene containing 0.4% 2,5diphenyloxazole (PPO) and 0.01% 1,4-bis-2- (5-phenyloxazolyl) benzene (POPOP) . Procedure
One to ten milligrams of protein was hydrolyzed in 6 N HCl for 3.5 hr at 14O”C, in sealed tubes. Hydrolyzates were then decolorized with activated charcoal, filtered, and evaporated to dryness under a stream of nitrogen. Excess nitrous acid reagent (1 ml/mg amino acids) was added to the hydrolyzate residue, allowed to stand at room temperature for 10 min, and then heated at 140’ until the color was only faintly yellow. The acid mixture was evaporated off in a steam bath and the residue extracted once with 5 ml of ether to remove deamination products. Imino acids are insoluble and loss of nitroso derivatives was slight. After ether extraction, the imino acids were taken up in 0.1 ml of 70% ethanol. This left much of the salt behind. A portion of this solution was chromatographed. Chromatography
Cellulose powder (without CaSO, binding agent) was suspended in distilled water and plates were spread with the applicator. Forty grams of cellulose in 200 ml of distilled water was found suitable for filling the applicator and getting an even flow of suitable thickness. Plates were air-dried and stored in a desiccator. The unknowns and marker spots of nitroso imino acids were applied to thin plates in 10-20 ~1 volumes containing 100-200 pg of each imino acid. After ascending chromatography for 5 hr in butanol: acetic acid: water solvent (63:27:10 v/v), plates were dried at 110°C for 10-15 min. Marker spots were sprayed with 0.2% ninhydrin in acetone or 0.4%
IMINO
ACID
195
SEPARATION
isatin in n-butanol containing 4% acetic acid and heated a few minutes at 110°C. Positions of the unknowns were marked by reference to the sprayed spots; a slightly larger area approximately 1 cm beyond the apparent spot was outlined in pencil and cut out with a razor blade. The cellulose was collected in a tube and eluted by two successive extractions with 10 ml of boiling distilled water. Pooled and filtered eluates were made to 25 ml with distilled water. Determinations of proline and hydroxyproline and radioactive counting were then carried out on this eluatc. Proline determinations were by the Troll and Lindsley modification of t,he Chinard ninhydrin reaction (4) and hydroxyproline determinations by the method of Woessner (5). A known volume of eluate was evaporated to dryness in a glass vial, the residue dissolved in Hyamine hydroxide 10-X, scintillator solution added, and the radioactivity measured by liquid scintillation counting in a Packard Tri-Garb model 314 automat,ic scintillation counter. Benzoic-Cl4 acid was used as internal standard to correct the counts to disint.egrations per nlinute.3 TABLE RI
VALUES
OF
1 AMINO
ACIDS
Color
RI
L-Proline N-Nitroso-cproline L-Hydroxyproline N-Nitroso-chydroxyproline
0.70 0.64 0.36 0.35 RESULTS
AND
with
ninhydrin
Yellow Reddish-yellow Yellow Orange
DISCUSSION
Table 1 shows that conversion of the imino acids to their nitroso does not affect their Rf values significantly. Calorimetric determinations and radioactive counting of the nitroso imino acids also give similar results to those of the imino acids themselves. Although separation on thin layers is adequate, Rf values are not as reproducible as on filter paper; therefore, it is advisable to include a reference on each chromatogram. Because of the excess nitrous acid and the conditions used, practically all of the amino acids are deaminated. Salt and deamination products may be removed using Dowex 50 (H’) ion-exchange resin but extraction with ether is rapid and convenient. Complete desalting was not carried out since thin layers are less sensitive to the presence of salt than are filter paper chromatograms. However, the salt concentration was kept derivatives
3Benzoic-C’* acid and L-proline-Cl* England Nuclear Corporation.
uniformly
labeled
were obtained
from New
196
MYHILL
AND
JACKSON
low by the use of 70% ethanol as solvent preparatory to chromatography. The effect of nitrous acid treatment upon a hydrolyzate of gelatin is shown in Fig. 1, in which the amounts of hydrolyzate and isolated imino
FIG. 1. Effect of nitrous acid treatment upon a hydrolyzate of gelatin. (,T) Gelatin hydrolyzate untreated. (,TN) Nitrous acid treated hydrolyzate. (,TNE) Nitrous acid treated and ether extracted hydrolyzate. (N-Pro, N-Hypro) Marker spots of nitrosoproline and nitrosohydroxyproline.
