Peter J. DeJong and Philip 1. Kumler Saginow Volley College University Center, Michigan 48710
I
Preparation of Immobilized Enzymes, and Determination of their pH Activity Profile An integrated organic-biology experiment
One of the problems facing those in charge of undergraduate organic laboratories is finding or devising experiments appropriate for classes that have a high percentage of biological science majors. Recently a number of experiments have appeared with these goals in mind (1-5). We have devised an experiment which fulfills a number of our objectives for experiments of this type. The described experiment: (a) combines organic, biological, and quantitative techniques, (b) is capable of completion in 2-3 3-hr laboratory periods, (c) does not require elahorate apparatus, (d) introduces the use of enzymatic techniques, and (e) shows the effect of an important parameter (pH) on an enzymatic hydrolysis. The experiment involves the preparation of an immohilized enzyme, hydrolysis of a protein using the immobilized enzyme, and a brief study of the effect of pH on the rate of enzymatic hydrolysis. One of the major drawbacks to the use of enzymes to effect chemical modifications is the relatively high cost involved in either purchasing or isolating enzymes. In addition, after the enzymes have been utilized their recovery presents formidahle prohlems. The use of immohilized enzymes to effect various chemical modifications is a technique which can overcome some of these difficulties. In this technique the enzyme is coupled (mechanically or chemically) to an insoluble support and utilized in this immobilized form which allows easy recovery and reuse. Such techniques seemed attractive as a means to introduce enzymatic methods into a typical undergraduate organic laboratory sequence. A variety of types of insoluble supports have been reported and include polyacrylamide beads, nylon net, glass beads, cellulose particles, and others (61. The immohilized enzyme may be added to an aqueous solution of its substrate and, after a sufficient time, the immobilized enzyme is easily removed from the reaction mixture and the products are more easily isolated. A slight variation of this technique is utilized in the current experiment; the immobilized enzyme is packed in a column, substrate is allowed to flow through the column, and the column effluent is monitored for the products of the enzymatic reaction. Discussion of the Integrated Experiment The specific experiment presented here makes use of the enzyme trypsin, which is a proteolytic enzyme of molecular weight -24,000, secreted by the pancreas and active in the small intestine of many animals. Weetal has previously reported the successful immohilization of trypsin on porous glass (7). Trypsin functions by breaking down large proteins to smaller peptides, cleaving the peptide hond adjacent to lysine or arginine residues.
The experiment involves four distinct parts: (a) glass is converted to a reactive form suitable for coupling to the enzyme, (h) immobilization of the enzyme (without loss of enzymatic activity) by chemically coupling it to the glass, (c) hydrolysis of a protein by allowing an aqueous solution of protein to slowly flow through a column packed with immohilized enzyme, and (d) determination of the extent of enzymatic hydrolysis by colorimetric procedures. Glass Preparation. Reactive OH groups on the surface of the glass are first linked to a silane which contains a reactive amino group (eqn. (1)). OEt @OH
I
+ Em-Si-CH,CH&H,NH, I
OEt
OEt
EtOH
11)
I OEt
0 @NH.
+ Cl-c
Enzyme Immobilization. The actual c o u ~ l i n zof the enzyme to the modified glass surface is performe2 by reacting the enzyme with the activated glass in a typical diazonium coupling reaction. The enzyme is now covalently linked, through an azo linkage, to the glass surface hut still maintains its enzymatic activity, i.e., it is immohilized (eqn. (5)).
OEt
OEt
/ Journal ot Chemical Education
I + @O-S~-CWH,CH,NH,@ -[ N -H ],
In our particular situation we have found it convenient to perform this step in advance of the actual laboratory usage; porous silica glass is refluxed in a 5% solution of y-aminopropyltriethoxysilane in toleune for 16 hr. The reactive amine group is converted to an amide by reaction with p-nitrohenzoyl chloride (eqn. (2)). The aromatic nitro group is reduced to an aromatic amine by reaction with sodium dithionite (eqn. (3)) and subsequently converted to a diazonium salt by reaction with NaNOzJHCI (eqn. (4)). The glass has now been modified such that it is ready to be coupled with the enzyme.
