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[24] Techniques in enzymic hydrolysis
214
CLEAVAGE OF PEPTIDE CHAINS
[24]
[24] T e c h n i q u e s in E n z y m i c H y d r o l y s i s B y D . G. SNIYTH
The classical studies of Fruton, 1 which revealed that the proteolytie enzyme trypsin possesses a high specificity in its catalysis of peptide bond hydrolysis, opened the way for application of enzymes in the specific cleavage of polypeptides. The use of enzymes now fills a central role in determination of protein structure. Planning of the practical techniques involved requires attention to the general principles that underlie enzyme action, and close attention to the particular purpose for which the enzyme is being employed. (1) Specificity of Enzyme. Ideally the enzyme is required to act at a limited number of sites in the polypeptide substrate. If this is achieved, relatively few fragments are released and these may be separated easily. It is an advantage when the specificity of an enzyme is well characterized because the number of peptides to be liberated by it can be anticipated. The enzyme selected for the specific cleavage of a large polypeptide is in general required to provide smaller peptides amenable to direct determination of amino acid sequence. In addition, where the enzyme has a narrow specificity, each peptide produced may be expected to terminate in a predictable amino acid residue, and this information is of value in the assignment of sequence. (2) Purity of Enzyme. The enzyme should be available, or be prepared, as free as possible of contaminating enzyme in order that the polypeptide substrate be attacked only at sites determined by the fundamental specificity. Even traces of contaminating enzymes may cause reduced yield of the required products and give rise to complex mixtures of peptides difficult to resolve. (3) Preparation o] Enzyme. A weighed amount of the crystalline enzyme should be dissolved immediately before use, and appropriate dilution carried out to obtain a suitable concentration; the dilute solution of enzyme is then added to a solution of substrate. A typical ratio of enzyme to substrate is in the region 1:100 parts by weight. Proteolytic enzymes are not usually stored in solution because of the possibility of autodigestion or irreversible denaturation, resulting in loss of enzyme activity. The enzyme should, of course, never be added in solid form to the solution of substrate because of adverse effects caused by the local excess. In some cases it is advantageous to expose an enzyme before use to conditions that inactivate contaminating enzymes more 1M. Bergmann, J. S. Fruton, and H. Pollock, J. Biol. Chem. 127, 643 (1939); M. Bergmann and J. S. Fruton, Advan. Enzymol. 1, 63 (1941).
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USE OF PROTEOLYTIC ENZYMES
215
rapidly than the enzyme itself; details of these procedures are included in the section on each enzyme. The activity of an enzyme should be checked against a standard substrate immediately before the enzyme is used. Proteolytic activity may be assayed with a small synthetic peptide as substrate, or with a large, naturally occurring polypeptide. For example, with the B-chain of insulin as a reference substrate, the rate of liberation of alanine provides a measure of the activity of trypsin, whereas the rate of liberation of tyrosine and the COOH-terminal tetrapeptide Thr-Pro-Lys-Ala gives a measure of the activity of chymotrypsin. The concentrations of these fragments can be accurately determined by amino acid analysis, and a measure of both activity and specificity is obtained. (4) Preparation o] Substrate. The peptide or protein substrate should be pure and in an unfolded form before enzymic hydrolysis is undertaken. Thus multiehain proteins are separated into their component polypeptides, and these are individually digested by the enzyme. In addition, the substrate must be physically available to the enzyme. Residues situated in the interior of a tightly folded protein molecule may be inaccessible, but can be exposed by physical denaturation (this volume [23]), chemical procedures involving cleavage of the disulfide bridges (this volume [19-22]), or cleavage at methionine residues (this volume [27]). Although digestions have been performed successfully in heterogeneous solution, the reaction cannot be carefully controlled. A product formed by partial digestion may possess an intrinsic low solubility and separate from solution as a "core," or a peptide released during the digestion may aggregate with itself or with other peptides and form a precipitate. These difficulties, as well as the problem of attempting to digest insoluble starting material, may be overcome by performing reactions in 2 M urea, in which the formation of aggregates is reduced and in which some enzymes retain activity for short periods. Protein disulfide bonds are in general cleaved prior to enzyme digestion. Disulfide bonds are labile under the neutral and alkaline conditions employed in structural studies, and readily undergo base-catalyzed rearrangements. Enzymes that function at acid pH, as pepsin does, are A-- S I ~ B--S
OH t
C--S
A.S' +
A.S'
+
B-S--OH
A--S
l
D--S
~
I +
C.S'
D--S
therefore much preferred for the liberation of peptides in which the native disulfide bonds are required intact.
216
CLEAVAGE OF PEPTIDE CHAINS
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(5) Optimum Digestion Periods. Although brief exposure to an enzyme can result in rapid cleavage of a particularly sensitive peptide bond, prolonged digestion may be necessary to ensure cleavage of resistant linkages. Extended periods of digestion, however, often result in fragmentation of the required products, due to the activity of small amounts of contaminating enzyme which slowly causes additional cleavage, or to an inherent lack of specificity in the principal enzyme. The substrate is exposed to the enzyme for the minimum period consistent with completion of the required cleavage. (6) Termination o] E n z y m e Action. Enzymes function over narrow ranges of pH. When a digestion proceeds at neutral pH, the most usual method of stopping the reaction is to acidify the reaction mixture; or when the digestion proceeds at acid pH, to neutralize the solution. It should be noted that, if the pH of the solution is subsequently restored to the initial value, the enzyme may recover its activity and an alternative method may be required to terminate the reaction. For example, when trypsin is used at pH 7 to produce fragments from bradykininogen, and the resulting mixture of peptides is tested at pH 7.4 for bradykinin activity, it is unsatisfactory to arrest the reaction by acidification; on neutralization, the trypsin may itself interfere with the biological assay. However, the reaction can be stopped by addition of trypsin inhibitor or by boiling. While proteolytic enzymes are destroyed by boiling, vigorous treatments of this type are permissible only if they leave the substrate unaffected. The optimal procedure for a particular enzymic digestion clearly depends on the enzyme and the substrate concerned, and on the degree of cleavage required. The methods presented are intended to serve as a general guide i'n experimental procedure. Trypsin Principle Trypsin catalyzes the hydrolysis of the peptide bond between the C 0 0 H group of lysine, or the C 0 0 H group of arginine, and the NH2 group of the adjacent amino acid. 1 Cleavage takes place more slowly when the basic residue is adjacent in sequence to an acidic residue or cystine, and does not occur at all when the residue is followed by proline. 2-4 2C. H. W. Hits, S. Moore, and W. H. Stein, J. Biol. Chem. 219, 623 (1956). 8A. Tsugita, D. T. Gish, J. Young, H. Fraenkel-Gonrat, C. A. Knight, and W. M. Stanley, Proe. Nat. Aead. Sei. U.S. 46~ 1463 (1960). rE. Margoliash, E. L. Smith, G. Kreil, and H. Tuppy, Nature 19'2, 1125 (1961).
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USE OF PROTEOLYTIC ENZYMES
217
~NH--CH - / ? R NH-(~H2)4 \ N H - - CIH - - C / NHz
--NI-I-- CH-- C I
~OH
\kO
+
R
NII --
I / NI-I2--CH--C,
%
NIt2
The sites of cleavage can be restricted: when the r-amino group of lysine is blocked, for example by earbamylation 5,~ or by trifluoroacetylation, 7 the residue becomes immune to the action of trypsin, and cleavage occurs only at arginine residues--as illustrated in the hydrolysis of the carbamylated S-peptide of ribonuclease by trypsin. ~ By application of a 6 ~ 12 •.-Ala- Lys -Phe -Glu-Arg-Glu-His...
~
Ic~a~ ~
""Ala-Lys-Phe-Glu-Arg-Glu-His... NH.CONI~ ltrypsin
NH~ I
•"-Ala-I{ys-Phe-Glu-Arg-COOH + Glu-His... NI-I.CONH2 reversible blocking agent to the substrate7 ,8 trypsin can be directed to act first at arginine residues, and then, on removing the blocking group, at lysine residues. The longer peptides contain intact sequences around lysine and provide information necessary for aligning the shorter peptides. The sites of cleavage can be extended: modification of the SH group of cysteine residues in the substrate by addition of ethylenimine results in the formation of new basic sites susceptible to the action of trypsin 9,1° D. G. 7R. T.
G. Smyth, W. H. Stein, and S. Moore, J. Biol. Chem. 237, 1845 (1962). 1%. Stark, W. H. Stein, and S. Moore, J. Biol. Chem. 235, 3177 (1960). F. Goldberger and C. B. Anfinsen, Biochemistry 1, 401 (1962). C. Merigan, W. J. Dreyer, and A. Berger, Biochim. Biophys. Aeta 62, 122
(1962). H. A. Lindley, Nature 178, 647 (1956). toM . A. 1%artery and 1%. D. Cole, Bioehem. Biophys. Res. Commun. I0, 467 (1963).
218
Ct,EiVteE OF PEPTIDE CHAINS
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(this volume [33]). A polypeptide prepared in this manner will undergo cleavage at arginine, lysine, and the modified cysteine residues; if the polypeptide is treated first with cyanate, cleavage is restricted to arginine and cysteine residues only. --?ys c~,s ~ CI'~ cH. i I \1,m CH~
--?ys -c~sH
c~ /
Materials and Apparatus Trypsin, 3 times crystallized, salt-free, and lyophilized (available from Worthington Biochemical Corporation, Freehold, New Jersey); automatic titrator, type TTT1, and chart recorder 11 (available from Radiometer, Copenhagen, Denmark); glass titration vessel, fitted with jacket for circulating water at constant temperature, internal volume 20--30 ml, with a flat base on which a glass coated magnet rotates freely (good thermal contact between the reaction bottle and the titration vessel maintained by filling the air space between the two with distilled water, see Fig. 1); constant temperature water bath with circulating pump (available from Haake Werk, Karlsruhe-Durlaeh, West Germany). When digestions are performed at pH 8.5 or above, it may be necessary to pass a slow stream of nitrogen, freed of COs by passage through
f/.-Glass electrode(coupledto pH meter) Flexiblecapillary from microsyringeH20~t~!
Glosscoated stirringbar
\
~
.-~,,.. Waterat constanttemperature Reactionbottle
Rotatingmagnet Fie. 1. Titration vessel for performing enzymic digestion under controlled conditions [A. M. Crestfield,Anal. Chem. 28~ 117 (1956)]. 11C. F. Jacobsen, J. I_~onis, K. Linderstrg~m-Lang,and M. Ottesen, in "Methods of Biochemical Analysis" (D. Glick, ed.), Vol. IV, p. 359. Wiley (Interscience), New York, 1955.
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USE OF PROTEOLYTIC ENZYMES
219
KOH solution, over the top of the titration vessel,~2 and contamination of the reaction mixture by NH~ or C02 is minimized. Procedure Treatment o/ Trypsin to Reduce Contamination by Chymotrypsin. A typical degree of chymotryptic activity exhibited by crystalline trypsin is that which would arise from about 0.1-0.5% contamination by chymotrypsin. 1~ It has been reported that this activity, assessed by measurement of hydrolysis of acetyltyrosine ethyl ester, 14 can be eliminated by treating the enzyme with a specific inhibitor of chymotrypsin, diphenylcarbamyl chloride15 or N-tosylphenylalanine chloromethyl ketone~ (this volume [26]) without seriously impairing the trypsin activity. A simple procedure employing the former reagent is described below. Trypsin (58 mg) is dissolved in 1.6 ml 0.05 M Tris-chloride buffer at pH 7.6. To this solution is added 0.2 ml diphenylcarbamyl chloride solution (5.2 mg, Distillation Products Industries, recrystallized from ethanol, in 25 ml isopropanol). The slightly turbid solution is maintained at 25 ° for 10 minutes and is centrifuged. The supernatant solution is assayed for trypsin activity. This procedure involves a reaction between a 1.4 X 10-s M solution of trypsin and approximately one tenth the concentration of diphenylcarbamyl chloride. According to kinetic studies, ~5 chymotrypsin present in quantities less than 5% of the trypsin is completely inactivated. While the practice of adding a chymotrypsin inhibitor to trypsin is recommended, trypsin treated in this manner still exhibits some chymotryptic activity on a polypeptide substrate. ~ The level of the ehymotryptic activity, however, is considerably reduced. Trypsin has been reported to possess a higher degree of stability than chymotrypsin when exposed to dilute acid, and solutions of trypsin at 4 ° in 0.001 N hydrochloric acid maintain enzyme activity over a period of several days. It appears advantageous therefore to dissolve the crystalline enzyme in 0.001 N HC1 a few hours before use. Trypsin possesses the highest fundamental specificity of all known proteolytic enzymes. This feature, coupled with the knowledge that the specificity can be redirected, commends the use of trypsin as the principal enzyme for the initial fragmentation of a protein.