acids seen on the chromatogram are each related to 1 mg of gelatin initially. Mixtures containing 500 pg of each imino acid applied in up to 50 ,~l of 70% ethanol may be separated as shown in Fig. 2. The usual working range on the plates involves 100-250 ,ug of each imino acid, separation of which is quite good. Should larger quantities of imino acids be required, use of the new variable thickness applicator may increase the amounts that can be handled by a large factor. Recovery from the chromatograms by elut.ing &th boiling distillecl
IMINO
FIc. 2. Quantitati\,c> imino avid.
separation
ACID
197
SEPARATIOK
of a mixture
containing
up
to
500 gg
of each
water was initially only in the region of 60%; however, with the large amounts separated, 100-200 pg is still easily obtainable, as indicated in Table 2. In subsequent, work, the elution recovery was increased by takTABLE RECOVERY Imino
acid
Proline Hydroxyproline a Nitrous acid b Two elutions
Untreated (fig)
200 200
2
OF IMINO
ACIDS
Treateda (PP) 172
166
treatment followed by a single extraction with 10 ml of boiling distilled water.
Elutedh ha)
63 58
125 117 with
5 ml of ether.
ing a larger spot for elution than was indicated by the apparent area of the reference spot. Only two elutions of 10 ml are considered necessary because virtually all recoverable imino acid was found in these fractions. The thin-layer chromatography method described here has been used
198
MYHILL
AND
JACKSON
by Bentley and Jackson (6) in studies on the in vivo incorporation of labeled amino acids during early stages of collagen biosynthesis. The results agree well with three previous sets of experiments using different isotopes and techniques. SUMMARY
Thin-layer chromatography allows rapid separation of proline and hydroxyproline over a wide quantitative range. The present method utilizes the nitrous acid reaction to destroy amino acids in protein hydrolyzates. After ether extraction of amino acid derivatives, proline and hydroxyproline are present unchanged or as unstable n-nitroso derivatives and can be rapidly separated by thin-layer chromatography on cellulose. Mixtures including up to 500 pg of each imino acid are separable on a 250- p thick layer. The imino acids can be eluted and determined calorimetrically. Studies using isotopically labeled proline and hydroxyproline are possible even when low specific activities are involved. This simple and rapid technique has proved useful in determination of the specific activity of radioactive protein bound hydroxyproline in crude collagen extracts. REFERENCES 1. STETTRN, 2.
3. 4. 5.
6.
M. R., J. Biol. Chem. 181, 31 (1949). PROCKOP, D. J., UDEPI’FRIEND, S., AND LINDSTEDT, S., J. Biol. (1961). STAHL, E., Pharmazie 11, 633 (1956). TROLL, W., AND LINDSLEY, J., J. Biol. Chem. 215, 655 (1955). WOESSNER, J. F., Arch. Biochem. Biophys. 93, 440 (1961). BENTLEY, J. P., AND JACKSON, D. S., Biochem. Biophys. Res. (1963).
Chem.
Commwz.
