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[35] Detection of peptides by chemical methods
[35]
DETECTION" OF PEPTIDF~S
325
[35] D e t e c t i o n of P e p t i d e s b y C h e m i c a l M e t h o d s 1 B y C. H. W. Hms
Introduction
Since they are general, the reactions with ninhydrin2 and with the Folin-Lowry3 reagent have both been used for the detection and estimation of peptides in solution. Occasionally reactions more selective for a particular type of side chain may have an advantage (e.g., the Sakaguchi reaction for arginine). The reaction of ninhydrin with peptides is complex because, in addition to reaction at a- and e-amino groups, hydrolysis may also occur. The rates of these various processes are a function of the structure of the peptide. The variability of color yield is decreased and the overall sensitivity of the procedure is increased when the peptide is first subjected to hydrolysis. Moreover, pyrrolidonecarboxylic acid does not react with ninhydrin to a significant degree under the usual conditions, and peptides that contain residues of this substance at the amino-terminal end [the consequence of cyclization of glutamine residues (cf. this volume [44])] may not be detected if hydrolysis is omitted. The problem presented by the absorption of ammonia from the atmosphere (particularly in cities) when acid hydrolysis is conducted under conditions that allow access of air, has focused attention on suitable procedures that involve alkaline treatment? While alkaline hydrolysis leads to the destruction of certain amino acids (e.g., serine and cystine) and complete cleavage of all peptide bonds requires prolongation of the treatment to an inconvenient degree, the procedure has the advantage of simplicity and lends itself well to the routine analysis of large numbers of samples. The Folin-Lowry procedure (cf. Vol. III, p. 448) may be used for the detection and quantitation of peptides. The color developed depends on the relative proportion of phenolic side chains and peptide bonds in the molecule. However, the sensitivity of the method does not compare favorably with that attainable in ninhydrin-oriented procedures. 1Research at Brookhaven National Laboratory performed under the auspices of the U.S. Atomic Energy Commission. S. Moore and W. H. Stein, J. Biol. Chem. 9.11, 907 (1954). 80. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 4C. H. W. Hirs, S. Moore. and W. H. Stein, J. Biol. Chem. 219, 623 (1956).
326
SEP~.RATION OF P]]PTIDES
[35]
Ninhydrin Reagent (a) Methyl CellosoIve. It is important that a peroxide-free grade be used. In situations where extensive analytical work with ninhydrin is contemplated, purchase of peroxide-free Methyl Cellosolve in 66-gallon barrels directly from the manufacturer (Union Carbide) is recommended. The peroxide conten~ should be checked regularly by addition of 5 ml Cellosolve to 1 ml 10% potassium iodide solution. As soon as the presence of peroxide is indicated by a detectable faint yellow color in this test, the Cellosolve should be either discarded or redistilled in the presence of zinc-copper couple. Distillation and removal of peroxide are conveniently accomplished in a 5-liter flask equipped with a 50-cm Vigreux column. The flask is charged with 3.5 liters of Cellosolve, and 20 g zinc-copper couple is added. All but 200 ml of the Cellosolve may be recovered. Zinc-copper couple suitable for the distillation may be prepared in bulk and stored for later use. A convenient procedure is to add 100 g zinc foil, cut into approximately 2-cm squares with scissors, to 4 liters of 0.25M copper sulfate solution. When the reaction has ceased, the powder is collected in a Biichner funnel and washed copiously in succession with water, 95% ethanol, and ether. It is dried in air. (b) Buyer Solution 4M, pH 5. 5 A 5-gallon polyethylene bottle is calibrated to a volume of 20 liters with water. The bottle is emptied and 2945 g anhydrous potassium acetate is introduced. The salt is dissolved by stirring with about 8 liters of water. Now 1360 g sodium acetate ~rihydrate is added. When the salt has dissolved, 1000 ml glacial acetic acid is introduced and the solution is diluted to the mark with water. The solution is filtered through sintered glass into a storage bottle. This operation is conveniently conducted with a siphon that incorporates a filter tube. When diluted 4-fold with water, the pH of the solution should have a value of 5.1. (c) Preparation and Storage of Reaqent. All operations should be conducted in a hood. An 18/7 semi-ball joint is attached to a 4-liter aspirator bottle, mounted on a magnetic stirring unit, with a convenient length (about 50 cm) of plastic tubing (e.g., Tygon, 8-ram I.D.). The joint, which should be clamped above the neck of the bottle, permits attachment to a cylinder of prepurified nitrogen. A large magnetic stirring bar (about 8 X 1.5 cm) should be used. Approximately 2 liters of peroxide-free Methyl Cellosolve are introduced and a slow stream of The solution used in the procedure described by Moore and Stein~ was a sodium acetate buffer. It has been found in their laboratory that incorporation of the potassium salt facilitates preparation of the buffer. The author is indebted to Dr. Stanford Moore for directions pertinent to the preparation of the solution.