A. M. Crestfield, Anal. Chem. 28, 117 (1956). l~j. D. Young and F. H. Carpenter, J. Biol. Chem. 236, 743 (1961). 14G. W. Schwert, H. Neurath, S. Kanffman, and J. E. Snoke, J. Biol. Chem. 172, 221 (1948). ~SB. F. Erlanger and W. Cohen, J. Am. Chem. Soc. 85, 348 (1963). 1,G. Schoellman and E. Shaw, Biochemistry 9., 252 (1963). 17K. Takahashi, J. Biol. Chem. 240, 4117 (1965).
220
CLEAVAGE OF PEPTIDE CHAIN'S
[24]
To illustrate alternative procedures in the use of trypsin, four examples are presented. (1) Digestion per]ormed in a pH-stat--hydrolysis o] the B-chain o] human hemoglobin: is The fl-chain is dissolved in water to provide a concentration of 1%, and the solution at 30 ° is adjusted to pH "9.0 by addition of 0.1 N sodium hydroxide. Vigorous stirring is employed to keep the protein, insoluble above pH 7.0, as a fine suspension. A portion of a 1% solution of trypsin in 0.001 N hydrochloric acid is added to give a trypsin concentration of 0.005%, and the solution is maintained automatically at pH 9.0. The protein suspension clarifies after approximately 30 minutes. The course of the reaction is monitored by automatic recording of alkali uptake. After 4 hours, when the reaction slows, a second portion of trypsin solution is added to give a total enzyme concentration of 0.01~, and the hydrolysis is allowed to continue for 16 hours until the base addition almost ceases. The reaction mixture is acidified to pH 4.5 with glacial acetic acid and is lyophilized. (2) Digestion per]ormed in a huller solution--hydrolysis o] oxidized ribonuclease by trypsin: 2 The reaction is carried out at 25 ° in 0.2M sodium phosphate at pH 7.0. The buffer is prepared from 8.28 g NaH2PO4"H20 and 19.88 g Na2HP04 (anhydrous salt) dissolved in 1 liter. The pH of the solution remains constant throughout the hydrolysis. The substrate concentration is 0.50%, and separate digestions are performed at enzyme concentrations of 0.0025% and 0.025%. The course of the hydrolysis is monitored by ninhydrin analysis 89 of portions of the reaction mixture (Fig. 2). The digestions are arrested by adjusting the solutions to pH 2.2 by addition ol~2 N hydrochloric acid. I00 A o,.,
~o~60 ._~ ~. 40 ~ .1:::: n," C
Time (hours)
Fro. 2, Rates of hydrolysis of oxidized ribonuclease (0.50% solution of protein at pH 7.0, 25°), according to Hirs [C. H. W. Hirs, S. Moore, and W. H. Stein, J. Biol. Chem. 219, 623 (1956)]. Curve A: trypsin concentration 0.025%. Curve B: trypsin concentration 0.0025%. '" G. Guidotti, R. J. Hill, and W. Konigsberg, J. Biol. Chem. 237, 2184 (1962). wS. Moore and W. H. Stein, J. Biol. Chem. 211, 907 (1954).
[24]
use OF PROTEOL~rlC ENZYMES
221
(3) Digestion in the presence o] 2 M uma--hydrolysis o] streptocvccal proteinase: 2° The reduced carboxymethylated protein (150 rag) is dissolved in 0.1 M T r i s - 8 M urea buffer, pH 8.0. The clear solution is diluted with 3 volumes of the Tris buffer containing 0.1% thiodiglycol, which reduces the urea concentration to 2 M; the concentration of protein is 1%. The protein precipitates as a fine suspension in 2 M urea solution. Trypsin (0.75 mg in 0.1 ml 0.001 N HC1) is added and the suspension clears after a few minutes. After 4 hours at 25 ° another 0.75 mg trypsin is added, and hydrolysis is allowed to proceed at 25 ° for 16 hours. (4) Digestions per]ormed with trypsin supported in a columnP 1 Although not yet in widespread use, this method potentially offers several advantages. The enzyme column allows precise regulation of the extent of hydrolysis of a substrate by varying the rate of flow of substrate solution through the resin, and by varying the concentrations of substrate and enzyme and the height of the column. Thus the first products to be formed by limited cleavage of a polypeptide substrate can be rapidly isolated by elution from the column. Full details of the preparation of a water-insoluble form of trypsin bound to a resin are presented below. POLYTYROSYL TRYPSIN. This derivative is prepared from trypsin by the procedure of Glazer, Bar-Eli, and Katchalski. 22 A polymer containing 9.6% enrichment of tyrosine exhibits 70-75% of the enzyme activity of the original trypsin. When polytyrosyl trypsin is coupled to a diazotized copolymer of DL-phenylalanine and leucine (1:2), the resulting water-insoluble trypsin derivative retains 15-30% of the activity of crystalline trypsin. COPOLYMER OF p-AMIN0-DL-PHENYLALANINE AND L-LEUCINE. 21 p-NCarbobenzoxyamino-a-N-carboxy-DL-phenylalanine anhydride (3.5 g) and N-carboxy-L-leucine anhydride (1.5 g) are copolymerized in anhydrous dioxane (100 ml) with triethylamine (0.1 ml) as the initiator. The reaction mixture is stored for 3 days at room temperature, and finally is heated under reflux for 1 hour. The product precipitates on addition of water (300 ml) to the cooled solution. Removal of the carbobenzoxy groups is effected by treating the dried copolymer (3.5 g) with anhydrous hydrogen bromide in glacial acetic acid (30 ml) for ] hour at room temperature. The copolymer hydrobromide is precipitated from solution in acetic acid by addition of ether (500 ml). The precipitate is washed repeatedly with ether to remove benzyl bromide, and is dried in vacuo over NaOH and over H2SO4 ; yield, 2.5 g. DIAZONIUM SALT OF COPOLYMER OF DL-PHENYLALANINE AND L-LEUCINE.
a T . Y. Liu, W. H. Stein, S. Moore, and S. D. Elliott, J. Biol. Chem. 240, 1143 (1965). a A. Bar-Eli and E. Katchalski, J. Biol. Chem. 238, 1960 (1963). ~A. N. Glazer, A. Bar-Eli, and E. Katchalski, J. Biol. Chem. 237, 1832 (1962).
222
CLEAVAGE OF PEPTIDE CHAINS
[24]
Diazotization is carried out as follows: 0.5 M NAN02 (0.6 ml) is added dropwise to an ice-cooled solution of copolymer (100 mg) in 0.5 N HC1 (3 ml). After 1 ~ hours at 4 ° a 10% sodium acetate solution (10 ml) is added, and the pH of the reaction mixture is adjusted to 7.5 with 2 N NaOH. The light brown polydiazonium salt which separates is centrifuged and washed 3-4 times with 15-ml portions of cold 10% sodium acetate. The wet diazonium salt (dry weight, 70 rag) is used as a waterinsoluble carrier in the coupling experiments with polytyrosyl trypsin. COUPLING OF POLYTYROSYL TRYPSIN TO THE COPOLYMER OF DL-PHENYLALANINE AND L-LEUCINE. A polytyrosyl trypsin solution containing 3.0 mg modified enzyme per ml 0.0025N HC1 is mixed with an ice-cooled suspension of the polydiazonium salt (100 mg) in 0.1 M phosphate buffer, pH 7.6 (6 ml). The suspension is stirred magnetically and the coupling reaction is allowed to proceed for 1½ hours at 4 °. The pH is adjusted to 6.8 and the reaction mixture is left for 20 hours at 4 °. The light brown product is isolated by centrifugation. Final purification is attained by washing with 0.0025 N HC1 until the washings show no proteolytic activity toward casein. PREPARATION OF THE TRYPSIN COLUMN.A trypsin column (0.6 X 3 cm) is prepared from a mixture of polytyrosyltrypsin coupled to a diazotized copolymer of p-amino-DL-phenylalanine and leucine (1:2) (6 mg resinbound polytyrosyl trypsin, corresponding in esteratic activity to 0.25 mg crystalline trypsin) with an inert polyvinyl resin, Geon 426 (0.5 g) in 0.0025N hydrochloric acid (10 ml), by pouring into a glass tube. The elutions are performed at 25 °. Before use the column is washed with a suitable buffer, and at the end of each experiment with 0.0025 N hydrochloric acid, which stabilizes the enzyme. The column is kept at 4 ° until needed for further use. U S E OF TRYPSIN COLUMN IN T H E DIGESTION OF OXIDIZED INSULIN.21 Oxidized insulin is dissolved in 0.1 M phosphate buffer, pH 7.6, and the insulin solution (5 mg/ml) is passed through the trypsin column (0.6 X 3 cm), prepared as above, at a flow rate of 1 ml/170 minutes. The effluent contains free alanine in 85% yield. Chymotrypsin Specificity. In synthetic substrates, chymotrypsin catalyzes the hydrolysis of the peptide bonds between the COOH group of tyrosine, phenylalanine, or tryptophan, and the NH2 group of the adjacent amino acid? ~ Knowledge of the specificity against large polypeptides has come from studies in which the enzyme has been used in determinations of
23M. Bergma~n and J. S. Fruton, J. Biol. Chem. 118, 405 (1937).
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USE OF PROTEOLYTIC ENZYMES
223
amino acid sequence. 2.-2¢ Slow hydrolysis has been found to occur at the COOH group of numerous amino acids, including asparagine, glutamine, histidine, leucine, lysine, methionine, serine, and threonine---in addition to rapid cleavages at aromatic residues--but cleavage at these sites is usually significant only at a high concentration of enzyme or when prolonged digestion is employed. The rates of chymotryptic attack are influenced by the nature of the amino acid residue adjacent to the susceptible bond. Thus, cleavage at the COOH group of asparagine appears to be favored when this residue is adjacent in sequence to Lys, His, or Arg. The enzyme does not significantly attack bonds formed by the imino group of proline. The reactivity of a susceptible site is influenced, in addition, by the general environment along the polypeptide chain. For example, the peptide bond between a serine and an alanine residue at position 18 in ribonuclease is readily cleaved when the tryptic peptide containing residues 11-31 is digested by chymotrypsin~,2T 14 15 16 17 18~19 20 21 22 23 -Asp-Ser-Ser-Thr-Ser-Ala-Ala-Ser-Ser-SerOn the other hand, negligible cleavage occurs at this position when the S-peptide (residues 1-20) is treated with chymotrypsin. Although the intrinsic specificity of chymotrypsin is rather broad, some advantage may be gained by adding soybean trypsin inhibitor to the chymotrypsin.2s The inhibitor abolishes the activity of any trypsin that may contaminate the chymotrypsin without seriously impairing the activity of the latter. It should be emphasized again that enzymic cleavages rarely occur on an "all-or-none" basis. Therefore experimental conditionsmthat is, temperature, pH, time of hydrolysis, substrate concentration, and molar ratio of chymotrypsin to substrate--are critical to optimum use of the enzyme. In practice, chymotrypsin has been employed under a wide variety of experimental conditions in studies on the structure of proteins. Thus, incubation has been carried out at pH values 7.054 and 9.0, 56 with temperatures between 20 °59 and 60°, ~° with digestion for 2 hours 55 or 30 hours, 59 and with enzyme-to-substrate molar ratios between 1:40054 and C. H. W. Hirs, W. H. Stein, and S. Moore, J. Biol. Chem. 221, 151 (1956). R. E. Canfield and C. B. Anfinsen,J. Biol. Chem. 238, 2684 (1963). R. J. Hill and W. Konigsberg, J. Biol. Chem. 2,37, 3151 (1962). =7C. H. W. Hirs, J. Biol. Chem. 23,5, 625 (1960). B. Keil and F. $orm, "Structure and Activity of Enzymes" (T. W. Goodwin, J. I. l=[arris, and B. S. Hartley, eds.), 1st Symp. Federation European Biochem. Soc., London 1964, p. 37. Academic Press, New York, 1964. E. Margoliash, J. Biol. Chem. 237, 2161 (1962). T. Ooi, J. A. Rupley, and H. Scheraga, Biochemiztry 2~ 421 (1963).