236,
1395
10, 271
BIOCHEMISTRY
Separation
6,
of
193-198
Proline
Thimn-Layer DEREK Prom
the Depart,ments
of
and
Hydroxyproline
Using
Chromatography’
MYHILL IWedical
(1963)
DAVID
AND
S. JACKSON
Surgery and Biochemistry, School, Portland, Oregon
Received
November
17nizjetxity
of OregorL
26, 1962
INTRODUCTION
Hydroxyproline is unique to collagen and is derived biosynthetically from proline (1). Thus, there are many problems of collagen and hydroxyproline biosynthesis that require a simple rapid method of isolating pure samples of proline and hydroxyproline, particularly where radioactive isotopes are involved. Several methods of achieving this objective have been described that use filter paper chromatography but these involve preliminary desalting and can handle only small amounts of material; they are, hence, time consuming and unsuitable for multiple samples. The most successful method is that of Prockop et al. (2) in which the pyrrole formed by oxidation of hydroxyproline is extracted into toluene and determined calorimetrically in this form and is also immediately available for radioactivity measurement by scintillation counting. However, not only is 335% of the tritium label lost from hydroxyproline during oxidation but proline cannot be separated simultaneously by this met,hod, a feature often to be desired.? Thin-layer chromatography is a rapid adsorption chromatographic method originally applied to partitioning of oils and lipids and recently extended to many organic compounds including amino acids (3). Simplicity of method and apparatus, speed of operation, and quantities handled are the main advantages of applying Stahl’s thin-layer technique to imino acid separation. ‘This investigation was supported by a PHS Research Grant RG-6483 from the Division of General Medical Sciences. ‘Since preparation of this manuscript, Peterkofsky and Prockop have modified this method to allow simultaneous measurement of the radioactivity of proline-C” hydroxyproline-Cl4 in biological materials [Anal. Biochem. 4, 400 (1962)l. 193
194
MYHILL
.4ND
JACKSON
EXPERIMENTAL
Apparatus
Desaga-Brinkmann apparatus was used for thin-layer chromatography. The applicator was nonadjustable, giving a standard layer of 250 p thickness. The glass plates used were 8” X 8#‘. Reagents
Nitrous acid was prepared by slowly adding 3 ml of 40% aqueous sodium nitrite to 10 ml of concentrated hydrochloric acid, in an ice bath. Proline and hydroxyproline standards were purified by chromatography on Dowex 50 X 8 (H+) resin with an elution gradient from 1.5 to 4 N HCl. Scintillator solution: reagent-grade toluene containing 0.4% 2,5diphenyloxazole (PPO) and 0.01% 1,4-bis-2- (5-phenyloxazolyl) benzene (POPOP) . Procedure
One to ten milligrams of protein was hydrolyzed in 6 N HCl for 3.5 hr at 14O”C, in sealed tubes. Hydrolyzates were then decolorized with activated charcoal, filtered, and evaporated to dryness under a stream of nitrogen. Excess nitrous acid reagent (1 ml/mg amino acids) was added to the hydrolyzate residue, allowed to stand at room temperature for 10 min, and then heated at 140’ until the color was only faintly yellow. The acid mixture was evaporated off in a steam bath and the residue extracted once with 5 ml of ether to remove deamination products. Imino acids are insoluble and loss of nitroso derivatives was slight. After ether extraction, the imino acids were taken up in 0.1 ml of 70% ethanol. This left much of the salt behind. A portion of this solution was chromatographed. Chromatography
Cellulose powder (without CaSO, binding agent) was suspended in distilled water and plates were spread with the applicator. Forty grams of cellulose in 200 ml of distilled water was found suitable for filling the applicator and getting an even flow of suitable thickness. Plates were air-dried and stored in a desiccator. The unknowns and marker spots of nitroso imino acids were applied to thin plates in 10-20 ~1 volumes containing 100-200 pg of each imino acid. After ascending chromatography for 5 hr in butanol: acetic acid: water solvent (63:27:10 v/v), plates were dried at 110°C for 10-15 min. Marker spots were sprayed with 0.2% ninhydrin in acetone or 0.4%
IMINO
ACID
195
SEPARATION
isatin in n-butanol containing 4% acetic acid and heated a few minutes at 110°C. Positions of the unknowns were marked by reference to the sprayed spots; a slightly larger area approximately 1 cm beyond the apparent spot was outlined in pencil and cut out with a razor blade. The cellulose was collected in a tube and eluted by two successive extractions with 10 ml of boiling distilled water. Pooled and filtered eluates were made to 25 ml with distilled water. Determinations of proline and hydroxyproline and radioactive counting were then carried out on this eluatc. Proline determinations were by the Troll and Lindsley modification of t,he Chinard ninhydrin reaction (4) and hydroxyproline determinations by the method of Woessner (5). A known volume of eluate was evaporated to dryness in a glass vial, the residue dissolved in Hyamine hydroxide 10-X, scintillator solution added, and the radioactivity measured by liquid scintillation counting in a Packard Tri-Garb model 314 automat,ic scintillation counter. Benzoic-Cl4 acid was used as internal standard to correct the counts to disint.egrations per nlinute.3 TABLE RI
VALUES
OF
1 AMINO
ACIDS
Color
RI
L-Proline N-Nitroso-cproline L-Hydroxyproline N-Nitroso-chydroxyproline
0.70 0.64 0.36 0.35 RESULTS
AND
with
ninhydrin
Yellow Reddish-yellow Yellow Orange
DISCUSSION
Table 1 shows that conversion of the imino acids to their nitroso does not affect their Rf values significantly. Calorimetric determinations and radioactive counting of the nitroso imino acids also give similar results to those of the imino acids themselves. Although separation on thin layers is adequate, Rf values are not as reproducible as on filter paper; therefore, it is advisable to include a reference on each chromatogram. Because of the excess nitrous acid and the conditions used, practically all of the amino acids are deaminated. Salt and deamination products may be removed using Dowex 50 (H’) ion-exchange resin but extraction with ether is rapid and convenient. Complete desalting was not carried out since thin layers are less sensitive to the presence of salt than are filter paper chromatograms. However, the salt concentration was kept derivatives
3Benzoic-C’* acid and L-proline-Cl* England Nuclear Corporation.
