246 Biochem. J. (1960) 76, 246 The Mechanism of Carbohydrase Action 5. ACTION OF HUMAN SALIVARY a-AMYLASE ON AMYLOPECTIN AND GLYCOGEN* By P. J. P. ROBERTSt AND W. J. W-HELANt Chemistry Department, University College of North Wales, Bangor (Received 11 January 1960) When human salivary oc-amylase acts on whole if any, esterified phosphorus (Schoch, 1953). It was pre- starch (amylose+amylopectin) there is produced pared from hand-sorted single-cross Tapicorn seed, (Bear a mixture of fermentable and unfermentable Hybrid Co., Decatur, Illinois, U.S.A.) as by Schoch (1957). sugars (Myrback, 1948). The fermentable sugars Buffers. These were 0-2 m-sodium acetate-acetic acid consist of maltose, maltotriose and/or glucose (see (pH 7.0) and 0-2M-sodiunm citrate-citric acid (pH 7-0). below). The non-fermentable sugars (a-limit Methods dextrins) are a mixture of higher oigosaccharides. Digests. Waxy-maize starch was moistened with ethanol Since the amylose component of starch, which and dissolved in 0-6N-sodium hydroxide by heating in a consists essentially only of 1:4-linked a-glucose boiling-water bath for 5 min. After cooling, the alkali was units, can be converted by the amylase completely neutralized to phenolphthalein either with sulphuric acid into maltose and maltotriose (Whelan & Roberts, [digests (1) and (2), see below] or hydrochloric acid [digest 1953) the a-limit dextrins must arise from the (3)]. Glycogen and the enzymes were dissolved in water. amylopectin component and may be assumed to After the substrate had dissolved, the buffer was added and contain the a-1:6-bonds which constitute the points then the enzyme. The digest was covered with toluene or of branching in the dendritic structure of this poly- toluene-nitrobenzene (3:1, v/v) and incubated at 35°. After incubation the enzyme was inactivated by heating in saccharide. The object of this investigation was to a boiling-water bath for 3 min. characterize the a-limit dextrins and thereby to Reducing power, developed during the digestions, was define the action of salivary a-amylase on amylo- measured with the Somogyi (1945 a) reagent, the time of pectin and the related polysaccharide glycogen. heating with the reagent being 60 min., in order to develop A preliminary account of this work has already full reducing power (Whelan, Bailey & Roberts, 1953). appeared (Whelan & Roberts, 1952). When R-enzyme was present the digest portion was treated with Somogyi's (1945b) deproteinizing reagents before measuring reducing power. A digest containing buffer and EXPERIMENTAL enzyme but no substrate was used in each case to estimate the reducing power of the non-substrate components. Materials Chromatography. Sugars were examined by chromato- Enzymes. oc-Amylase was prepared from human saliva graphy on Whatman no. 54 paper in one or more of the as by Whelan & Roberts (1953), by the method of Meyer, following solvents: water-saturated phenol (Partridge, Fischer, Staub & Bernfeld (1948). R-Enzyme was prepared 1948), pyridine-fusel oil-water (1:1:1, by vol.), propanol- from broad beans ('Prolific Longpod', Sutton and Sons, ethyl acetate-water (6:1:3, by vol.) and butanol-acetic Reading) as by Hobson, Whelan & Peat (1951). acid-water (4:1:5, by vol.). The sugar spots were revealed Substrates. Glycogen was prepared from the livers of with aniline hydrogen phthalate (Partridge, 1949), benzi- pregnant rabbits. The livers were boiled with water as soon dine-trichloroacetic acid (Bacon & Edelman, 1951) or as possible after removal from the animal. After cooling, silver nitrate-sodium hydroxide (Trevelyan, Procter & the liver was ground with acid-washed sand and re- Harrison, 1950). extracted with the same liquid by boiling for 20 min. Then Charcoal-Celite columns were prepared and used as by the solution was cooled, centrifuged, and the supernatant Whelan et al. (1953). All column dimensions given are solution filtered through paper. The glycogen was precipi- those of the adsorbent. Sugars were detected in the tated with ethanol (2 vol.) and washed with 66% (v/v) fractions by measurement of optical rotation in a 4 dm. ethanol, and ether. Finally it was purified by precipitation tube. All weights of products quoted were estimated by with acetic acid (Bell & Young, 1934; Schlamovitz, 1951). reducing power, where the product was glucose, or by acid The amylopectin was a gift from Dr T. J. Schoch. This hydrolysis to glucose, as by Pirt & Whelan (1951). The was waxy-maize starch, which is almost pure amylopectin weights are quoted in terms of combined glucose (CeHI005). in the sense of being free from amylose and contains little, Characterization of products. The products of enzyme action were characterized by one or more of the following * Part 4: Walker & Whelan (1957). methods: measurement of [a]D (in water) (4 dm. tube), t Present addresses. (P.J. P.R.): Imperial Chemical concentration being estimated by acid hydrolysis to Industries, Rocksavage Works, Weston, Runcorn, Cheshire; glucose; Rp and co-chromatography with authentic (W. J. W.): The Lister Institute of Preventive Medicine, material; formation of the ,-acetate (Whelan & Roberts, Chelsea Bridge Road, London, S.W. 1. 