224
CLEAVAGE OF PEPTIDE CHAINS
[24]
1:2021 Three examples are selected to illustrate the application of chymotrypsin. (1) Digestion ]or 2~ hours (at pH 7.0, 25 °, enzyme-to-substrate molar ratio 1:360)--hydrolysis o] per]ormic acid-oxidized ribonuclease by chymotrypsin: 2~ Oxidized ribonuclease (200 mg) is dissolved in 20 ml 0.2 M phosphate buffer at pH 7.0 and a solution of chymotrypsin (crystalline, salt-free, from Worthington Biochemical Corp.) in the same buffer is added, providing a substrate concer:tration of 1.0% and an enzyme concentration of 0.005%. The digestion is allowed to proceed at 25 ° for 24 hours; the course of the reaction is followed by direct ninhydrin analysis 19 of small portions of the reaction mixture. The reaction is terminated by addition of 1 N hydrochloric acid to pH 2.2. The peptide mixture is stored at --15 °. (2) Digestion ]or 2 hours (at pH 8.0, 37 °, enzyme-to-substrate molar ratio 1:90)--hydrolysis o] reduced carboxymethylated lysozyme by chymotrypsin: 25 The reduced carboxymethylated protein (1.0 g) is suspended in 100 ml distilled H20 at 37 °, and dilute NH~OH is added to pH 8.0. Chymotrypsin (20 mg) in a small volume of water is added, and the solution is maintained at pH 8.0 by further addition of NH40H. The suspension clears after 20 minutes, and the digestion is allowed to proceed for 2 hours. The solution is lyophilized. (3) Partial digestion o] a native protein--limited hydrolysis o] ribonuclease: 3° Digestions are performed with a Radiometer pH-stat, and progress of the reaction is followed by ninhydrin analysis, decrease in ribonuclease activity, and measurement of the volume of base required to maintain the pH. Reaction mixtures contain 10 mg per milliliter of ribonuelease, 0.01 M KC1 or CaC12, and 0.05-0.5 mg chymotrypsin (Sigma Chemical Corporation) per milliliter of solution. The pH is maintained at 6.5 and the temperature at 25-60 ° . In the presence of 0.01 M CaC12 and in the absence of substrate, chymotrypsin loses 50% of its activity at 60 ° in less than 5 minutes. In the absence of calcium, inactivation is more rapid. Several consecutive additions of enzyme are necessary to ensure a moderate degree of hydrolysis. Associated with the inactivation of chymotrypsin under these conditions is the development of turbidity which increases with time. The reaction is terminated by the addition of diisopropyl phosphorofluoridate to a concentration of 0.01 M, and by cooling to 0 ° ; the mixture is lyophilized. Pepsin In synthetic substrates, pepsin catalyzes the hydrolysis of the peptide bonds formed by the amino or carboxyl groups of phenylalanine, tyrosine, 81A. Light and E. L. Smith, J. Biol. Chem. 237, 2537 (1962).
[24]
USE OF PROTEOLYTIC ENZYMES
225
leucine, alanine, glutamic acid, cystine, and cysteine~2; the enzyme can split the bond on either side of these residues. In its action on proteins, pepsin has been found to catalyze the hydrolysis of peptide bonds formed by all amino acids except proline and isoleucine. The most susceptible sites, however, are those formed by the aromatic amino acids, by leucine, and to a lesser extent by alanine. As the sites and rates of attack on a polypeptide cannot be predicted with accuracy, the optimal reaction conditions for a particular application of pepsin are selected on the basis of exploratory experiments carried out on a small scale. A detailed analysis of the specificity of pepsin in its action on the ~- and fl-chains of hemoglobin has been provided by Konigsberg and HHill.83,s4 Digestion of certain peptides from the B-chain, with a low relative concentration of pepsin, resulted in complete hydrolysis of a limited number of peptide bonds within a few minutes, and only after much longer digestion did extensive cleavage take place at other sites: 8~ 105 120 NH2Leu-Leu-Gly-Asn-Val-Leu-Val-CySO3H-Val-Leu-Ala-His-His-Phe-Gly-Lys (0.002% pepsin 25°, pH 2, 4 min) 120 NH2Leu-Leu-Gly-Asn-Val-Leu + Val-CySOsH-Val-Leu-Ala-His-His-Phe-Gly-Lys The presence of a free a-amino group at a site potentially susceptible to pepsin appears to reduce the rate of attack. The peptide shown above is rapidly cleaved by pepsin at Leu-110, whereas the NH2-terminal Leu-105 is not affected, even on prolonged digestion. Similarly, the NI-I2-terminal lysine residue in ribonuclease is liberated by trypsin only to a very small extent under conditions where cleavage at 9 of the 10 internal lysine residues is complete; the remaining lysine is present in a Lys-Pro sequence. 2S Pepsin can also catalyze transpeptidation reactions, s5 If this process were to occur to a significant extent during use of the enzyme in determinations of amino acid sequence, confusing results would be obtained. However, in analytical studies so far reported, no evidence has been obtained that this reaction interferes during application of pepsin to the cleavage of polypeptides. It has been reported that, when pepsinogen is activated at pH 3.0 instead of at pH 2.0, transpeptidation activity is eliminated without alteration of hydrolytic activity, s6 =J. S. Fruton and M. Bergmann, J. Biol. Chem. 127, 627 (1939). ~sW. Konigsberg and R. J. Hill, J. Biol. Chem. 237, 3157 (1962). W. Konigsberg, J. Goldstein, and R. J. Hill, J. Biol. Chem. 238, 2028 (1963). H. Newmann, N. Sharon, and E. Katchalski, Bioehem. J. 73, 33 (1959). H. Newmann and N. Sharon, Bioehim. Biophys. Acta 41, 370 (1960).
226
CLEAVAGE OF PEPTIDE CHAINS
[24]
Three examples illustrating the use of pepsin are presented. (1) Digestion o] fl-chain o] human hemoglobin with pepsinP 4 The pepsin used is twice crystallized from ethanol (Worthington Biochemical Corporation). The enzyme is stored for no longer than 2 weeks as a 1% solution in pyridine acetate, pH 3.8, at --15 °, and is thawed immediately before use. Mono-S-carboxymethyl fl-ehain from hemoglobin (800 mg) is dissolved in 80 ml 0.01 N HC1, and the pH is adjusted to 2.0. A solution (0.8 ml) of 1 ~ pepsin in 0.2M pyridine acetate buffer, pH 4.2, is added and the mixture is kept at 25 ° for 1 hour. The reaction is stopped by addition of pyridine until the solution reaches pH 5, and the peptide mixture is frozen and lyophilized. In exploratory experiments, aliquots of the digest are adjusted to pH 5, and paper electrophoresis is used to assess the extent of hydrolysis.
(2) Digestion o] reduced carboxymethylated lysozyme with pepsinP 7 Reduced carboxymethylated lysozyme (550 mg) is dissolved in 50 ml 5 ~ aqueous formic acid. Pepsin (6.6 mg), dissolved in 2 ml 0.5 M NaCI, is added and the digestion allowed to proceed at 23 ° for 60 minutes. The solution is frozen and lyophilized. (3) Combined enzymatic hydrolysis o] diisopropylphosphoryl (DIP) trypsinP 8 A solution of 7.0 g DIP-trypsin in 700 ml water is adjusted to pH 2 with hydrochloric acid and the temperature is maintained at 37 °. To the solution is added slowly, with vigorous stirring, 216 mg pepsin (enzyme to substrate ratio----1:50), and the reaction mixture is maintained at pH 2.0 by addition of 0.4 M HC1 from an autotitrator. After 12 hours, peptic hydrolysis is terminated by addition of 1 M NaOH to pH 6.5. A solution of 72 mg trypsin in 12 ml water (enzyme to substrate ratio ~-- 1:100) is added, and the pH maintained at 6.5. After 1 hour, a second portion of trypsin (72 mg) in 12 ml water is added and the digestion is allowed to proceed for 4 hours. A solution of 72 mg chymotrypsin in 25 mt water (enzyme to substrate ratio = l:100) is added; after 7 hours, the digestion is arrested by addition of acetic acid to pH 4.0. The solution is filtered and lyophilized. The digestion with pepsin was performed under acidic conditions which reduced the incidence of disulfide interchange, as a problem discussed in this volume [53]. The resulting peptides retained the disulfide bonds present in the native protein. The subsequent digestions, performed at pH 6.5, served to pare the disulfide-containing peptides until each disulfide bond was present in relatively small peptides. After isolation of these 3~R. E. Canfield, J. Biol. Chem. 238, 2693 (1963). ~V. Holeysovsky, V. Tomasek, O. Miles, A. S. Danilova, and F. ~orm, Collection Czech. Chem. Commun. 30, 3936 (1965). nD. H. Spackman, W. H. Stein, and S. Moore, J. Biol. Chem. 23~ 648 (1960).
[24]
USE OF PROTEOLYTIC ENZYMES
227
peptides the disulfide bonds were cleaved in each, and the two derived peptides were identified. The positions of the disulfide bonds could then be assigned by comparison with the known positions of the cystine residues in the overall amino acid sequence. Papain
An enzyme that possesses a low order of specificity may be expected to produce a large number of fragmen~ when it acts on a long polypeptide. In using such an enzyme, advantage may be obtained by employing a low concentration and a short incubation period, which will reduce the multiplicity of products. Papain exhibits a wide specificity in its action both on small synthetic peptides and on large polypeptides. 4° This property makes the enzyme suitable for the further degradation of peptides obtained by the action of trypsin or chymotrypsin; the smaller fragments produced may be more tractable for determination of amino acid sequence. Peptide bonds formed by the carboxyl groups of arginine and lysine residues, provided these amino acids are not NH2-terminal, appear to be highly susceptible to papain. In addition, cleavage occurs readily at the C 0 0 H group of histidine, and also at glycine, glutamic acid, glutamine, leucine, and tyrosine. The rates of cleavage, however, show considerable variation; ~1 papain can therefore be used to produce overlapping fragments. This is illustrated in the cleavage of an octapeptide from ribonuclease2 The 11 18 Pyr-Hts-MeSO~-Asp-Ser-Ser-Thr -Ser
Pyr-His-MeSO~-Asp-Ser,
Asp-Ser-Ser-Thr-Ser
l (74% yield) Pyr-His-MeSO2 (81% yield)
Asp-Ser + Ser-Thr-Ser (7% yield)
(23% yield)
susceptibility to attack of a particular bond may be influenced by the ease with which other bonds in the immediate vicinity undergo hydrolysis. In the example illustrated above, the rapid prior cleavage which occurs between methionine sulfone and aspartic acid may reduce the rate of subsequent cleavage expected at His-12. M. Dixon and E. C. Webb, "Enzymes," 1st Ed., p. 251. Longmans, Green, London, 1958. J. R. Kimmel and E. L. Smith, Advan. Enzymol. 19, 267 (1957).