[35]
DETECTION OF P]~PTIDES
327
nitrogen is permitted to pass into the bottle. A 500-ml polypropylene beaker is used to weigh 80.0 g ninhydrin2 A quantitative transfer to the bottle is accomplished with several rinses of Cellosolve. A similar technique is used to weigh (100-ml beaker) and transfer 12.0 g hydrindantin6 into the bottle. Cellosolve to a total of 3000 ml is finally added, and stirring is continued until all the solids have dissolved. At this point 1000 ml of the 4 M buffer solution is added, and the bottle is closed with a silicone rubber stopper that carries an 18/7 socket joint. Nitrogen is passed through the solution for 10 minutes before transfer to a storage bottle. The ninhydrin reagent is stored in a 4-liter bottle. A suitable arrangement consists of a 4-liter aspirator bottle equipped with a silicone rubber stopper through which are inserted a vent tube and a feed tube that reaches almost to the bottom of the bottle. The feed tube is closed with a stopcock (Teflon plug) and carries an 18/7 socket joint through which connection may be established to the plastic tube attached to the bottle in which the reagent is prepared. The tubulation of the storage bottle leads to an automatic pipetting machine. The vent tube is connected to a constant-pressure nitrogen reservoir of at least 6-liter capacity. This reservoir is fabricated from two 9-liter aspirator bottles, the tubulations of which are connected by a short length of plastic tubing. One bottle is closed with a silicone rubber stopper which carries a flexible connection to the vent tube of the ninhydrin storage bottle. Sufficient light mineral oil is added to approximately half fill both bottles of the reservoir. Prepurified nitrogen is passed into the storage bottle through the feed tube. The oil in the closed half of the reservoir is displaced into the open half; excess nitrogen escapes to the atmosphere through the open bottle. When the unit is first assembled, it will be necessary to thoroughly flush with nitrogen for 2 hours to be certain that only negligible quantities of oxygen remain. Transfer of reagent to the storage bottle cannot proceed until the air, trapped between the stopcock on the feed tube and the joint that permits attachment of the preparation bottle, has been eliminated. For this purpose the flexible transfer tube is momentarily clamped tight and connection to the feed tube is established. Slight negative pressure is applied to the preparation bottle and, in succession, the flexible tube is unclamped and the stopcock opened. Reagent (or nitrogen) in the storage bottle is drawn up to displace all air in the transfer tube. Thereupon slight positive pressure is applied with the nitrogen cylinder, and the reagent is forced into the storage bottle. When the preparation bottle eNinhydrin and hydrindantin purchased from the Pierce Chemical Co., Rockford, Illinois, may be used directly.
