Evan R. Kantrowitz and George Eirele
Harvard University Cambridge, Massachusetts 02138
TWO Spectrophotometric Experiments with Alpha-Chymotrypsin
Alpha-Chymotrypsin has been one of the most widely studied enzymes, both in solution and in the crystal (1-3). It is a maior comnonent of mammalian digestive svstems. where it breaks large protein molecules into small& frag: ments suitable for further digestion by other proteolytic enzymes. Chymotrypsin pre?erentiall$ cleaves peptide bonds involving the carhoxyl groups of the aromatic amino acids tyrosine, phenylalanine, and tryptophan. A polymer of 241 amino acids, it has a molecular weight of 25,000 daltons and only one active site (4). Chymotrypsin also catalyzes the hydrolyses of amides and esters of aromatic amino acids, though much more slowly than it cleaves peptide bonds (51. Such reactions ha;e proven useful in the elucidation of the catalytic mechanism (6). The generally accepted reaction scheme consists of a reversible association step followed by two catalytic steps. After the enzyme and suhstrate associate to form a reversible enzyme-substrate complex, they react to form a covalent acyl-enzyme complex with the release of the first product. In the last step this acyl-enzyme complex is hydrolyzed to yield free enzyme and the second product. This scheme is illustrated below E+S+ES-EP,-E+P,
Figure 1. Reaction of N-trans-cinnamoyl-imidazole with chymotrypsin, 0.05 M phosphate buffer. pH 8 . 0 (Praflauine) = 2 X M, (chymotrypsin) = (cinnamoyl-imidazale) = 10.' M. Reference cell contained (Proflavine) = 2 X 1 0 - 5 M .
+
p,
where E is the enzyme, S the suhstrate, ES the reversible enzyme-substrate complex, EP2 the acyl-enzyme, and PI and Pp are the first and second products, respectively. If the suhstrate is glutaryl-L-phenylalanine p-nitroanilide, the first product (PI) is p-nitroanilide, and the second nroduct (Pa)is elutawl-L-nhenvlalanine. ~ecause-bf ihym&yp&'s -general availability, it has become practical to conduct exueriments involvinn this enzyme i n the undergraduate iahoratory. Such experiments include simple enzyme assays (7), as well as advanced experiments involving the interpretation of complex kinetic data (8). A potential problem in the development of new experiments is that chymotrypsin reacts so rapidly with good substrates that only stopped-flow techniques can adequately monitor the course of the reaction (9). However, simple spectrophotometric methods can he employed with a number of poor substrates such as methyl cinnamate (lo), iodoleacryloyl imidazole (11). and Ntrans-cinnamoyl-imidazole(12). A central question in the study of enzymes is the source of their fantastic catalytic efficiency. Although this question has yet no definitive answer, one important approach is to investigate the mechanisms by which enzymes function. The first of the experiments described below allows the student to examine the mechanism of chymotrypsin. The experiment utilizes the acridine dye proflavine (I) to
binds per enzyme molecule. tive inhibition with specific lieved to he at or near the does not hind dye, hut the by using a poor substrate, imidazole (11)
Since the dye shows competisubstrates, the hinding is beactive site. The acyl-enzyme free enzyme does. Therefore, such as N-tram-cinnamoyl-
A
"monitor" some of the reactions of chvmotmnsin. Bernhard, et al. (13) have studied the interaction bf this dye with chymotrypsin, and have found that one dye molecule 410 / Journal of Chemical Education