228
CLEAVAGE OF PEPTIDE CHAINS
[24]
Some differences in specificity have been observed in experiments in which digestions were performed at different pH values. An octapeptide from the ~-chain of hemoglobin, Val-His-Leu-Thr-Pro-Glu-Glu-Lys, on digestion with papain at pH 4.25 undergoes hydrolysis at either side of leucine, whereas at pH 6 cleavage occurs additionally at the a-carboxyl groups of the two glutamic acid residues23 The ability to redirect specificity can thus be exploited to provide different fragments, which m a y give useful information on amino acid sequence. The experimental use of papain involves addition of cysteine or cyanide to the digestion mixture in order to activate the enzyme. It is important that the reducing agent employed should not affect the substrafe or interfere with subsequent fractionation procedures. Two examples of the use of papain are described. (1) Hydrolysis o] Pyr-His-2~eSO~-Asp-Ser-Ser-Thr-Ser ~ derived ]rom oxidized ribonuclease: To a solution of 7 #moles of the octapeptide in 20 ml H~0 are added 7.4 mg sodium ethylenediaminetetraacetate, 10 mg sodium cyanide, and 100 ~1 papain suspension containing 3 mg enzyme per milliliter (papain, twice crystallized, from Worthington Biochemical Corp.). The resulting clear solution is brought to pH 7 by dropwise addition of 1 ~ aqueous ammonia, and is maintained at 37 ° for 16 hours. To terminate the reaction, 1 ml 0.1 M formic acid is added and the solution is frozen. (2) The use o] a water-insolubl~ ]orm of papain 42 Preparation o] water-insoluble papain: A copolymer of L-leucine and p-amino-DL-phenylalanine is prepared according to Bar-Eli and Katehalski, ~1 and the copolymer is diazotized as described above (p. 222). Coupling with papain is carried out by adding crystalline papain (Worthington, twice crystallized, 80 rag) suspended in 0.03 M cysteine (2 ml) to a suspension of the diazotized copolymer in aqueous solution (40 ml) at pH 7.6, 0.075 M in phosphate, 0.005 M in cysteine, and 0.002 M in EDTA. Oxygen is removed by flushing with nitrogen, and the coupling is allowed to proceed in a tightly stoppered vessel for 20 hours at 4 ° with magnetic stirring. The water-insoluble product is isolated by centrifugation, and washed several times with a solution of 0.005 M cysteine containing 0.002 M EDTA at pH 6.0. The resulting water-insoluble papain (250 mg) is stored at 4 ° under an aqueous solution of composition similar to that of the wash solution. The water-insoluble enzyme is homogenized in a Potter homogenizer fitted with a Teflon pestle. Storage of the product at 4 ° for 1 month results in a 3 0 ~ loss of activity. The activity of the insoluble papain can be assessed by measurement a j. j. Cebra, D. Givol, H. I. Sflman, and E. Katchalski, J. Biol. Chem. 236, 1720 (1961).
[24]
use OF PROTEOLYTIC ENZYMES
229
of the rate of hydrolysis of a well-stirred solution of benzoylarginine ethyl ester (BAEE) at pH 6.0, 30 °, in an autotitrator. The asasy solution (5 ml) contains 0.025M BAEE, 0.002M EDTA, and 0.005M cysteine at pH 6.0. The equivalent amount of active enzyme is calculated from the initial rate of alkali consumption by comparison with soluble papain; in the presence of 100 ~g enzyme, alkali is consumed at an initial rate of 1.6 ~moles per minute. By this assay, 100 ~g water-insoluble papain is equivalent to 20 ~g crystalline enzyme. Limited hydrolysis o] a protein by insoluble papain: ~2 y-Globulin is dissolved in 0.9% NaC1 at a concentration of 10-20 mg protein per milliliter, and is dialyzed against 0.9% NaC1. After dialysis, EDTA is added to a final concentration of 0.002M and the solution is adjusted to pH 8.5. An amount of the water-insoluble papain preparation (10 mg), corresponding in weight to 1/20 that of the protein substrate (200 rag), is washed with 5 volumes of 0.9% NaC1, centrifuged to remove the supernatant solution, and resuspended in the above v-globulin solution (10-20 ml). The enzyme is kept in suspension by stirring, and the course of the proteolysis at pH 8.5, 20 °, is followed by titration with 0.1 N NaOH from an autotitrator. The water-insoluble enzyme is removed by filtration through Whatman No. 1 filter paper; the filtrate contains the required fragments of v-globulin. Subtilisin Subtilisin was first isolated from a culture of Bacillus subtilis, and was obtained in crystalline formA ~ A second crystalline enzyme has been isolated from a different strain of the same organism ~4 and is currently available commercially (Sigma Chemical Corporation) under the trade name, Nagarse. The two enzymes exhibit very similar specificity in their action on polypeptides, but the specificity is so wide that a preference for bonds involving particular amino acids cannot be stated. As in the use of papain, the broad specificity renders subtilisin suitable for degrading peptides of intermediate size, containing about six to twelve amino acids, to provide small fragments suitable for direct determination of sequence by the Edman method. A detailed account of the many sites in peptides that have proved susceptible to the action of subtilisin has recently been presented. '5 Three procedures employed in the use of subtilisin are described. A. V. GSntelberg and M. Ottesen, Compt. Rend. Tray. Lab. Carlsberg, Set. chim. 29, 36 (1954). "B. Hagihara, in "The Enzymes" (P. D. Boyer, It. Lardy, and K. Myrb~ck, eds.), 2nd Ed., Vol. 4, p. 193. Academic Press, New York, 1960. R. L. Hill, Advan. Protein Chem. 2@, 37 (1965).
230
CLEAVAGE OF PEPTIDE CHAINS
[24]
(1) Hydrolysis o] Asp-Ser-CySO~H-Glu-Gly-Gly-Asp-Ser-Gly-ProVal-CySOsH-Ser-Gly-Lys (O-Tr-1) by subtilisin: 4~ Subtilisin was obtained according to the published method. "s The peptide O-Tr-1 (0.2 ~mole, isolated from performic acid-oxidized trypsin) is dissolved in 0.2 ml 0.1 M ammonium acetate at pH 8.0. A portion (10 ~l) of a solution of subtilisin (2.8 mg dissolved in 0.1 ml 0.1 M ammonium acetate, pH 8.0) is added, and the solution is held at 25 ° for 12 hours. The solution is acidified to pH 3.6, and the resulting peptide mixture is resolved by paper ionophoresis.
(2) Hydrolysis o] a peptide o] 15 residues ]rom lysozyme by subtilisin: 37 Subtilisin was obtained in crystalline form (from the Nagarse Co., Osaka, Japan). The peptide (0.5 #mole) is dissolved in 0.5 ml 0.1 M ammonium bicarbonate, and 25 ~l 1% solution of subtilisin in the same buffer is added. The mixture is kept at 37 ° for 6 hours, and the reaction is stopped by acidification to pH 3.6. The points of attack by the enzyme are indicated by the arrows: Ileu-Val-Ser-Asp-4~ly-Asp-Gly-Met-Asn-Ala-Try-Val-Ala-Try-Arg
T T T "( T (3) Partial hydrolysis o] native ribonuclease by subtilisin27 Ribonuclease A (730 rag) is dissolved in 5 ml 0.1 M potassium chloride, and the solution is placed in the cell of a pH-stat. The temperature is lowered to 3 ° and the pH adjusted to 8.0 with 0.1 N sodium hydroxide. Subtilisin (1 mg dissolved in 0.2 ml water; prepared by the method of Guntelberg and Ottesen) is added, and the rate of addition of 0.1 N sodium hydroxide is recorded. After 3 hours the digestion is stopped by addition of 1 N HC1 to pH 3, which rapidly denatures the enzyme. Pronase
The enzyme is isolated from a strain of Streptomyces griseus ~s and exhibits the broadest specificity of any known proteolytic enzyme. In its action on synthetic substrates ~9,s° almost all dipeptides, tripeptides, and amino acid amides undergo hydrolysis at least in part on prolonged incubation. Where extensive proteolysis of a peptide or protein is required, pronase is extremely effective. Thus the enzyme is suitable for the liberation of free asparagine and glutamine under conditions that preserve the integrity of their amide groups. The extensive degradation produced by this enzyme resembles that produced by partial acid hydrolysis, except that high yields of the hydrolytic products are obtained. G. H. Dixon, D. L. Kauffman, and H. Neurath, J. Am. Chem. Soc. 233, 1373 (1958). " F . M. Richards and P. J. Vithayathil, J. Biol. Chem. 234, 1459 (1959). M. Nomoto and Y. Narahashi, J. Biochem. 46, 653 (1959). ~' M. Nomoto, Y. Narahashi, and M. Murakami, J. Biochem. 48, 593 (1960). M. Nomoto, Y. Narahashi, and M. Murakami, J. Bioehem. 48~ 906 (1960).
[2S]
CHROMATOGRAPHY
OF TRYPSIN A N D CIIYMOTRYPSIN
231
It should be noted that pronase differs from prolidase or prolinase. Pronase is a bacterial enzyme that derives its name from its striking ability to digest proteins, whereas prolinase and prolidase are alternative titles for an enzyme that possesses an ability to cleave peptide bonds involving proline sl, 52 (a purified preparation of pronase is available from Sigma Chemical Corporation). A procedure for the use of Streptomyce~ griseus protease (pronase) is described. The enzyme was prepared according to published procedures. 4g To 1 ml enzyme solution (0A4 mg protein nitrogen per ml) is added 1 ml 0.1 M substrate solution, and the reaction is allowed to proceed at pH 7.0-7.2, 40 ° , for 24 hours. The reaction mixture is acidified and the products are separated by paper chromatography. ~1W. Grassmann, H. Dyekerhoff, and O. yon Schoenbeck, Bet. 62, 1307 (1929). mN. C. Davie and E. L. Smith, J. Biol. Chem. 224, 261 (1957).
[ 2 5 ] C h r o m a t o g r a p h y of T r y p s i n t o R e m o v e C h y m o t r y p s i n , a n d of C h y m o t r y p s i n t o R e m o v e T r y p s i n
By M m E m L E
ROVERY
The procedure for the commercial preparation of bovine trypsin and chymotrypsin is the same as far as the crystallization of chymotrypsinogen. Thus it is not surprising to find traces of one enzyme in preparations of the other. Therefore, when peptide bonds of aromatic amino acid residues are cleaved during tryptic hydrolysis it seems reasonable to invoke chymotryptic impurities. Conversely, when peptide bonds of basic residues are split during chymotryptic hydrolysis, trypsin contamination may be implicated. However, as trypsin and chymotrypsin have many similarities in structure of the active centers, they m a y also have limited eross-specificities for certain peptide bonds. In practice, contamination is responsible for lack of specificityin most cases. Thus, it is important to use highly purified enzymes to obtain the most specific and reproducible hydrolysis possible. Techniques of varying efficiency have been used previously to inhibit chymotrypsin-like impurities in trypsin preparations: incubation in dilute acid, 1 denaturation in 8 M urea, 2 and treatment with small quantities of diisopropyl phosphorofluoridate? In recent years two additional I j. H. Northrop and M. Kunitz, in "Handbuch der biologischen Arbeitsmethoden" (E. Abderhalden, ed.), Vol. IV, Part 2, p. 2213. Urban & Schwarzenberg, Berlin, 19~6. 2j. I. Harris, Nature 177, 471 (1956). ~J. T. Potts, A. Berger, J. Cooke, and C. B. Anfmsen, J. Biol. Chem. 237, 1851 (1962).