328
S~PAP~TION OF PEPTIDES
[35]
is nearly empty, the stopcock is closed and the transfer tube is disconnected. To prevent spilling of ninhydrin reagent, the storage bottle is suitably lowered prior to opening the ball joint. The excess reagent above the stopcock is removed by aspiration, and the open end of the feed tube is capped with a semi-ball stopper. If these precautions are observed there is little likelihood that contamination of the nitrogen barrier with air will occur. If a few bubbles of air are accidentally introduced during reagent transfer, the system m a y be flushed slowly with nitrogen for about 1 hour, at the expense, however, of a slight concentration of the reagent. In cases of major contamination it is advisable to clean the system completely and begin afresh. The reagent keeps satisfactorily for about 2 months if it is stored at 15°J I t is convenient to accommodate the storage bottle in a small refrigerator (Model PF-8, 1-cubic-foot refrigerator, manufactured by the United Refrigerator Co., 156 North Franklin Street, Hempstead, L. I., New York, and modified for operation at 15 ° by use of a Ranco No. 20710 temperature control). If the reagent is stored at room temperature it should be protected from light. This m a y be accomplished by spraypainting the bottle with black paint; a small vertical band should be left unpainted for observation of the liquid level. Connections between the storage bottle and the pipetting machine may be made with either Saran or thin-walled Teflon tubing. Alkaline Hydrolysis s The reaction is performed in polypropylene test tubes (18 X 120 mm). The samples to be analyzed are carefully pipetted into the bottoms of the tubes. The tubes, accommodated in sets of 50 in aluminum test tube racks, are placed in a well-ventilated oven at 110 °. The oven should be placed in a hood if the solvent contains substances such as formic acid, pyridine, etc. Heating is continued until the sample solutions have evaporated to dryness. The length of time required must be determined for the particular solvent used and the volume taken. As much as 1 ml of certain solvents or dilute salt solutions m a y be evaporated without difficulty. Each tube receives 0.15 ml 13.5 N N a O H (715 ml 50% reagent grade ~The improved storage qualities attained at 15° were discovered by Dr. Stanford Moore, to whom the author expresses appreciation for this information and for the specifications regarding a suitable refrigeration unit. 8The procedure given here is based on the experience of I)rs. A. B. Edmundson, T. H. Plummer, R. P. Carry, and M. E. Elzinga in the author's laboratory, and of Dr. A. M. Crestfield [A. M. Crestfield, W. H. Stein, and S. Moore, J. Biol. Chem. 238, 618 (1963)] of Rockefeller University.
[3S]
DETECTION OF PEPTIDES
329
NaOH solution is diluted under cooling to 1 liter with water). The alkali may be added with a calibrated, bulb-operated glass pipette, and should be dropped directly onto the residue in each tube. The tubes are placed in an autoclave and heated with steam under a pressure of 15 psi (approximately 121 °) for a period of 20 minutes2 After the pressure h a s been released and the tubes have cooled, the alkali is neutralized by addition of 0.25 ml glacial acetic acid} ° If large numbers of determinations are made it is convenient to add the acid from a pipetting machine. Ninhydrin
Reaction
Each tube receives a volume of 0.50 ml ninhydrin reagent. The tubes are gently shaken to mix the contents, individually covered with loosely fitting culture-tube caps, and heated for 15 minutes in a covered boiling water bath. (The heating time should be controlled as accurately as possible if reproducible color yields are to be obtained.) The tubes are cooled in a basin of cold water for about 10 minutes, the caps are removed, and 2.5 ml 50% ethanol is added to each tube, most satisfactorily with a pipetting machine. The tubes are shaken vigorously for 45 seconds. The absorbance of each solution at 570 m# is determined in a 1-cm cuvette against water with a Beckman DU spectrophotemeter.11 A blank prepared from reagents only should be prepared each day as a check on the procedure. The absorbance reading of an acceptable blank should not exceed 0.05. The molar absorptivity of the colored product, the anion of diketohydrindylidene-diketohydrindamine, at 570 mt~ is very nearly 2 X 104M -1 cm-1. The procedure may be checked with the aid of a 1.5 X 10-~M solution of leucine. Several aliquots (0.05 ml) of this solution should be carried' through the hydrolysis and color reaction steps.
In the author's laboratory an American Sterilizer Co. autoclave is used with the controls set for "fast exhaust." There is no alteration in volume of the samples. 1oThe procedure should be checked occasionally by measurement of the pH of the neutralized mixture. After addition of 3 ml water the solution should be at pH 4.9. 11It is convenient to use a device such as a Gilson Medical Electronics Corp. "Transferator" when many samples must be processed.