in conjunction with proflavine the deacylation and acylation steps in the catalytic mechanism can be distinguished and studied independently. This is accomplished by using the enzyme-proflavine difference spectrum, which can differentiate between the free and the acyl-enzyme. Under appropriate conditions the enzyme-proflavine difference spectrum undergoes a hiphasic change upon the addition of suhstrate, as shown in Figure 1. Within a few seconds after the addition of the substrate, the intensity of the difference spectrum is reduced to practically zero because the suhstrate displaces proflavine from the active site as the acyl-enzyme intermediate is formed. Then, over the course of the next few minutes, depending on the pH, the difference spectrum returns to its original intensity as a consequence of the hydrolysis of the acyl-enzyme intermediate and the reformation of the enzyme-proflavine complex. Since chymotrypsin is a protein, it would hydrolyze itself if i t were synthesized in an active form. To avoid this self-destruction, the pancreas synthesizes and secretes the precursor protein chymotrypsinogen. In the intestine another proteolytic enzyme, trypsin, cleaves a specific peptide bond of the precursor, converting it into active chy-
motrypsin (14, 15). Depending on the conditions of activation, a number of different forms of chymotrypsin, all biologically active, can he prcduced (16). The second experiment illustrates specifically the activation of chymotrypsinogen by trypsin.1 This experiment is made possible by the fact that proflavine does not hind to chymotrypsinogen (17). Therefore, as trypsin produces more chymotrypsin, proflavine binding, which can he followed spectmphotometrically, becomes possible. Numerous additional experiments or projects are possible using proflavine in cbnjunction w i t h ~ c h y m o t ~ s i n . Since little equipment beyond a dual-beam spectrophotometer is required, these experiments are ideal for the undergraduate lahoratory. The complexity of the experiments can easily he adjusted to the preparation and interest of the students. Experimental
Materials and Methods a-Chymotrypsin(bovine), a-chymotrypsinogen A, trypsin, proflavine hemi-sulfate, N-trans-cinnamoyl-imidazole, tris(hydroxymetbyl)aminomethane,z and potassium phosphate were obtained from Sigma Chemical Company, St. Louis, Mo. 63178. All other chemicals were of reagent grade. Spectrophotometric measurements were made on a Varian Techtron 635 dual-beam spectrophotometer, although any dual-beam spectrophotometer is sufficient. Proflavine solutions were prepared in the huffer appropriate for each experiment. The concentration of proflavine was determined spectrophotometrically at 444 nm using a molar absorptivity of 3.34 X lo4M 1 cm-' (13). Concentrations of chymotrypsin, chymotrypsinogen, and trypsin were determined either by direct weighing of the solid or by ahsorbance measurements a t 280 nm, using an extinction coefficient of 2.06 cmZ/mg for hoth chymotrypsin (18) and chymotrypsinogen (19), and 1.44 cmz/mg for trvusin (20). l \ r k c k . li ! . A r n l c m ~ rPnn, hew Yorr 1960, p 91 117 (ilarcr A U . h r \ a , . 4 w d ? I ' < 5 & 1.1 IWX 1 I , W J . H'adr. R 1, nnd N e u r a l , H . Arm d r h r m B v , l h ) l . 59. 145 CIC;
.
14-,1.
(191 Sehwert,G. W., andKaufman. S., J B i a l . Cham.. 190.807(1951). (20) Davie, E. W.,sndNcumth,H.. J. B i d Chrm., 212,515(1955). (211 Edanger, B. F., Edel, F.,and Cmper. A. G., Arch. Biorhsm. Biophys.. 115. 206
11966). 1221 Bander,M.L., J A m e r . Chsm. Soe. 86,2582(19621. (231 MeClum. W.O..andEdelman.G.M..Bloebmisfry. 6,567(19671. 1241 Bender,M.L., andKsiser,E.T.,ilAmer Chem. Soe., &1,2556(19621. (251 Bender, M.L.,sndHamiltan, G.A.,J.Amar Chem Soc., 84.2570(1962), (261 Wi1non.L.R.. J.CHEM.EDUC..46.449i19691. (271 Hopkina, Jr., H P . , andMather, J.H.,J.CHEM.EDUC.. 49,126(19721. (28) Kantm%rlfz.E.R.,J.CHEM.EDUC.Sl.202 (1970.
Volume 52, Number 6. June 1975 / 413
