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Pacific Southwest Association of Chemistry Teachers
John Leo Abernethy' Long Beach and Fresno State Colleges and Marvin Kientz2 Fresno State College California
Papain as an Enzyme Catalyst in Undergraduate Organic Chemistry
Papain has become a well-known commodity on the American market in reccnt years because of its meat tenderizing activity. To the chemist, this is known to he a consequence of the ability of this enzyme to catalyze the hydrolysis of special types of amide linhges, particularly those involved in polypeptides of protein molecules. Also, papain catalyzes the hydrolysis of other peptide-like bonds and esters of amido acids. The origin of the enzyme is the papaya fruit, grown in Ceylon, Burma, South Africa, and America in states such as Hawaii and Florida. Until recently, Ceylon was the principal source of the enzyme, from the juice of the Carica-papaya. At present, the best papain is obtained from papayas grown in South Africa. I n the preparation of commercial and more highly purified crystalline papain, the latex from unripe papayas is first collected while the fruit is still on the tree by making gashes in the fruit and allowing the latex to flow into appropriate receptacles. It is then dried. The resultant solid is treated according to various intermediate procedures. After this, it is often ground with water and sand in a mortar, filtered, and fract,ionallycrystallized with the aid of ammonium sulfate and sodium chloride. Crystals are allowed to dry ( I , 2) and are suitable for activation. Although most enzymes are not a t all adaptable to undergraduate laboratory work in organic chemistry, papain is an outstanding exception. It is readily available, easily activated, inexpensive, and conveniently stored by refrigeration for long periods of time. I t catalyzes many reactions a t a rate convenient for undergraduate observational work. These reactions can be selected to take place between reactants that are inexpensive and available in quantity. Products can be ohtained in good yield, purified by simple means, and identified by melting point determinations. If the melting points are high, a melting point bath of 60% concentrated sulfuric acid and 40y0 potassium sulfate (3) gives students experience in surmounting the difficulty of bath decomposition. This mixture is good for temperatures above 365% A Fisher-Johns melt-
' Present address: Laboratory of Nuclear Medicine and R a d k tion Biology, University of California Medical Center, Los Angeles 24. Fresna County Heart Association undergraduatefellow, 195758; winner of California Heart Association undergraduate research award, 1957. 582
/
Journal o f Chemical Education
ing point block is often unavailable to undergraduate students, but is usually satisfactory for most temperatures encountered. The use of papain in catalyzing the formation of peptide-like linkages, rather than their hydrolysis, was first demonstrated by Max Bergmann and Heinz Fraenkel-Conrat in 1937 in a series of reactions between amido acids and aniline or phenylhydrazine (4). Since that time, a large number of related investigations has followed (5, 6, 7). Hippuric acid (benzoylglycine) was shown to give a substantial yield of either the anilide.or the phenylhydrazide. 0
11
0
I
CeH,CNHCHICOH (Hippurie Acid)
Papain, 40°C. + HzKC6HapH "4.G5.0 -A
(Aniline)
0
0
II
/I
C6HaCNHCH&NHCeHjf H1O (Hippuric Anilide)
and 0
I1
0
CeH,CNHCHs OH
+ HaNNHCaHaPapain, 40°C. d
(Hippuric Aeid) (Phen~lhydra~ine) pH C 4.Fr5.0 0
I1
0
I1
+
C6HsCNHCH1CNHKHCeH5 H?O (Hippuric Phenylbydrazide)
The insolubility of the product is the chief driving force of this reaction, which is actually reversible. It has been demonstrated that separations of racemic mixtures of amido acids can he accomplished with ease (4). Carbobenzoxy-DL-leucineyielded primarily the anilide of the L-isomer as an insoluble product while the D-isomer remained unreacted upon in the solution as a consequence of the configuration of papain and therefore its specificity. C6HIC&OCONHCHC0OH
+ H2NC6HsPapain, 40°C ___f
(CHdHCHn (Cttrboben~ox~.-DL-leueine)
pH G 4.5-5.0
+ H20
CsHsCH20CONHCHCONHCaHs
4
(CH& HCHn (Anilide of Carbobeneoxy-L-leucine)
Carhobenzoxy-DL-leucine is rather insoluble itself,
a hydrazide from hydrazine and methyl or ethyl benzoate (14). With hydrazine playing such an important role in moderu chemistry, it is available in quantity for such work. The usual precautions must be shown in its usage. Papain-catalyzed reactions with hydrazides point up the difference between the acidity of an acylated amino group and the basicity of an amino group not joined to an acyl radical, both of which occur in benzhydrazide. Replacement of hippuric acid with carbobenzoxyn~-alauine provides a means for a demonstrable resolution of a racemic mixture. Carbobenzoxy-DLalanine (mp 114-15') is commercially available3 but is somewhat costly. Directions are given for the preparation of carbobensoxy-DL-alanine (15) from benzyl chloroformate, which is sold commercially under the name of carbobenzoxy chloride.( One person in the class can substitute carbobenzoxy-DL-alaninefor hippuric acid. The entire class can observe the negative rotation of the resultant N"-benzoyl-A'O-carbobenzoxyL-alanylhydrazine in a pyridine solution.