DETECTION" OF PEPTIDF~S
325
[35] D e t e c t i o n of P e p t i d e s b y C h e m i c a l M e t h o d s 1 B y C. H. W. Hms
Introduction
Since they are general, the reactions with ninhydrin2 and with the Folin-Lowry3 reagent have both been used for the detection and estimation of peptides in solution. Occasionally reactions more selective for a particular type of side chain may have an advantage (e.g., the Sakaguchi reaction for arginine). The reaction of ninhydrin with peptides is complex because, in addition to reaction at a- and e-amino groups, hydrolysis may also occur. The rates of these various processes are a function of the structure of the peptide. The variability of color yield is decreased and the overall sensitivity of the procedure is increased when the peptide is first subjected to hydrolysis. Moreover, pyrrolidonecarboxylic acid does not react with ninhydrin to a significant degree under the usual conditions, and peptides that contain residues of this substance at the amino-terminal end [the consequence of cyclization of glutamine residues (cf. this volume [44])] may not be detected if hydrolysis is omitted. The problem presented by the absorption of ammonia from the atmosphere (particularly in cities) when acid hydrolysis is conducted under conditions that allow access of air, has focused attention on suitable procedures that involve alkaline treatment? While alkaline hydrolysis leads to the destruction of certain amino acids (e.g., serine and cystine) and complete cleavage of all peptide bonds requires prolongation of the treatment to an inconvenient degree, the procedure has the advantage of simplicity and lends itself well to the routine analysis of large numbers of samples. The Folin-Lowry procedure (cf. Vol. III, p. 448) may be used for the detection and quantitation of peptides. The color developed depends on the relative proportion of phenolic side chains and peptide bonds in the molecule. However, the sensitivity of the method does not compare favorably with that attainable in ninhydrin-oriented procedures. 1Research at Brookhaven National Laboratory performed under the auspices of the U.S. Atomic Energy Commission. S. Moore and W. H. Stein, J. Biol. Chem. 9.11, 907 (1954). 80. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 4C. H. W. Hirs, S. Moore. and W. H. Stein, J. Biol. Chem. 219, 623 (1956).
326
SEP~.RATION OF P]]PTIDES
[35]
Ninhydrin Reagent (a) Methyl CellosoIve. It is important that a peroxide-free grade be used. In situations where extensive analytical work with ninhydrin is contemplated, purchase of peroxide-free Methyl Cellosolve in 66-gallon barrels directly from the manufacturer (Union Carbide) is recommended. The peroxide conten~ should be checked regularly by addition of 5 ml Cellosolve to 1 ml 10% potassium iodide solution. As soon as the presence of peroxide is indicated by a detectable faint yellow color in this test, the Cellosolve should be either discarded or redistilled in the presence of zinc-copper couple. Distillation and removal of peroxide are conveniently accomplished in a 5-liter flask equipped with a 50-cm Vigreux column. The flask is charged with 3.5 liters of Cellosolve, and 20 g zinc-copper couple is added. All but 200 ml of the Cellosolve may be recovered. Zinc-copper couple suitable for the distillation may be prepared in bulk and stored for later use. A convenient procedure is to add 100 g zinc foil, cut into approximately 2-cm squares with scissors, to 4 liters of 0.25M copper sulfate solution. When the reaction has ceased, the powder is collected in a Biichner funnel and washed copiously in succession with water, 95% ethanol, and ether. It is dried in air. (b) Buyer Solution 4M, pH 5. 5 A 5-gallon polyethylene bottle is calibrated to a volume of 20 liters with water. The bottle is emptied and 2945 g anhydrous potassium acetate is introduced. The salt is dissolved by stirring with about 8 liters of water. Now 1360 g sodium acetate ~rihydrate is added. When the salt has dissolved, 1000 ml glacial acetic acid is introduced and the solution is diluted to the mark with water. The solution is filtered through sintered glass into a storage bottle. This operation is conveniently conducted with a siphon that incorporates a filter tube. When diluted 4-fold with water, the pH of the solution should have a value of 5.1. (c) Preparation and Storage of Reaqent. All operations should be conducted in a hood. An 18/7 semi-ball joint is attached to a 4-liter aspirator bottle, mounted on a magnetic stirring unit, with a convenient length (about 50 cm) of plastic tubing (e.g., Tygon, 8-ram I.D.). The joint, which should be clamped above the neck of the bottle, permits attachment to a cylinder of prepurified nitrogen. A large magnetic stirring bar (about 8 X 1.5 cm) should be used. Approximately 2 liters of peroxide-free Methyl Cellosolve are introduced and a slow stream of The solution used in the procedure described by Moore and Stein~ was a sodium acetate buffer. It has been found in their laboratory that incorporation of the potassium salt facilitates preparation of the buffer. The author is indebted to Dr. Stanford Moore for directions pertinent to the preparation of the solution.