1953), measuring the [X]D (in chloroform) and m.p. (un- Vol. 76 oc-AMYLOLYSIS OF AMYLOPECTIN AND GLYCOGEN 247 corrected); degree of f-amylolysis (Whelan & Roberts, 24 hr. Thereafter an increase of about 0-5 % conversion/day 1954); measurement of degree of polymerization (DP), took place over 27 days and the reaction was terminated by based on the reducing power, relative to that of maltose inactivating the enzyme at 85-6 % conversion. The digest (Whelan et al. 1953). (960 ml., 9-56 g. of starch) was fractionated on charcoal- Celite (5 cm. x 118 cm.). Glucose (0-29 g., estimated by cz-Amyloly8i8 of amylopectin (1) optical rotation), maltose (4-64 g.) and maltotriose (1-88 g.) were obtained and their identities checked by measurement Waxy-maize starch (8-063 g., moisture content 13-1%, of Rp. The a-dextrins (2-60 g.) were eluted with 50% starch content 6-77 g. by acid hydrolysis) was incubated at ethanol. When maltotriose was incubated with the salivary 350 in 20 mm-sodium citrate buffer (pH 7-0, 1 1.) with a-amylase used in this experiment a slow increase in salivary a-amylase (1 g.). After 48 hr. the apparent per- reducing power took place, amounting to 10 % after59 days. centage conversion into maltose was 71-6 %. The enzyme The a-dextrins had DP 7-13 and were not further was inactivated and a portion of the digest (982 ml., attacked by cx-amylase, when treated under the same condi- 6-65 g. of starch) was fractionated on a charcoal-Celite tions as the original amylopectin. They were identical in column (4.2 cm. x 95 cm.). No monosaccharide was eluted composition with the first preparation, as revealed by with water (3-3 1.) but di- (2-42 g.), tri- (1.74 g.) and higher paper chromatography, and the two preparations were oligo-saccharide fractions (1.97 g.) were obtained, a combined. recovery of 92-4%. The di- and tri-saccharide fractions Enzymic debranching of amylopectin oc-limit dextrin8. The corresponded in Bp values to maltose and maltotriose combined cx-limit dextrins (see above, 3-47 g.) were incu- respectively. The disaccharide had DP 2-02, [a]D+ 1340, bated at 350 in 20 mM-sodium acetate buffer (pH 7-0, and its ,B-acetate m.p. 1590, [M]D+ 630. The trisaccharide 500 ml.) with R-enzyme (0-924 g.). After 140 and 168 hr. had DP 3-00, [aI]D + 1690, and its ,B-acetate m.p. 1360, the apparent conversion into maltose was constant at [M]D + 870. These are the properties of maltose, maltotriose 62-0%. The initial value was 28-3% and the ratio final: and their derivatives (Whelan & Roberts, 1953). initial reducing power was therefore 2-19:1. A portion of The higher oligosaccharides had DP 8-89. A small-scale the digest (8 ml.) was then incubated for 24 hr. with R- digestion with a-amylase showed an increase in reducing enzyme (50 mg.), when no further increase in reducing power. Accordingly, the dextrins (1-88 g.) were incubated power took place. Accordingly, the enzyme in the main at 350 in 8 mM-sodium citrate buffer (pH 7-0, 250 ml.) with digest was inactivated and a portion (465 ml., 3-22 g.) salivary a-amylase (0-881 g.). The reducing power of a fractionated on charcoal-Celite. No glucose was found. 0-5 ml. portion of digest rose from 3-90 ml. of 5 mN-thio- Maltose and maltotriose were obtained in pure form but the sulphate (45 min. heating) to 6-00, 6-14, 5-99 and 6-12 ml. last fractions containing the triose also contained malto- after 7, 9, 11 and 14 days respectively. The activity of the tetraose and similarly some of the tetraose was contami- enzyme remaining after this time was such that soluble nated with pentaose. The pentaose was followed by higher starch was rendered achromic after afewminutes incubation. oligosaccharides, mainly hexaose and heptaose, which were The enzyme was inactivated and a portion of the digest combined together. To estimate the yields of individual (234 ml., 1-76 g.) was fractioned on charcoal-Celite components of mixed fractions the mixtures were treated (4-0 cm. x 72 cm.). No monosaccharide was eluted with with ,B-amylase under conditions which would hydrolyse water (3-2 1.). Ethanol (7-5 %) eluted maltose (0-103 g.) maltotetraose and higher maltodextrins but not malto- but 15% ethanol (5-4 1.) did not elute any trisaccharide. triose (Whelan et al. 1953). The products were quanti- Ethanol (50 %) desorbed higher oligosaccharides (1-358 g.).