CLEAVAGE OF PEPTIDE CHAINS
[24]
[24] T e c h n i q u e s in E n z y m i c H y d r o l y s i s B y D . G. SNIYTH
The classical studies of Fruton, 1 which revealed that the proteolytie enzyme trypsin possesses a high specificity in its catalysis of peptide bond hydrolysis, opened the way for application of enzymes in the specific cleavage of polypeptides. The use of enzymes now fills a central role in determination of protein structure. Planning of the practical techniques involved requires attention to the general principles that underlie enzyme action, and close attention to the particular purpose for which the enzyme is being employed. (1) Specificity of Enzyme. Ideally the enzyme is required to act at a limited number of sites in the polypeptide substrate. If this is achieved, relatively few fragments are released and these may be separated easily. It is an advantage when the specificity of an enzyme is well characterized because the number of peptides to be liberated by it can be anticipated. The enzyme selected for the specific cleavage of a large polypeptide is in general required to provide smaller peptides amenable to direct determination of amino acid sequence. In addition, where the enzyme has a narrow specificity, each peptide produced may be expected to terminate in a predictable amino acid residue, and this information is of value in the assignment of sequence. (2) Purity of Enzyme. The enzyme should be available, or be prepared, as free as possible of contaminating enzyme in order that the polypeptide substrate be attacked only at sites determined by the fundamental specificity. Even traces of contaminating enzymes may cause reduced yield of the required products and give rise to complex mixtures of peptides difficult to resolve. (3) Preparation o] Enzyme. A weighed amount of the crystalline enzyme should be dissolved immediately before use, and appropriate dilution carried out to obtain a suitable concentration; the dilute solution of enzyme is then added to a solution of substrate. A typical ratio of enzyme to substrate is in the region 1:100 parts by weight. Proteolytic enzymes are not usually stored in solution because of the possibility of autodigestion or irreversible denaturation, resulting in loss of enzyme activity. The enzyme should, of course, never be added in solid form to the solution of substrate because of adverse effects caused by the local excess. In some cases it is advantageous to expose an enzyme before use to conditions that inactivate contaminating enzymes more 1M. Bergmann, J. S. Fruton, and H. Pollock, J. Biol. Chem. 127, 643 (1939); M. Bergmann and J. S. Fruton, Advan. Enzymol. 1, 63 (1941).
[24]
USE OF PROTEOLYTIC ENZYMES
215
rapidly than the enzyme itself; details of these procedures are included in the section on each enzyme. The activity of an enzyme should be checked against a standard substrate immediately before the enzyme is used. Proteolytic activity may be assayed with a small synthetic peptide as substrate, or with a large, naturally occurring polypeptide. For example, with the B-chain of insulin as a reference substrate, the rate of liberation of alanine provides a measure of the activity of trypsin, whereas the rate of liberation of tyrosine and the COOH-terminal tetrapeptide Thr-Pro-Lys-Ala gives a measure of the activity of chymotrypsin. The concentrations of these fragments can be accurately determined by amino acid analysis, and a measure of both activity and specificity is obtained. (4) Preparation o] Substrate. The peptide or protein substrate should be pure and in an unfolded form before enzymic hydrolysis is undertaken. Thus multiehain proteins are separated into their component polypeptides, and these are individually digested by the enzyme. In addition, the substrate must be physically available to the enzyme. Residues situated in the interior of a tightly folded protein molecule may be inaccessible, but can be exposed by physical denaturation (this volume [23]), chemical procedures involving cleavage of the disulfide bridges (this volume [19-22]), or cleavage at methionine residues (this volume [27]). Although digestions have been performed successfully in heterogeneous solution, the reaction cannot be carefully controlled. A product formed by partial digestion may possess an intrinsic low solubility and separate from solution as a "core," or a peptide released during the digestion may aggregate with itself or with other peptides and form a precipitate. These difficulties, as well as the problem of attempting to digest insoluble starting material, may be overcome by performing reactions in 2 M urea, in which the formation of aggregates is reduced and in which some enzymes retain activity for short periods. Protein disulfide bonds are in general cleaved prior to enzyme digestion. Disulfide bonds are labile under the neutral and alkaline conditions employed in structural studies, and readily undergo base-catalyzed rearrangements. Enzymes that function at acid pH, as pepsin does, are A-- S I ~ B--S
OH t
C--S
A.S' +
A.S'
+
B-S--OH
A--S
l
D--S
~
I +
C.S'
D--S
therefore much preferred for the liberation of peptides in which the native disulfide bonds are required intact.
216
CLEAVAGE OF PEPTIDE CHAINS
[24]
(5) Optimum Digestion Periods. Although brief exposure to an enzyme can result in rapid cleavage of a particularly sensitive peptide bond, prolonged digestion may be necessary to ensure cleavage of resistant linkages. Extended periods of digestion, however, often result in fragmentation of the required products, due to the activity of small amounts of contaminating enzyme which slowly causes additional cleavage, or to an inherent lack of specificity in the principal enzyme. The substrate is exposed to the enzyme for the minimum period consistent with completion of the required cleavage. (6) Termination o] E n z y m e Action. Enzymes function over narrow ranges of pH. When a digestion proceeds at neutral pH, the most usual method of stopping the reaction is to acidify the reaction mixture; or when the digestion proceeds at acid pH, to neutralize the solution. It should be noted that, if the pH of the solution is subsequently restored to the initial value, the enzyme may recover its activity and an alternative method may be required to terminate the reaction. For example, when trypsin is used at pH 7 to produce fragments from bradykininogen, and the resulting mixture of peptides is tested at pH 7.4 for bradykinin activity, it is unsatisfactory to arrest the reaction by acidification; on neutralization, the trypsin may itself interfere with the biological assay. However, the reaction can be stopped by addition of trypsin inhibitor or by boiling. While proteolytic enzymes are destroyed by boiling, vigorous treatments of this type are permissible only if they leave the substrate unaffected. The optimal procedure for a particular enzymic digestion clearly depends on the enzyme and the substrate concerned, and on the degree of cleavage required. The methods presented are intended to serve as a general guide i'n experimental procedure. Trypsin Principle Trypsin catalyzes the hydrolysis of the peptide bond between the C 0 0 H group of lysine, or the C 0 0 H group of arginine, and the NH2 group of the adjacent amino acid. 1 Cleavage takes place more slowly when the basic residue is adjacent in sequence to an acidic residue or cystine, and does not occur at all when the residue is followed by proline. 2-4 2C. H. W. Hits, S. Moore, and W. H. Stein, J. Biol. Chem. 219, 623 (1956). 8A. Tsugita, D. T. Gish, J. Young, H. Fraenkel-Gonrat, C. A. Knight, and W. M. Stanley, Proe. Nat. Aead. Sei. U.S. 46~ 1463 (1960). rE. Margoliash, E. L. Smith, G. Kreil, and H. Tuppy, Nature 19'2, 1125 (1961).
[24]
USE OF PROTEOLYTIC ENZYMES
217
~NH--CH - / ? R NH-(~H2)4 \ N H - - CIH - - C / NHz
--NI-I-- CH-- C I
~OH
\kO
+
R
NII --
I / NI-I2--CH--C,
%
NIt2
The sites of cleavage can be restricted: when the r-amino group of lysine is blocked, for example by earbamylation 5,~ or by trifluoroacetylation, 7 the residue becomes immune to the action of trypsin, and cleavage occurs only at arginine residues--as illustrated in the hydrolysis of the carbamylated S-peptide of ribonuclease by trypsin. ~ By application of a 6 ~ 12 •.-Ala- Lys -Phe -Glu-Arg-Glu-His...
~
Ic~a~ ~
""Ala-Lys-Phe-Glu-Arg-Glu-His... NH.CONI~ ltrypsin
NH~ I
•"-Ala-I{ys-Phe-Glu-Arg-COOH + Glu-His... NI-I.CONH2 reversible blocking agent to the substrate7 ,8 trypsin can be directed to act first at arginine residues, and then, on removing the blocking group, at lysine residues. The longer peptides contain intact sequences around lysine and provide information necessary for aligning the shorter peptides. The sites of cleavage can be extended: modification of the SH group of cysteine residues in the substrate by addition of ethylenimine results in the formation of new basic sites susceptible to the action of trypsin 9,1° D. G. 7R. T.
G. Smyth, W. H. Stein, and S. Moore, J. Biol. Chem. 237, 1845 (1962). 1%. Stark, W. H. Stein, and S. Moore, J. Biol. Chem. 235, 3177 (1960). F. Goldberger and C. B. Anfinsen, Biochemistry 1, 401 (1962). C. Merigan, W. J. Dreyer, and A. Berger, Biochim. Biophys. Aeta 62, 122
(1962). H. A. Lindley, Nature 178, 647 (1956). toM . A. 1%artery and 1%. D. Cole, Bioehem. Biophys. Res. Commun. I0, 467 (1963).
218
Ct,EiVteE OF PEPTIDE CHAINS
[24]
(this volume [33]). A polypeptide prepared in this manner will undergo cleavage at arginine, lysine, and the modified cysteine residues; if the polypeptide is treated first with cyanate, cleavage is restricted to arginine and cysteine residues only. --?ys c~,s ~ CI'~ cH. i I \1,m CH~
--?ys -c~sH
c~ /
Materials and Apparatus Trypsin, 3 times crystallized, salt-free, and lyophilized (available from Worthington Biochemical Corporation, Freehold, New Jersey); automatic titrator, type TTT1, and chart recorder 11 (available from Radiometer, Copenhagen, Denmark); glass titration vessel, fitted with jacket for circulating water at constant temperature, internal volume 20--30 ml, with a flat base on which a glass coated magnet rotates freely (good thermal contact between the reaction bottle and the titration vessel maintained by filling the air space between the two with distilled water, see Fig. 1); constant temperature water bath with circulating pump (available from Haake Werk, Karlsruhe-Durlaeh, West Germany). When digestions are performed at pH 8.5 or above, it may be necessary to pass a slow stream of nitrogen, freed of COs by passage through
f/.-Glass electrode(coupledto pH meter) Flexiblecapillary from microsyringeH20~t~!
Glosscoated stirringbar
\
~
.-~,,.. Waterat constanttemperature Reactionbottle
Rotatingmagnet Fie. 1. Titration vessel for performing enzymic digestion under controlled conditions [A. M. Crestfield,Anal. Chem. 28~ 117 (1956)]. 11C. F. Jacobsen, J. I_~onis, K. Linderstrg~m-Lang,and M. Ottesen, in "Methods of Biochemical Analysis" (D. Glick, ed.), Vol. IV, p. 359. Wiley (Interscience), New York, 1955.
[24]
USE OF PROTEOLYTIC ENZYMES
219
KOH solution, over the top of the titration vessel,~2 and contamination of the reaction mixture by NH~ or C02 is minimized. Procedure Treatment o/ Trypsin to Reduce Contamination by Chymotrypsin. A typical degree of chymotryptic activity exhibited by crystalline trypsin is that which would arise from about 0.1-0.5% contamination by chymotrypsin. 1~ It has been reported that this activity, assessed by measurement of hydrolysis of acetyltyrosine ethyl ester, 14 can be eliminated by treating the enzyme with a specific inhibitor of chymotrypsin, diphenylcarbamyl chloride15 or N-tosylphenylalanine chloromethyl ketone~ (this volume [26]) without seriously impairing the trypsin activity. A simple procedure employing the former reagent is described below. Trypsin (58 mg) is dissolved in 1.6 ml 0.05 M Tris-chloride buffer at pH 7.6. To this solution is added 0.2 ml diphenylcarbamyl chloride solution (5.2 mg, Distillation Products Industries, recrystallized from ethanol, in 25 ml isopropanol). The slightly turbid solution is maintained at 25 ° for 10 minutes and is centrifuged. The supernatant solution is assayed for trypsin activity. This procedure involves a reaction between a 1.4 X 10-s M solution of trypsin and approximately one tenth the concentration of diphenylcarbamyl chloride. According to kinetic studies, ~5 chymotrypsin present in quantities less than 5% of the trypsin is completely inactivated. While the practice of adding a chymotrypsin inhibitor to trypsin is recommended, trypsin treated in this manner still exhibits some chymotryptic activity on a polypeptide substrate. ~ The level of the ehymotryptic activity, however, is considerably reduced. Trypsin has been reported to possess a higher degree of stability than chymotrypsin when exposed to dilute acid, and solutions of trypsin at 4 ° in 0.001 N hydrochloric acid maintain enzyme activity over a period of several days. It appears advantageous therefore to dissolve the crystalline enzyme in 0.001 N HC1 a few hours before use. Trypsin possesses the highest fundamental specificity of all known proteolytic enzymes. This feature, coupled with the knowledge that the specificity can be redirected, commends the use of trypsin as the principal enzyme for the initial fragmentation of a protein.