[35]
DETECTION OF P]~PTIDES
327
nitrogen is permitted to pass into the bottle. A 500-ml polypropylene beaker is used to weigh 80.0 g ninhydrin2 A quantitative transfer to the bottle is accomplished with several rinses of Cellosolve. A similar technique is used to weigh (100-ml beaker) and transfer 12.0 g hydrindantin6 into the bottle. Cellosolve to a total of 3000 ml is finally added, and stirring is continued until all the solids have dissolved. At this point 1000 ml of the 4 M buffer solution is added, and the bottle is closed with a silicone rubber stopper that carries an 18/7 socket joint. Nitrogen is passed through the solution for 10 minutes before transfer to a storage bottle. The ninhydrin reagent is stored in a 4-liter bottle. A suitable arrangement consists of a 4-liter aspirator bottle equipped with a silicone rubber stopper through which are inserted a vent tube and a feed tube that reaches almost to the bottom of the bottle. The feed tube is closed with a stopcock (Teflon plug) and carries an 18/7 socket joint through which connection may be established to the plastic tube attached to the bottle in which the reagent is prepared. The tubulation of the storage bottle leads to an automatic pipetting machine. The vent tube is connected to a constant-pressure nitrogen reservoir of at least 6-liter capacity. This reservoir is fabricated from two 9-liter aspirator bottles, the tubulations of which are connected by a short length of plastic tubing. One bottle is closed with a silicone rubber stopper which carries a flexible connection to the vent tube of the ninhydrin storage bottle. Sufficient light mineral oil is added to approximately half fill both bottles of the reservoir. Prepurified nitrogen is passed into the storage bottle through the feed tube. The oil in the closed half of the reservoir is displaced into the open half; excess nitrogen escapes to the atmosphere through the open bottle. When the unit is first assembled, it will be necessary to thoroughly flush with nitrogen for 2 hours to be certain that only negligible quantities of oxygen remain. Transfer of reagent to the storage bottle cannot proceed until the air, trapped between the stopcock on the feed tube and the joint that permits attachment of the preparation bottle, has been eliminated. For this purpose the flexible transfer tube is momentarily clamped tight and connection to the feed tube is established. Slight negative pressure is applied to the preparation bottle and, in succession, the flexible tube is unclamped and the stopcock opened. Reagent (or nitrogen) in the storage bottle is drawn up to displace all air in the transfer tube. Thereupon slight positive pressure is applied with the nitrogen cylinder, and the reagent is forced into the storage bottle. When the preparation bottle eNinhydrin and hydrindantin purchased from the Pierce Chemical Co., Rockford, Illinois, may be used directly.