so that in general practice other amido acids are sometimes preferably employed as substrates. The specificity of the enzyme was explained by Max Bergmann and Joseph S. Fruton (8) to result from a three-point contact between the amido acid substrate and the enzyme surface. The mechanism is largely explained as a contact with an energy-rich thiolactone structure a t the active site of papain (9, 10) and is in accord with the general principles of enzyme action (11, 12). Organic Laboratory Experiments
A rather large number of papain-catalyzed reactions can be selected for use in undergraduate organic laboratory work. Variations of the basic, amino-containing reactant involve less expensive reagents. Most of the amido acids are quite expensive, thus imposing rather drastic restrictions in their usage. The behavior of hippuric acid toward aniline (4), p-aminophenol (IS), pehnylhydrazine (41, p-anisidine (18), m-aminoacetophenone (IS), and other substituted anilines (18) provides a series of substrate combina-
C6H6CH20CONH[ a ] r = -38.31' (2 per cent in pyridine) I CHTC-CONHNHCOC& H20
CaHaCHIOCONH T CHsCCOOH
A
H
+
Precipitate, mp 20&5'
+ I
CH3-CCOOH
A
C6HsCHnOCONH
(Racemic Carbobenzoxy-DL-Alanine)
In Solution Unreaoted Upon
(nesolved Carbobenzoxy-wahine in solution: N"-Benaoyl-Nkarboheneoxy-lralanylhydrazine precipitated
Nomenclature is a bit tedious, but with patience the students soon manage this with ease. These reactions proceed very rapidly and are often largely completed within 12 hours. The optimum pH is about 4.0, in contrast with a pH of 5.0 for anilide formation. This has an added feature of involving an easy synthesis of
Activation of the enzyme by means of hydrogen sulfide is carried out by a very simple modification (18) of the original method of Grassmanu (16). Papain is dissolved in ice-cold water and the solution is placed in an ice bath while hydrogen sulfide is passed through the solution overnight. Precipitation of the enzyme with methanol or ethanol is followed by centrifugation a t 2000 rpm. The enzyme can then be used directly as a paste, without bothering to dry it. It must be stored under refrigeration to prevent molding. If prepared considerably beforehand, the paste can be dried one week over phosphorus pentoxide. It is usually necessary t o decant the alcohol which separates during the drying operation a t the end of each of the first two days. An amber or light tan, brittle, solid results which is crushed very lightly and stored in stoppered vials that are kept in a brown bottle with a screw cap in a refrigerator. I n our experience me have found that activated papain stored in this way has retained a very high degree of activity even after two years of storage. Two excellent sources of unactivated, dried, papain latex are the Wallerstein Laboratoriess and the Schwarz Laboratories.~ It should be emphasized that papain
a K and K Laboratories, Inc., 177-10 93rd Avenue, Jamaica 33, N.Y. ' Mann Research Laboratories, Inc., 136 Liberty Street, New York 6, N. Y. Wdlerstein Company, Inc., 180 Madison Avenue, New York 16, N. Y.
