Krisna Rungruangsak and Bhinyo Panijpan Department of Biochemistry Faculty of Science Mahidol University Rama VI Road Bangkok 4. Thailand
I The Mechanism of Action
I of salivary Amylase 1
I
A simple undergraduate experiment
a-Amylase in t h e diluted h u m a n saliva is used commonly for a n earlv-vear undergraduate experiment o n enzyme catalysis. I n this the enzymatic activity is followed b y t h e t i m e of non-appearance of the purplish color of the iodine-starch charge-transfer complex formed on a d d i n g iodine solution, at various times, to different aqueous d r o p s containing starch a n d saliva (1-3). T h e experiment is c h e a p and c a n h e d o n e easily. However, we wish to suggest an e x p e r i m e n t o n t h e kinetics of a-amylase digestion of s t a r c h which yields more information o n the activity and mechanism of the enzyme action t h a n t h e t v o e mentioned above. Moreover. t h e s t u d e n t will learn t o appreciate that, in a kinetic study, ieaction rates c a n h e obtained bv following either the disapvearance of the s u b s t r a t e o r thk appearance of the product 6r both. Since i t has been shown a b u n d a n t l y that a-amylase cleaves between t h e mostly a 1,4-linked glucopyranoside residues in starch in a r a n d o m m a n n e r and the final e n d product is predominantly maltose (4-7),i t seems a p p r o p r i a t e t h a t a simple underg r a d u a t e experiment should h e conducted to illustrate t h i s mechanism.'
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Experimental A 2% potato starch suspension in 0.05 M Tris buffer pH 6.9, boiled for exactly 1min and allowed to cool to roam temperature was used for substrate andlor serially diluted to give a standard curve of ahsorhance (A) a t 650 nm ( A s d versus percent starch concentration. T o start the enzymatic reaction for the preliminary test of enzymatic activity 0.25 ml of the Tris buffer and 0.25 ml of an appropriately diluted saliva solution were added in this order t o 2 ml of 2% starch resulting in 1.6% starch. An appropriately diluted saliva solution (saliva in 0.05 M Tris buffer pH 6.9) was one that when added to the starch would bring Assa down from 0.5-0.6 to about 0.3 in 2-3 min. A dilution of 1 in 5M)(saliva in buffer v:v) was often found satisfactory. The diluted saliva kept very well for several hours in ice. The dinitrosalieylie acid (DNS) reagent used to detect maltose was prepared by dissolving 100 mg of DNS in 20 ml of 2 N NaOH and 50 ml of water; 30 g of sadium-potassium tartrate was added and the solution was made up to 100 ml with water (8). This reagent kept well in the dark for weeks. The DNS reagent was added to 20 tubes (0.5 mlltube). Meanwhile the water in a bath was boiling, and Spectronic 20 spectrophotometer was zeroed a t 650 nm. 20 ml of 2% starch solution was placed in a 50 ml beaker, 2.5 ml of Tris buffer was added, 2.5 ml of the diluted saliva followed and the mixture was stirred vigorously. Immediately the timing was started and 3 5 ml of the incubation mixture was pipetted into a cuvette which was placed in the spectrophotameter and the absorbance was read as soon as possible (not exceeding 30 sec after mixing) and the reading was continued a t 15sec intervals thereafter for 5 mi". Simultaneously0.5 ml aliquots were taken from the beaker and added to the tuhescontaining DNS reagent a t 15 sec intervals also for 5 min (See Footnote). Mixing of the tube contents was ensured before the tubes were put into the boiling water and kept there for exactly 5 min each. 4 ml of water was then added to and mixed well with the content of each DNS assay tube. The spectro-
photometer was by now zerwd a t 470 nm and A m of the DNS assay tubes was read. A maltose standard curve was prepared in a similar way using serially diluted maltose solutions to react with DNS reagent (8,9). (If absolute concentrations are not required the standard curves far starch and maltose need not be prepared. However they are described in the Results andDiscussion below). It was established alsa that diluted saliva kept a t room temperature remained equally active for several hours. The buffer added to the starch solution before the addition of enzyme may contain ions for studies on the effects of ions if it is so desired.
Results and Discussion On addition of the appropriately diluted saliva solution to start the of the incubation mixenzyme-catalyzed breakdown of starch, &so ture, which measures the structural integrity of the starch substrate, decreased linearly for a t least 2 min (Fig. 1). But the maltose assay showed no significant change in A m for 1.5 min. A linear A m versus time plot was observed only after about 2 min a t which time the A m = 0.05. The linear rate of the production of the reducing disaccharide lasted for a t least 8 mi". The rapid linear drop of A m in the first 2 min of incubation and the relative invariance of the A m over the same period clearly indicate the internal and probably random cleavage of the starch substrate by the enzyme to yield smaller polysaccharide units during this time. The subsequent linear kinetics of reducing products released and the flattening of the Assa curve indicate a further breakdown of the smaller oolvsaccharides to vield mainlv maltose. , I t may IIP argued that a reaction mechanirm in which the starch rutstrate issystematically broken down from m e end or more to give maltme may still t,t proposed ns an alternative tu the internal and random cleavage mechanism deduced from the evidence above. However a closer look a t the expected maltose released a t 2 min should eliminate the systematic end cleavage mechanism. At this time point Asso indicates a drop of 40% scattering power of the original incubation mixture. This should correspond to a maltose concentration m last was in the order of 6 melml in the reaction mixture. if all the A .,.... t ~ ~ g i rice v e tomaltwe through s)\temattcend cleavage. But the D M array gave Ar70uiunly tIUSat thlr tinw,an absurbance \ d u e hardly above the invariant background reading, and thus should correspond to 0.1 mglml maltose a t the highest or more likely no maltose a t all. It is recognized that the preceding argument is based on a simpleminded interpretation of the results. But, in spite of this consideration, given the evidence shown in Figure 1, the internal cleavage mechanism is the more prohahle mechanism for a-amylase action on
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'l'hr fullowing poinu are north emphasizing. Two or more atudrnts ahodd furm a tmm. The eupmment ihould be rehear-rd mentally in advance and t hr rule of carh worker specified. Care must be exercised in the preparation of starch used as substrate. The saliva should be diluted and tested as suggested first, preferably by an instructor on apooled saliva sample tosave time, before the actualex~ e r i m e nin t order to eive results that will illustrate the mechanism 01 r l r a v ~ eThe . t m e intervals usrd tor mnlt