uniformly
labeled
were obtained
from New
196
MYHILL
AND
JACKSON
low by the use of 70% ethanol as solvent preparatory to chromatography. The effect of nitrous acid treatment upon a hydrolyzate of gelatin is shown in Fig. 1, in which the amounts of hydrolyzate and isolated imino
FIG. 1. Effect of nitrous acid treatment upon a hydrolyzate of gelatin. (,T) Gelatin hydrolyzate untreated. (,TN) Nitrous acid treated hydrolyzate. (,TNE) Nitrous acid treated and ether extracted hydrolyzate. (N-Pro, N-Hypro) Marker spots of nitrosoproline and nitrosohydroxyproline.
acids seen on the chromatogram are each related to 1 mg of gelatin initially. Mixtures containing 500 pg of each imino acid applied in up to 50 ,~l of 70% ethanol may be separated as shown in Fig. 2. The usual working range on the plates involves 100-250 ,ug of each imino acid, separation of which is quite good. Should larger quantities of imino acids be required, use of the new variable thickness applicator may increase the amounts that can be handled by a large factor. Recovery from the chromatograms by elut.ing &th boiling distillecl
IMINO
FIc. 2. Quantitati\,c> imino avid.
separation
ACID
197
SEPARATIOK
of a mixture
containing
up
to
500 gg
of each
water was initially only in the region of 60%; however, with the large amounts separated, 100-200 pg is still easily obtainable, as indicated in Table 2. In subsequent, work, the elution recovery was increased by takTABLE RECOVERY Imino
acid
Proline Hydroxyproline a Nitrous acid b Two elutions
Untreated (fig)
200 200
2
OF IMINO
ACIDS
Treateda (PP) 172
166
treatment followed by a single extraction with 10 ml of boiling distilled water.
Elutedh ha)
63 58
125 117 with
5 ml of ether.
ing a larger spot for elution than was indicated by the apparent area of the reference spot. Only two elutions of 10 ml are considered necessary because virtually all recoverable imino acid was found in these fractions. The thin-layer chromatography method described here has been used
198
MYHILL
AND
JACKSON
by Bentley and Jackson (6) in studies on the in vivo incorporation of labeled amino acids during early stages of collagen biosynthesis. The results agree well with three previous sets of experiments using different isotopes and techniques. SUMMARY
Thin-layer chromatography allows rapid separation of proline and hydroxyproline over a wide quantitative range. The present method utilizes the nitrous acid reaction to destroy amino acids in protein hydrolyzates. After ether extraction of amino acid derivatives, proline and hydroxyproline are present unchanged or as unstable n-nitroso derivatives and can be rapidly separated by thin-layer chromatography on cellulose. Mixtures including up to 500 pg of each imino acid are separable on a 250- p thick layer. The imino acids can be eluted and determined calorimetrically. Studies using isotopically labeled proline and hydroxyproline are possible even when low specific activities are involved. This simple and rapid technique has proved useful in determination of the specific activity of radioactive protein bound hydroxyproline in crude collagen extracts. REFERENCES 1. STETTRN, 2.
3. 4. 5.
6.
M. R., J. Biol. Chem. 181, 31 (1949). PROCKOP, D. J., UDEPI’FRIEND, S., AND LINDSTEDT, S., J. Biol. (1961). STAHL, E., Pharmazie 11, 633 (1956). TROLL, W., AND LINDSLEY, J., J. Biol. Chem. 215, 655 (1955). WOESSNER, J. F., Arch. Biochem. Biophys. 93, 440 (1961). BENTLEY, J. P., AND JACKSON, D. S., Biochem. Biophys. Res. (1963).
Chem.
Commwz.
236,
1395
10, 271