A. M. Crestfield, Anal. Chem. 28, 117 (1956). l~j. D. Young and F. H. Carpenter, J. Biol. Chem. 236, 743 (1961). 14G. W. Schwert, H. Neurath, S. Kanffman, and J. E. Snoke, J. Biol. Chem. 172, 221 (1948). ~SB. F. Erlanger and W. Cohen, J. Am. Chem. Soc. 85, 348 (1963). 1,G. Schoellman and E. Shaw, Biochemistry 9., 252 (1963). 17K. Takahashi, J. Biol. Chem. 240, 4117 (1965).
220
CLEAVAGE OF PEPTIDE CHAIN'S
[24]
To illustrate alternative procedures in the use of trypsin, four examples are presented. (1) Digestion per]ormed in a pH-stat--hydrolysis o] the B-chain o] human hemoglobin: is The fl-chain is dissolved in water to provide a concentration of 1%, and the solution at 30 ° is adjusted to pH "9.0 by addition of 0.1 N sodium hydroxide. Vigorous stirring is employed to keep the protein, insoluble above pH 7.0, as a fine suspension. A portion of a 1% solution of trypsin in 0.001 N hydrochloric acid is added to give a trypsin concentration of 0.005%, and the solution is maintained automatically at pH 9.0. The protein suspension clarifies after approximately 30 minutes. The course of the reaction is monitored by automatic recording of alkali uptake. After 4 hours, when the reaction slows, a second portion of trypsin solution is added to give a total enzyme concentration of 0.01~, and the hydrolysis is allowed to continue for 16 hours until the base addition almost ceases. The reaction mixture is acidified to pH 4.5 with glacial acetic acid and is lyophilized. (2) Digestion per]ormed in a huller solution--hydrolysis o] oxidized ribonuclease by trypsin: 2 The reaction is carried out at 25 ° in 0.2M sodium phosphate at pH 7.0. The buffer is prepared from 8.28 g NaH2PO4"H20 and 19.88 g Na2HP04 (anhydrous salt) dissolved in 1 liter. The pH of the solution remains constant throughout the hydrolysis. The substrate concentration is 0.50%, and separate digestions are performed at enzyme concentrations of 0.0025% and 0.025%. The course of the hydrolysis is monitored by ninhydrin analysis 89 of portions of the reaction mixture (Fig. 2). The digestions are arrested by adjusting the solutions to pH 2.2 by addition ol~2 N hydrochloric acid. I00 A o,.,
~o~60 ._~ ~. 40 ~ .1:::: n," C
Time (hours)
Fro. 2, Rates of hydrolysis of oxidized ribonuclease (0.50% solution of protein at pH 7.0, 25°), according to Hirs [C. H. W. Hirs, S. Moore, and W. H. Stein, J. Biol. Chem. 219, 623 (1956)]. Curve A: trypsin concentration 0.025%. Curve B: trypsin concentration 0.0025%. '" G. Guidotti, R. J. Hill, and W. Konigsberg, J. Biol. Chem. 237, 2184 (1962). wS. Moore and W. H. Stein, J. Biol. Chem. 211, 907 (1954).
[24]
use OF PROTEOL~rlC ENZYMES
221
(3) Digestion in the presence o] 2 M uma--hydrolysis o] streptocvccal proteinase: 2° The reduced carboxymethylated protein (150 rag) is dissolved in 0.1 M T r i s - 8 M urea buffer, pH 8.0. The clear solution is diluted with 3 volumes of the Tris buffer containing 0.1% thiodiglycol, which reduces the urea concentration to 2 M; the concentration of protein is 1%. The protein precipitates as a fine suspension in 2 M urea solution. Trypsin (0.75 mg in 0.1 ml 0.001 N HC1) is added and the suspension clears after a few minutes. After 4 hours at 25 ° another 0.75 mg trypsin is added, and hydrolysis is allowed to proceed at 25 ° for 16 hours. (4) Digestions per]ormed with trypsin supported in a columnP 1 Although not yet in widespread use, this method potentially offers several advantages. The enzyme column allows precise regulation of the extent of hydrolysis of a substrate by varying the rate of flow of substrate solution through the resin, and by varying the concentrations of substrate and enzyme and the height of the column. Thus the first products to be formed by limited cleavage of a polypeptide substrate can be rapidly isolated by elution from the column. Full details of the preparation of a water-insoluble form of trypsin bound to a resin are presented below. POLYTYROSYL TRYPSIN. This derivative is prepared from trypsin by the procedure of Glazer, Bar-Eli, and Katchalski. 22 A polymer containing 9.6% enrichment of tyrosine exhibits 70-75% of the enzyme activity of the original trypsin. When polytyrosyl trypsin is coupled to a diazotized copolymer of DL-phenylalanine and leucine (1:2), the resulting water-insoluble trypsin derivative retains 15-30% of the activity of crystalline trypsin. COPOLYMER OF p-AMIN0-DL-PHENYLALANINE AND L-LEUCINE. 21 p-NCarbobenzoxyamino-a-N-carboxy-DL-phenylalanine anhydride (3.5 g) and N-carboxy-L-leucine anhydride (1.5 g) are copolymerized in anhydrous dioxane (100 ml) with triethylamine (0.1 ml) as the initiator. The reaction mixture is stored for 3 days at room temperature, and finally is heated under reflux for 1 hour. The product precipitates on addition of water (300 ml) to the cooled solution. Removal of the carbobenzoxy groups is effected by treating the dried copolymer (3.5 g) with anhydrous hydrogen bromide in glacial acetic acid (30 ml) for ] hour at room temperature. The copolymer hydrobromide is precipitated from solution in acetic acid by addition of ether (500 ml). The precipitate is washed repeatedly with ether to remove benzyl bromide, and is dried in vacuo over NaOH and over H2SO4 ; yield, 2.5 g. DIAZONIUM SALT OF COPOLYMER OF DL-PHENYLALANINE AND L-LEUCINE.
a T . Y. Liu, W. H. Stein, S. Moore, and S. D. Elliott, J. Biol. Chem. 240, 1143 (1965). a A. Bar-Eli and E. Katchalski, J. Biol. Chem. 238, 1960 (1963). ~A. N. Glazer, A. Bar-Eli, and E. Katchalski, J. Biol. Chem. 237, 1832 (1962).
222
CLEAVAGE OF PEPTIDE CHAINS
[24]
Diazotization is carried out as follows: 0.5 M NAN02 (0.6 ml) is added dropwise to an ice-cooled solution of copolymer (100 mg) in 0.5 N HC1 (3 ml). After 1 ~ hours at 4 ° a 10% sodium acetate solution (10 ml) is added, and the pH of the reaction mixture is adjusted to 7.5 with 2 N NaOH. The light brown polydiazonium salt which separates is centrifuged and washed 3-4 times with 15-ml portions of cold 10% sodium acetate. The wet diazonium salt (dry weight, 70 rag) is used as a waterinsoluble carrier in the coupling experiments with polytyrosyl trypsin. COUPLING OF POLYTYROSYL TRYPSIN TO THE COPOLYMER OF DL-PHENYLALANINE AND L-LEUCINE. A polytyrosyl trypsin solution containing 3.0 mg modified enzyme per ml 0.0025N HC1 is mixed with an ice-cooled suspension of the polydiazonium salt (100 mg) in 0.1 M phosphate buffer, pH 7.6 (6 ml). The suspension is stirred magnetically and the coupling reaction is allowed to proceed for 1½ hours at 4 °. The pH is adjusted to 6.8 and the reaction mixture is left for 20 hours at 4 °. The light brown product is isolated by centrifugation. Final purification is attained by washing with 0.0025 N HC1 until the washings show no proteolytic activity toward casein. PREPARATION OF THE TRYPSIN COLUMN.A trypsin column (0.6 X 3 cm) is prepared from a mixture of polytyrosyltrypsin coupled to a diazotized copolymer of p-amino-DL-phenylalanine and leucine (1:2) (6 mg resinbound polytyrosyl trypsin, corresponding in esteratic activity to 0.25 mg crystalline trypsin) with an inert polyvinyl resin, Geon 426 (0.5 g) in 0.0025N hydrochloric acid (10 ml), by pouring into a glass tube. The elutions are performed at 25 °. Before use the column is washed with a suitable buffer, and at the end of each experiment with 0.0025 N hydrochloric acid, which stabilizes the enzyme. The column is kept at 4 ° until needed for further use. U S E OF TRYPSIN COLUMN IN T H E DIGESTION OF OXIDIZED INSULIN.21 Oxidized insulin is dissolved in 0.1 M phosphate buffer, pH 7.6, and the insulin solution (5 mg/ml) is passed through the trypsin column (0.6 X 3 cm), prepared as above, at a flow rate of 1 ml/170 minutes. The effluent contains free alanine in 85% yield. Chymotrypsin Specificity. In synthetic substrates, chymotrypsin catalyzes the hydrolysis of the peptide bonds between the COOH group of tyrosine, phenylalanine, or tryptophan, and the NH2 group of the adjacent amino acid? ~ Knowledge of the specificity against large polypeptides has come from studies in which the enzyme has been used in determinations of
23M. Bergma~n and J. S. Fruton, J. Biol. Chem. 118, 405 (1937).
[24]
USE OF PROTEOLYTIC ENZYMES
223
amino acid sequence. 2.-2¢ Slow hydrolysis has been found to occur at the COOH group of numerous amino acids, including asparagine, glutamine, histidine, leucine, lysine, methionine, serine, and threonine---in addition to rapid cleavages at aromatic residues--but cleavage at these sites is usually significant only at a high concentration of enzyme or when prolonged digestion is employed. The rates of chymotryptic attack are influenced by the nature of the amino acid residue adjacent to the susceptible bond. Thus, cleavage at the COOH group of asparagine appears to be favored when this residue is adjacent in sequence to Lys, His, or Arg. The enzyme does not significantly attack bonds formed by the imino group of proline. The reactivity of a susceptible site is influenced, in addition, by the general environment along the polypeptide chain. For example, the peptide bond between a serine and an alanine residue at position 18 in ribonuclease is readily cleaved when the tryptic peptide containing residues 11-31 is digested by chymotrypsin~,2T 14 15 16 17 18~19 20 21 22 23 -Asp-Ser-Ser-Thr-Ser-Ala-Ala-Ser-Ser-SerOn the other hand, negligible cleavage occurs at this position when the S-peptide (residues 1-20) is treated with chymotrypsin. Although the intrinsic specificity of chymotrypsin is rather broad, some advantage may be gained by adding soybean trypsin inhibitor to the chymotrypsin.2s The inhibitor abolishes the activity of any trypsin that may contaminate the chymotrypsin without seriously impairing the activity of the latter. It should be emphasized again that enzymic cleavages rarely occur on an "all-or-none" basis. Therefore experimental conditionsmthat is, temperature, pH, time of hydrolysis, substrate concentration, and molar ratio of chymotrypsin to substrate--are critical to optimum use of the enzyme. In practice, chymotrypsin has been employed under a wide variety of experimental conditions in studies on the structure of proteins. Thus, incubation has been carried out at pH values 7.054 and 9.0, 56 with temperatures between 20 °59 and 60°, ~° with digestion for 2 hours 55 or 30 hours, 59 and with enzyme-to-substrate molar ratios between 1:40054 and C. H. W. Hirs, W. H. Stein, and S. Moore, J. Biol. Chem. 221, 151 (1956). R. E. Canfield and C. B. Anfinsen,J. Biol. Chem. 238, 2684 (1963). R. J. Hill and W. Konigsberg, J. Biol. Chem. 2,37, 3151 (1962). =7C. H. W. Hirs, J. Biol. Chem. 23,5, 625 (1960). B. Keil and F. $orm, "Structure and Activity of Enzymes" (T. W. Goodwin, J. I. l=[arris, and B. S. Hartley, eds.), 1st Symp. Federation European Biochem. Soc., London 1964, p. 37. Academic Press, New York, 1964. E. Margoliash, J. Biol. Chem. 237, 2161 (1962). T. Ooi, J. A. Rupley, and H. Scheraga, Biochemiztry 2~ 421 (1963).