328
S~PAP~TION OF PEPTIDES
[35]
is nearly empty, the stopcock is closed and the transfer tube is disconnected. To prevent spilling of ninhydrin reagent, the storage bottle is suitably lowered prior to opening the ball joint. The excess reagent above the stopcock is removed by aspiration, and the open end of the feed tube is capped with a semi-ball stopper. If these precautions are observed there is little likelihood that contamination of the nitrogen barrier with air will occur. If a few bubbles of air are accidentally introduced during reagent transfer, the system m a y be flushed slowly with nitrogen for about 1 hour, at the expense, however, of a slight concentration of the reagent. In cases of major contamination it is advisable to clean the system completely and begin afresh. The reagent keeps satisfactorily for about 2 months if it is stored at 15°J I t is convenient to accommodate the storage bottle in a small refrigerator (Model PF-8, 1-cubic-foot refrigerator, manufactured by the United Refrigerator Co., 156 North Franklin Street, Hempstead, L. I., New York, and modified for operation at 15 ° by use of a Ranco No. 20710 temperature control). If the reagent is stored at room temperature it should be protected from light. This m a y be accomplished by spraypainting the bottle with black paint; a small vertical band should be left unpainted for observation of the liquid level. Connections between the storage bottle and the pipetting machine may be made with either Saran or thin-walled Teflon tubing. Alkaline Hydrolysis s The reaction is performed in polypropylene test tubes (18 X 120 mm). The samples to be analyzed are carefully pipetted into the bottoms of the tubes. The tubes, accommodated in sets of 50 in aluminum test tube racks, are placed in a well-ventilated oven at 110 °. The oven should be placed in a hood if the solvent contains substances such as formic acid, pyridine, etc. Heating is continued until the sample solutions have evaporated to dryness. The length of time required must be determined for the particular solvent used and the volume taken. As much as 1 ml of certain solvents or dilute salt solutions m a y be evaporated without difficulty. Each tube receives 0.15 ml 13.5 N N a O H (715 ml 50% reagent grade ~The improved storage qualities attained at 15° were discovered by Dr. Stanford Moore, to whom the author expresses appreciation for this information and for the specifications regarding a suitable refrigeration unit. 8The procedure given here is based on the experience of I)rs. A. B. Edmundson, T. H. Plummer, R. P. Carry, and M. E. Elzinga in the author's laboratory, and of Dr. A. M. Crestfield [A. M. Crestfield, W. H. Stein, and S. Moore, J. Biol. Chem. 238, 618 (1963)] of Rockefeller University.
[3S]
DETECTION OF PEPTIDES
329
NaOH solution is diluted under cooling to 1 liter with water). The alkali may be added with a calibrated, bulb-operated glass pipette, and should be dropped directly onto the residue in each tube. The tubes are placed in an autoclave and heated with steam under a pressure of 15 psi (approximately 121 °) for a period of 20 minutes2 After the pressure h a s been released and the tubes have cooled, the alkali is neutralized by addition of 0.25 ml glacial acetic acid} ° If large numbers of determinations are made it is convenient to add the acid from a pipetting machine. Ninhydrin
Reaction
Each tube receives a volume of 0.50 ml ninhydrin reagent. The tubes are gently shaken to mix the contents, individually covered with loosely fitting culture-tube caps, and heated for 15 minutes in a covered boiling water bath. (The heating time should be controlled as accurately as possible if reproducible color yields are to be obtained.) The tubes are cooled in a basin of cold water for about 10 minutes, the caps are removed, and 2.5 ml 50% ethanol is added to each tube, most satisfactorily with a pipetting machine. The tubes are shaken vigorously for 45 seconds. The absorbance of each solution at 570 m# is determined in a 1-cm cuvette against water with a Beckman DU spectrophotemeter.11 A blank prepared from reagents only should be prepared each day as a check on the procedure. The absorbance reading of an acceptable blank should not exceed 0.05. The molar absorptivity of the colored product, the anion of diketohydrindylidene-diketohydrindamine, at 570 mt~ is very nearly 2 X 104M -1 cm-1. The procedure may be checked with the aid of a 1.5 X 10-~M solution of leucine. Several aliquots (0.05 ml) of this solution should be carried' through the hydrolysis and color reaction steps.
In the author's laboratory an American Sterilizer Co. autoclave is used with the controls set for "fast exhaust." There is no alteration in volume of the samples. 1oThe procedure should be checked occasionally by measurement of the pH of the neutralized mixture. After addition of 3 ml water the solution should be at pH 4.9. 11It is convenient to use a device such as a Gilson Medical Electronics Corp. "Transferator" when many samples must be processed.