R
200
-
Protein Hydrolysis. T h e immobilized enzyme, after appropriate washing, is transferred to a small column consisting of a Pasteur pipet with a n analytical funnel secured t o the t o p of the pipet by a short length of rubber tubing. A protein (we have used casein) in a n appropriate buffer is then slowly passed through the column to allow t h e enzymatic hydrolysis t o occur. Determining the Extent of Hydrolysis. The effluent from the column (and a control containing the buffer-protein mixture which bas not been through the column) is treated with trichloroacetic acid to precipitate unhydrolyzed protein, and the supernatant is mixed with Folin reagent which detects tyrosine and other aromatic amino acid residues. The intensity of the developed blue color is proportional t o the concentration of these aromatic amino acids and can be estimated visually (or preferably determined calorimetrically) by comparison with a standard working curve relating tyrosine equivalents to absorbance a t 660 n m . In actual practice i t is desirable t o have each student (or group of students) perform the hydrolysis a t a number of p H values. Thus, they can easily construct a pH profile of enzymatic activity by plotting absorbance uersw pH. For the hydrolysis of casein using immobilized trypsin, typical student d a t a is summarized in the table. In general the student results agree quite well with literature data which cite the p H optimum for trypsin activity a t p H 8-9 (8). Comments. An average student may complete the above project in 6-9 h r of laboratory work. Directions by the instructor a s t o convenient "stopping places" are usually desirable a n d the samples should be stored in a refrigerator between laboratory periods. Although we have performed this experiment in its entirety (with t h e exception of the first step a s noted above) other instructors may wish to shorten this experiment by previously carrying out some of the early steps in the sequence. Experimental Details Glass Preparation A l-g sample of activated glass beads1 is gently refluxed for 1 hr in a solution containing 10 ml of chloroform, 100 mg pnitrohenzoyl chloride, and 50 mg triethylamine; magnetic stirring is desirable but not essential during this operation. After cooling, the supernatant is decanted and the beads washed (by deeantation) with three 20-ml portions of chloroform. The heads are dried at 80°C for 15 min and then refluxed for 1 hr in 10 ml of water containing 100 mg sodium dithionite; again, magnetic stirring is desirable. After cooling, the supernatant is decanted and the beads washed three times with 20-ml portions of distilled water. A 10-ml portion of 2 N HC1 is added to the heads and the flask cmled to -5°C in an ice bath. Sodium nitrite (250 mg) is added partionwise with mixing to the flask maintained in an ice bath; the flask is evacuated (water aspirator) for 30 min at 5'C. The supernatant is removed, the beads washed three times with 20-ml portions of cold water and excess nitrous acid then destroyed by the addition of 10 ml of a eold 1% aqueous solution of sulfamic
' Corning@ Controlled Pore Glass CPG-10-700 (Corning Glass Works) which has been refluxed far 16 hr in toluene containing 5% by weight of y-aminapropyltriethoxysilane (Union Carbide A-11001. Available commercially as Fisher Scientific Phenol Reagent. 3For t h experiment ~ a convenient work~ngcurve may be ohtained by preparing solutions of tyrosine varyng from 20-400 pg/ ml.