' Schware Laboratories, Inc., 230 Washington Street, Mount Vernon, N. Y. The senior author (J. L. A.) is indebted t o Dr. Heinz FraenkelConrat of the Virus Laboratory, University of California, Berkb ley 4, California, far this warning through private cammunication.
tions of easy application and moderate cost. The melting points of the products are: hippuric anilide, mp 212.5'; hippuric phenylhydrazide, mp 184.5'; hippuric p-hydroxyanilide, mp 2434"; hippuric pmethoxyanilide,. mp m-acetylanilide, . 217-18'; . hippuric - mp 229-300. Another such series of combinations would be the reactions between amido acids like hinnuric acid and benzhydrazide or other hydrazides (10jio yield N", Nodiacylhydrazines. n "
11
R-C-NH-NH; (Hydraeide)
n
n "
I Papain, 40°C CHsNHCCsHa d (Hippuric Acid) pH 4.0
+ HO
Volume 36, Number
1 I, November 1959 / 583
from some sources' cannot be satisfactorily activated for this work. A small amount of L-cysteine hydrochloride is used as a promotor for these reactions. Temperature control around 3%40°C should be possible in a crudely devised incubator consisting of an electric light bulb and appropriate packing in a box, in the event an incubator is not available. The enzyme should not be heated above 50°C, and even keeping it below 45'C is probably wise, during any of the procedures involving its preparation and use. This avoids any possibility of deactivation. Solutions can be buffered a t pH 4.5 to 5.0 with the use of sodium acetate and acetic acid and a t pH 4.0 by means of sodium lactate and lactic acid. The total salt and acid concentration used as the buffer should be about 0.5 M. Partial neutralization of lactic acid is a convenient way of getting this latter buffer mixture. Acknowledgment Research related to this paper was supported by the Sigma Xi Society and RESA, the Research Corporation, the Fresno County Heart Association, and the California Heart Association. Dr. Kendall Holmes, Dr. Robert D. Beech, and Mrs. Joyce Richardson of the Fresno County Heart Association, and Dr. John J. Sampson, Dr. Robert Maybury, and Miss Phyllis Hecker of the California Heart Association were particularly instrumental in securing these grants. Students in the undergraduate organic laboratories practiced on some of these reactions. Vice Chancellor William G . Young of the University of California, Los Angeles, suggested avenues of approach for securing research funds. Professor Charles D. Hurd of Northwestern University and Professor Carl Niemann of the California Institute of Technology gave certain advice in connection with the work.
584
/
Journal o f Chemical Education
Professor Ennis B. Womack of Fresno State College made available certain equipment. Assistance was also given by Mr. Calvin Johnson, Mr. Dennis Karle, Mr. Ronald Johnson, and Mr. Rodney Johnson. Literature Cited (1) KIMMEL,J . R., 515 (1954).
AND
SMITH,E. L., J . Biol. C h m . 207,
(2) Ibid., "Crystalline Papain," in "Biochemical Preparations," VESTLING, CARLS., editor&-chief, Volume 6, John Wiley &Sons, Inc., New York, 1958, p. 61. (3) MORTON,A. V., "L~boratory Teohniqne in Organic Chemistry," MeGrsw-Hill Book Co., Inc., New York, 1038. r. n 21 --(4) BERGMANN, M., A N D FRAENHEL-CONRAT, H., J . B i d . Chem., 119, 707 (1937). (5) BENNETT,E. L., A N D NIEMANN, C., J . Am. Chem. Soc.,
----.
72, 1789 (1950). (6) MILNE.H. B.. A N D STEVENS, C. M., J . Am. Chem. Soc., D O H ~ R T Y , D.
G.;
AND
POPENOE, JR., E. A., J.Biol. Chem.,
189, 447 (1951).
BERGMANN, M., AND FRUTON,J . S., Advances in Enaymology, 1, 63 (1941). SMITH,E. L., J . Biol. C h m . , 233, 1382 (1958). ABERNETHY, J. L., KIENTZ,M., JOHNSON, R.>A N D JOHNSON, R., J. Am. C h m . Soc., 81,3994, (1959). DIXON, M., AND WEBB, E. C., "Enzymes," Academia Press, Inc., New York, 1958, p. 63. REINER. , J. M.. "Behavior of Enevme Svstems." Bureess Publishing Co., Minneapolis, Minnesota, 1959, p. 9. ABERNETHY, J. L.,NAKAMURA, J., A N D COLLINS, BRO.M., Org. C h . , 23, 586 (1958). ~ ~ c a r ~ R o TW. r oJ., ~ "Reactions , of Organic Compounds," Longmans, Green, and Co., Inc., New Yark, 1936, p. ~~
199.(15) CARTER,H. E., FRANK,R. L.,
A N D JOHNSTON, R. W., "Organic Synthesis," Collective Volume 111, HORNING, E. C. ed,itol;in-chief, John Wiley & Sons, Inc., New York, 1955, p. 169. W., Biochem. Z., 279, 131 (1953). (16) GRASSMINN,