224
CLEAVAGE OF PEPTIDE CHAINS
[24]
1:2021 Three examples are selected to illustrate the application of chymotrypsin. (1) Digestion ]or 2~ hours (at pH 7.0, 25 °, enzyme-to-substrate molar ratio 1:360)--hydrolysis o] per]ormic acid-oxidized ribonuclease by chymotrypsin: 2~ Oxidized ribonuclease (200 mg) is dissolved in 20 ml 0.2 M phosphate buffer at pH 7.0 and a solution of chymotrypsin (crystalline, salt-free, from Worthington Biochemical Corp.) in the same buffer is added, providing a substrate concer:tration of 1.0% and an enzyme concentration of 0.005%. The digestion is allowed to proceed at 25 ° for 24 hours; the course of the reaction is followed by direct ninhydrin analysis 19 of small portions of the reaction mixture. The reaction is terminated by addition of 1 N hydrochloric acid to pH 2.2. The peptide mixture is stored at --15 °. (2) Digestion ]or 2 hours (at pH 8.0, 37 °, enzyme-to-substrate molar ratio 1:90)--hydrolysis o] reduced carboxymethylated lysozyme by chymotrypsin: 25 The reduced carboxymethylated protein (1.0 g) is suspended in 100 ml distilled H20 at 37 °, and dilute NH~OH is added to pH 8.0. Chymotrypsin (20 mg) in a small volume of water is added, and the solution is maintained at pH 8.0 by further addition of NH40H. The suspension clears after 20 minutes, and the digestion is allowed to proceed for 2 hours. The solution is lyophilized. (3) Partial digestion o] a native protein--limited hydrolysis o] ribonuclease: 3° Digestions are performed with a Radiometer pH-stat, and progress of the reaction is followed by ninhydrin analysis, decrease in ribonuclease activity, and measurement of the volume of base required to maintain the pH. Reaction mixtures contain 10 mg per milliliter of ribonuelease, 0.01 M KC1 or CaC12, and 0.05-0.5 mg chymotrypsin (Sigma Chemical Corporation) per milliliter of solution. The pH is maintained at 6.5 and the temperature at 25-60 ° . In the presence of 0.01 M CaC12 and in the absence of substrate, chymotrypsin loses 50% of its activity at 60 ° in less than 5 minutes. In the absence of calcium, inactivation is more rapid. Several consecutive additions of enzyme are necessary to ensure a moderate degree of hydrolysis. Associated with the inactivation of chymotrypsin under these conditions is the development of turbidity which increases with time. The reaction is terminated by the addition of diisopropyl phosphorofluoridate to a concentration of 0.01 M, and by cooling to 0 ° ; the mixture is lyophilized. Pepsin In synthetic substrates, pepsin catalyzes the hydrolysis of the peptide bonds formed by the amino or carboxyl groups of phenylalanine, tyrosine, 81A. Light and E. L. Smith, J. Biol. Chem. 237, 2537 (1962).
[24]
USE OF PROTEOLYTIC ENZYMES
225
leucine, alanine, glutamic acid, cystine, and cysteine~2; the enzyme can split the bond on either side of these residues. In its action on proteins, pepsin has been found to catalyze the hydrolysis of peptide bonds formed by all amino acids except proline and isoleucine. The most susceptible sites, however, are those formed by the aromatic amino acids, by leucine, and to a lesser extent by alanine. As the sites and rates of attack on a polypeptide cannot be predicted with accuracy, the optimal reaction conditions for a particular application of pepsin are selected on the basis of exploratory experiments carried out on a small scale. A detailed analysis of the specificity of pepsin in its action on the ~- and fl-chains of hemoglobin has been provided by Konigsberg and HHill.83,s4 Digestion of certain peptides from the B-chain, with a low relative concentration of pepsin, resulted in complete hydrolysis of a limited number of peptide bonds within a few minutes, and only after much longer digestion did extensive cleavage take place at other sites: 8~ 105 120 NH2Leu-Leu-Gly-Asn-Val-Leu-Val-CySO3H-Val-Leu-Ala-His-His-Phe-Gly-Lys (0.002% pepsin 25°, pH 2, 4 min) 120 NH2Leu-Leu-Gly-Asn-Val-Leu + Val-CySOsH-Val-Leu-Ala-His-His-Phe-Gly-Lys The presence of a free a-amino group at a site potentially susceptible to pepsin appears to reduce the rate of attack. The peptide shown above is rapidly cleaved by pepsin at Leu-110, whereas the NH2-terminal Leu-105 is not affected, even on prolonged digestion. Similarly, the NI-I2-terminal lysine residue in ribonuclease is liberated by trypsin only to a very small extent under conditions where cleavage at 9 of the 10 internal lysine residues is complete; the remaining lysine is present in a Lys-Pro sequence. 2S Pepsin can also catalyze transpeptidation reactions, s5 If this process were to occur to a significant extent during use of the enzyme in determinations of amino acid sequence, confusing results would be obtained. However, in analytical studies so far reported, no evidence has been obtained that this reaction interferes during application of pepsin to the cleavage of polypeptides. It has been reported that, when pepsinogen is activated at pH 3.0 instead of at pH 2.0, transpeptidation activity is eliminated without alteration of hydrolytic activity, s6 =J. S. Fruton and M. Bergmann, J. Biol. Chem. 127, 627 (1939). ~sW. Konigsberg and R. J. Hill, J. Biol. Chem. 237, 3157 (1962). W. Konigsberg, J. Goldstein, and R. J. Hill, J. Biol. Chem. 238, 2028 (1963). H. Newmann, N. Sharon, and E. Katchalski, Bioehem. J. 73, 33 (1959). H. Newmann and N. Sharon, Bioehim. Biophys. Acta 41, 370 (1960).
226
CLEAVAGE OF PEPTIDE CHAINS
[24]
Three examples illustrating the use of pepsin are presented. (1) Digestion o] fl-chain o] human hemoglobin with pepsinP 4 The pepsin used is twice crystallized from ethanol (Worthington Biochemical Corporation). The enzyme is stored for no longer than 2 weeks as a 1% solution in pyridine acetate, pH 3.8, at --15 °, and is thawed immediately before use. Mono-S-carboxymethyl fl-ehain from hemoglobin (800 mg) is dissolved in 80 ml 0.01 N HC1, and the pH is adjusted to 2.0. A solution (0.8 ml) of 1 ~ pepsin in 0.2M pyridine acetate buffer, pH 4.2, is added and the mixture is kept at 25 ° for 1 hour. The reaction is stopped by addition of pyridine until the solution reaches pH 5, and the peptide mixture is frozen and lyophilized. In exploratory experiments, aliquots of the digest are adjusted to pH 5, and paper electrophoresis is used to assess the extent of hydrolysis.
(2) Digestion o] reduced carboxymethylated lysozyme with pepsinP 7 Reduced carboxymethylated lysozyme (550 mg) is dissolved in 50 ml 5 ~ aqueous formic acid. Pepsin (6.6 mg), dissolved in 2 ml 0.5 M NaCI, is added and the digestion allowed to proceed at 23 ° for 60 minutes. The solution is frozen and lyophilized. (3) Combined enzymatic hydrolysis o] diisopropylphosphoryl (DIP) trypsinP 8 A solution of 7.0 g DIP-trypsin in 700 ml water is adjusted to pH 2 with hydrochloric acid and the temperature is maintained at 37 °. To the solution is added slowly, with vigorous stirring, 216 mg pepsin (enzyme to substrate ratio----1:50), and the reaction mixture is maintained at pH 2.0 by addition of 0.4 M HC1 from an autotitrator. After 12 hours, peptic hydrolysis is terminated by addition of 1 M NaOH to pH 6.5. A solution of 72 mg trypsin in 12 ml water (enzyme to substrate ratio ~-- 1:100) is added, and the pH maintained at 6.5. After 1 hour, a second portion of trypsin (72 mg) in 12 ml water is added and the digestion is allowed to proceed for 4 hours. A solution of 72 mg chymotrypsin in 25 mt water (enzyme to substrate ratio = l:100) is added; after 7 hours, the digestion is arrested by addition of acetic acid to pH 4.0. The solution is filtered and lyophilized. The digestion with pepsin was performed under acidic conditions which reduced the incidence of disulfide interchange, as a problem discussed in this volume [53]. The resulting peptides retained the disulfide bonds present in the native protein. The subsequent digestions, performed at pH 6.5, served to pare the disulfide-containing peptides until each disulfide bond was present in relatively small peptides. After isolation of these 3~R. E. Canfield, J. Biol. Chem. 238, 2693 (1963). ~V. Holeysovsky, V. Tomasek, O. Miles, A. S. Danilova, and F. ~orm, Collection Czech. Chem. Commun. 30, 3936 (1965). nD. H. Spackman, W. H. Stein, and S. Moore, J. Biol. Chem. 23~ 648 (1960).
[24]
USE OF PROTEOLYTIC ENZYMES
227
peptides the disulfide bonds were cleaved in each, and the two derived peptides were identified. The positions of the disulfide bonds could then be assigned by comparison with the known positions of the cystine residues in the overall amino acid sequence. Papain
An enzyme that possesses a low order of specificity may be expected to produce a large number of fragmen~ when it acts on a long polypeptide. In using such an enzyme, advantage may be obtained by employing a low concentration and a short incubation period, which will reduce the multiplicity of products. Papain exhibits a wide specificity in its action both on small synthetic peptides and on large polypeptides. 4° This property makes the enzyme suitable for the further degradation of peptides obtained by the action of trypsin or chymotrypsin; the smaller fragments produced may be more tractable for determination of amino acid sequence. Peptide bonds formed by the carboxyl groups of arginine and lysine residues, provided these amino acids are not NH2-terminal, appear to be highly susceptible to papain. In addition, cleavage occurs readily at the C 0 0 H group of histidine, and also at glycine, glutamic acid, glutamine, leucine, and tyrosine. The rates of cleavage, however, show considerable variation; ~1 papain can therefore be used to produce overlapping fragments. This is illustrated in the cleavage of an octapeptide from ribonuclease2 The 11 18 Pyr-Hts-MeSO~-Asp-Ser-Ser-Thr -Ser
Pyr-His-MeSO~-Asp-Ser,
Asp-Ser-Ser-Thr-Ser
l (74% yield) Pyr-His-MeSO2 (81% yield)
Asp-Ser + Ser-Thr-Ser (7% yield)
(23% yield)
susceptibility to attack of a particular bond may be influenced by the ease with which other bonds in the immediate vicinity undergo hydrolysis. In the example illustrated above, the rapid prior cleavage which occurs between methionine sulfone and aspartic acid may reduce the rate of subsequent cleavage expected at His-12. M. Dixon and E. C. Webb, "Enzymes," 1st Ed., p. 251. Longmans, Green, London, 1958. J. R. Kimmel and E. L. Smith, Advan. Enzymol. 19, 267 (1957).