Tvrtical Student Data for the Hydrolysis of Casein
Stvdent A Student B
Student C
p g tymsine/m1 pH 9
pH 1
PH 8
31 1W
288
60
146
303 330 180
100
pH 10
234 280 130
acid. After 15 min the supernatant is decanted and the beads washed three times with 20-ml portions of cold distilled water. The diazotized glass is ready for immediate coupling to an appropriate enzyme. Enzyme Immobilization and Column Preparation To the diazotized glass sample is added 10 n j of phosphate buffer (0.05 M, p H 7.0) containing 100 mg of trypsm (Sigma Chemical Ca.). The mixture is maintained in an ice bath for 60 min, the solution decanted, and the beads washed three times with 20-ml pqrtions of eold distilled water. When the student is ready to prepare the column, the heads should be allowed to come to room temperature. A small plug of glass wool is inserted into a Pasteur pipet, the pipet is filled with phosphate buffer (0.05 M, pH 7.0) at roam temperature, and sufficient immobilized enzyme is transferred to the column to give a height of about 2 cm. Do not allow the column to run dry during this procedure (a small piece of rubber tubing and a pinch clamp may be used at the lower end of the pipet). Protein Hydrolysis The column prepared as above is rinsed with 25 ml of phosphate buffer (0.05 M, pH 7.0) and then with 25 ml of tris buffer (0.05 M, pH 7.0) containing 1% casein (Matheson, Coleman, and Bell). Attachment of a 45-mm analytical funnel to the tap of the column by a piece of rubber tubing facilitates this procedure. The column is drained to a level just below the top of the Pasteur pipet and 15 ml d a 1% casein solution in tris buffer (0.05 M.p H 7.0) is added to the column. The first 10 ml of effluent is discarded and the next 5 ml is added to a test tube containing 1 ml of 1.5 M aqueous trichloroacetic acid. A 15-ml sample of 1% casein solution in tris buffer (0.05 M, p H 8.0) is then added to the column, the first 10 ml of effluent discarded. and 5 ml added to a test tuhe cuntnininp. I ml oi trirhlnmnreric acid. The nhovr proerdure is npeatcd usin:: carran in I r i s butfer a t pH'> Y and 10. A control is prepared by adding 5 rnl of case)" m r r buffer ~ !pH 7 . 0 , ru trichloroacetic acid Determining Relative Extent of Hydrolysis Centrifuge each of the five tubes for 5 min to remove unhydrolyzed protein. Transfer 0.5 ml of supernatant from each of the tubes to clean test tubes. Mia equal volumes of Folin reagents dl and 82 181 and add 5 ml of the mixture to each tube. After 15 min add 0.5 ml of Folin reagent $3 (9A2 mix by inverting the tubes, and allow 15 min for full color development. The control tube is transferred to a Speetronic 20 cuvet (or any appropriate colorimeter) and used to set the meter at 100% transmittance on a Speetmnic 20.at 660 nm. Each of the other tubes is successivelv read at the same wavelength. The optimal pH for the enzymat& hydrolvsis can be determined .. eraohicallv bv olottine % transmit. l a n c e or absurhance versw pH. If desired the spertmphnromerrie data ma" be converted to equi\alentr tymsine hy comparison wirh asrandar? curve prov~ded11). the mstrurtor
.
Literature Cited I11 Mil1ar.J.F.. and COW..^. G., J . CHEM. EDUC..48.475119711. I21 Je~aifia,R.G.,andKrantz,A., J.CHEM. EDUC.. 18. 137 1197L). I31 C1omenf.G.E.. andPatter,R.,J. CHEM.EDUC.,48.695 11971). (4) Hopkins. H. P..andMather. J . H . . J . CHEM. EDUC.. 19. 126,1912). 15) Je~aitla.R.G.,andKranrz, A,, J. CHEM. EDUC.. 19.436 11972,. 16) Winprd, Jr., L. R., (Editor), "Enzyme Engineering." Wiloy-Interscience, York. 1912.00 177-217. (71 ~ e e r a ~ ; ~ . H . : S c i c n 166, r a . 61511969). 181 Barker. R.. "Organic Cheminfw of Biological Compounds." Prcnfice Hall. Englew w d Cliffs. NewJer~ey.1 9 7 1 . ~ 9 6 . (91 Damm. H. C.. Bench. P. ti.. Couri, D., and Goldwyn. A. .I.. "Methods and Refere n c e in Biochemistry and Biophysics," The W d d Publishing Co., Cleveland, 1 9 S 6 . p ~19-21.
Volume 51, Number 3, March 1974
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