228
CLEAVAGE OF PEPTIDE CHAINS
[24]
Some differences in specificity have been observed in experiments in which digestions were performed at different pH values. An octapeptide from the ~-chain of hemoglobin, Val-His-Leu-Thr-Pro-Glu-Glu-Lys, on digestion with papain at pH 4.25 undergoes hydrolysis at either side of leucine, whereas at pH 6 cleavage occurs additionally at the a-carboxyl groups of the two glutamic acid residues23 The ability to redirect specificity can thus be exploited to provide different fragments, which m a y give useful information on amino acid sequence. The experimental use of papain involves addition of cysteine or cyanide to the digestion mixture in order to activate the enzyme. It is important that the reducing agent employed should not affect the substrafe or interfere with subsequent fractionation procedures. Two examples of the use of papain are described. (1) Hydrolysis o] Pyr-His-2~eSO~-Asp-Ser-Ser-Thr-Ser ~ derived ]rom oxidized ribonuclease: To a solution of 7 #moles of the octapeptide in 20 ml H~0 are added 7.4 mg sodium ethylenediaminetetraacetate, 10 mg sodium cyanide, and 100 ~1 papain suspension containing 3 mg enzyme per milliliter (papain, twice crystallized, from Worthington Biochemical Corp.). The resulting clear solution is brought to pH 7 by dropwise addition of 1 ~ aqueous ammonia, and is maintained at 37 ° for 16 hours. To terminate the reaction, 1 ml 0.1 M formic acid is added and the solution is frozen. (2) The use o] a water-insolubl~ ]orm of papain 42 Preparation o] water-insoluble papain: A copolymer of L-leucine and p-amino-DL-phenylalanine is prepared according to Bar-Eli and Katehalski, ~1 and the copolymer is diazotized as described above (p. 222). Coupling with papain is carried out by adding crystalline papain (Worthington, twice crystallized, 80 rag) suspended in 0.03 M cysteine (2 ml) to a suspension of the diazotized copolymer in aqueous solution (40 ml) at pH 7.6, 0.075 M in phosphate, 0.005 M in cysteine, and 0.002 M in EDTA. Oxygen is removed by flushing with nitrogen, and the coupling is allowed to proceed in a tightly stoppered vessel for 20 hours at 4 ° with magnetic stirring. The water-insoluble product is isolated by centrifugation, and washed several times with a solution of 0.005 M cysteine containing 0.002 M EDTA at pH 6.0. The resulting water-insoluble papain (250 mg) is stored at 4 ° under an aqueous solution of composition similar to that of the wash solution. The water-insoluble enzyme is homogenized in a Potter homogenizer fitted with a Teflon pestle. Storage of the product at 4 ° for 1 month results in a 3 0 ~ loss of activity. The activity of the insoluble papain can be assessed by measurement a j. j. Cebra, D. Givol, H. I. Sflman, and E. Katchalski, J. Biol. Chem. 236, 1720 (1961).
[24]
use OF PROTEOLYTIC ENZYMES
229
of the rate of hydrolysis of a well-stirred solution of benzoylarginine ethyl ester (BAEE) at pH 6.0, 30 °, in an autotitrator. The asasy solution (5 ml) contains 0.025M BAEE, 0.002M EDTA, and 0.005M cysteine at pH 6.0. The equivalent amount of active enzyme is calculated from the initial rate of alkali consumption by comparison with soluble papain; in the presence of 100 ~g enzyme, alkali is consumed at an initial rate of 1.6 ~moles per minute. By this assay, 100 ~g water-insoluble papain is equivalent to 20 ~g crystalline enzyme. Limited hydrolysis o] a protein by insoluble papain: ~2 y-Globulin is dissolved in 0.9% NaC1 at a concentration of 10-20 mg protein per milliliter, and is dialyzed against 0.9% NaC1. After dialysis, EDTA is added to a final concentration of 0.002M and the solution is adjusted to pH 8.5. An amount of the water-insoluble papain preparation (10 mg), corresponding in weight to 1/20 that of the protein substrate (200 rag), is washed with 5 volumes of 0.9% NaC1, centrifuged to remove the supernatant solution, and resuspended in the above v-globulin solution (10-20 ml). The enzyme is kept in suspension by stirring, and the course of the proteolysis at pH 8.5, 20 °, is followed by titration with 0.1 N NaOH from an autotitrator. The water-insoluble enzyme is removed by filtration through Whatman No. 1 filter paper; the filtrate contains the required fragments of v-globulin. Subtilisin Subtilisin was first isolated from a culture of Bacillus subtilis, and was obtained in crystalline formA ~ A second crystalline enzyme has been isolated from a different strain of the same organism ~4 and is currently available commercially (Sigma Chemical Corporation) under the trade name, Nagarse. The two enzymes exhibit very similar specificity in their action on polypeptides, but the specificity is so wide that a preference for bonds involving particular amino acids cannot be stated. As in the use of papain, the broad specificity renders subtilisin suitable for degrading peptides of intermediate size, containing about six to twelve amino acids, to provide small fragments suitable for direct determination of sequence by the Edman method. A detailed account of the many sites in peptides that have proved susceptible to the action of subtilisin has recently been presented. '5 Three procedures employed in the use of subtilisin are described. A. V. GSntelberg and M. Ottesen, Compt. Rend. Tray. Lab. Carlsberg, Set. chim. 29, 36 (1954). "B. Hagihara, in "The Enzymes" (P. D. Boyer, It. Lardy, and K. Myrb~ck, eds.), 2nd Ed., Vol. 4, p. 193. Academic Press, New York, 1960. R. L. Hill, Advan. Protein Chem. 2@, 37 (1965).
230
CLEAVAGE OF PEPTIDE CHAINS
[24]
(1) Hydrolysis o] Asp-Ser-CySO~H-Glu-Gly-Gly-Asp-Ser-Gly-ProVal-CySOsH-Ser-Gly-Lys (O-Tr-1) by subtilisin: 4~ Subtilisin was obtained according to the published method. "s The peptide O-Tr-1 (0.2 ~mole, isolated from performic acid-oxidized trypsin) is dissolved in 0.2 ml 0.1 M ammonium acetate at pH 8.0. A portion (10 ~l) of a solution of subtilisin (2.8 mg dissolved in 0.1 ml 0.1 M ammonium acetate, pH 8.0) is added, and the solution is held at 25 ° for 12 hours. The solution is acidified to pH 3.6, and the resulting peptide mixture is resolved by paper ionophoresis.
(2) Hydrolysis o] a peptide o] 15 residues ]rom lysozyme by subtilisin: 37 Subtilisin was obtained in crystalline form (from the Nagarse Co., Osaka, Japan). The peptide (0.5 #mole) is dissolved in 0.5 ml 0.1 M ammonium bicarbonate, and 25 ~l 1% solution of subtilisin in the same buffer is added. The mixture is kept at 37 ° for 6 hours, and the reaction is stopped by acidification to pH 3.6. The points of attack by the enzyme are indicated by the arrows: Ileu-Val-Ser-Asp-4~ly-Asp-Gly-Met-Asn-Ala-Try-Val-Ala-Try-Arg
T T T "( T (3) Partial hydrolysis o] native ribonuclease by subtilisin27 Ribonuclease A (730 rag) is dissolved in 5 ml 0.1 M potassium chloride, and the solution is placed in the cell of a pH-stat. The temperature is lowered to 3 ° and the pH adjusted to 8.0 with 0.1 N sodium hydroxide. Subtilisin (1 mg dissolved in 0.2 ml water; prepared by the method of Guntelberg and Ottesen) is added, and the rate of addition of 0.1 N sodium hydroxide is recorded. After 3 hours the digestion is stopped by addition of 1 N HC1 to pH 3, which rapidly denatures the enzyme. Pronase
The enzyme is isolated from a strain of Streptomyces griseus ~s and exhibits the broadest specificity of any known proteolytic enzyme. In its action on synthetic substrates ~9,s° almost all dipeptides, tripeptides, and amino acid amides undergo hydrolysis at least in part on prolonged incubation. Where extensive proteolysis of a peptide or protein is required, pronase is extremely effective. Thus the enzyme is suitable for the liberation of free asparagine and glutamine under conditions that preserve the integrity of their amide groups. The extensive degradation produced by this enzyme resembles that produced by partial acid hydrolysis, except that high yields of the hydrolytic products are obtained. G. H. Dixon, D. L. Kauffman, and H. Neurath, J. Am. Chem. Soc. 233, 1373 (1958). " F . M. Richards and P. J. Vithayathil, J. Biol. Chem. 234, 1459 (1959). M. Nomoto and Y. Narahashi, J. Biochem. 46, 653 (1959). ~' M. Nomoto, Y. Narahashi, and M. Murakami, J. Biochem. 48, 593 (1960). M. Nomoto, Y. Narahashi, and M. Murakami, J. Bioehem. 48~ 906 (1960).
[2S]
CHROMATOGRAPHY
OF TRYPSIN A N D CIIYMOTRYPSIN
231
It should be noted that pronase differs from prolidase or prolinase. Pronase is a bacterial enzyme that derives its name from its striking ability to digest proteins, whereas prolinase and prolidase are alternative titles for an enzyme that possesses an ability to cleave peptide bonds involving proline sl, 52 (a purified preparation of pronase is available from Sigma Chemical Corporation). A procedure for the use of Streptomyce~ griseus protease (pronase) is described. The enzyme was prepared according to published procedures. 4g To 1 ml enzyme solution (0A4 mg protein nitrogen per ml) is added 1 ml 0.1 M substrate solution, and the reaction is allowed to proceed at pH 7.0-7.2, 40 ° , for 24 hours. The reaction mixture is acidified and the products are separated by paper chromatography. ~1W. Grassmann, H. Dyekerhoff, and O. yon Schoenbeck, Bet. 62, 1307 (1929). mN. C. Davie and E. L. Smith, J. Biol. Chem. 224, 261 (1957).
[ 2 5 ] C h r o m a t o g r a p h y of T r y p s i n t o R e m o v e C h y m o t r y p s i n , a n d of C h y m o t r y p s i n t o R e m o v e T r y p s i n
By M m E m L E
ROVERY
The procedure for the commercial preparation of bovine trypsin and chymotrypsin is the same as far as the crystallization of chymotrypsinogen. Thus it is not surprising to find traces of one enzyme in preparations of the other. Therefore, when peptide bonds of aromatic amino acid residues are cleaved during tryptic hydrolysis it seems reasonable to invoke chymotryptic impurities. Conversely, when peptide bonds of basic residues are split during chymotryptic hydrolysis, trypsin contamination may be implicated. However, as trypsin and chymotrypsin have many similarities in structure of the active centers, they m a y also have limited eross-specificities for certain peptide bonds. In practice, contamination is responsible for lack of specificityin most cases. Thus, it is important to use highly purified enzymes to obtain the most specific and reproducible hydrolysis possible. Techniques of varying efficiency have been used previously to inhibit chymotrypsin-like impurities in trypsin preparations: incubation in dilute acid, 1 denaturation in 8 M urea, 2 and treatment with small quantities of diisopropyl phosphorofluoridate? In recent years two additional I j. H. Northrop and M. Kunitz, in "Handbuch der biologischen Arbeitsmethoden" (E. Abderhalden, ed.), Vol. IV, Part 2, p. 2213. Urban & Schwarzenberg, Berlin, 19~6. 2j. I. Harris, Nature 177, 471 (1956). ~J. T. Potts, A. Berger, J. Cooke, and C. B. Anfmsen, J. Biol. Chem. 237, 1851 (1962).