ACTION OF, 3-AMYLASE ON BRANCHED OLIGOSACCHARIDES*

Transcription

1 ACTON OF, 3-AMYLASE ON BRANCHED OLGOSACCHARDES* BY RUSS SUMMER AND DEXTER FRENCH (From the Department of Chemistry, owa State College, Ames, owa) (Received for publication, January 3, 1956) t has been shown by methylation studies that the /$amylase limit dextrin of amylopectin contains all the end-groups, and consequently all the branch points, found in the original amylopectin molecule (1). From this work it was concluded that the end-groups of the B-limit dextrin contained, on the average, 1.5 glucose units. More recently other workers have subjected the p-limit dextrin to the action of R-enzyme (2), and, from the nature of the resulting products, it was suggested that on the average the P-limit dextrin end-groups were 2.5 glucose units in length. n this laboratory a study of the action of,&amylase as it approaches an a-l,6 linkage has been carried out. Branched oligosaccharides of low molecular weight were prepared and subjected to the action of p-amylase. These model substrates are analogous to the branch points found in amylopectin or glycogen, and were somewhat easier to study than the natural molecules of high molecular weight. Experiments indicate that the rate of P-amylase action gradually falls as the a-l,6 linkage is approached, and that it might be impractical, if not impossible, to attain an absolute limit dextrin. Evidence is also presented that, on the average, the end-groups of the limit dextrin would never be any shorter than 2.5 glucose units. Apparently the glucose units on one chain have an effect on the ability of /3-amylase to act on the glucosidic linkages of the opposite chain. Materials and Methods Enzymes-Crystalline sweet potato p-amylase, kindly supplied by Dr. Schwimmer, was used. The Bacillus macerans amylase was prepared and purified according to a method described by Norberg (3), and the R-enzyme was prepared from broad beans (4). Paper Chromatography-Eaton and Dikeman No. 613 filter paper (8 X 8s inches) was used in the preparation of chromatograms by the ascending method. The solvent system consisted of three parts water, four parts pyridine, and six parts 1-butanol by volume (3 : 4: 6). The alkaline copper spray reagent was followed by the phosphomolybdic acid as described by Wild (5). * Journal Paper No. J-2836 of the owa Agricultural Experiment Station, Project Supported in part by a grant from the Corn ndustries Research Foundation. Presented at the 128th meeting of the American Chemical Society, Division of Biological Chemistry, September,

2 47 /~-A~LAsE ON OLGO~ACCHARDES After each chromatogram had been developed as indicated, a recording was made with a Photovolt densitometer, model No. 425, which was equipped with a scanning stage for semiautomatic plotting, a slit aperture 1 X 15 mm. being used. Since the intensity of the background of a chromatogram was extremely variable, the background was set at an optical density of 1. X 1-l in each case. Such recording is actually a plot of the distance from the point of application of the spot against the optical density, and, although it did not prove to be particularly helpful in a quantitative estimation of concentration, it was of value as a permanent nonfading record. Panose-Coupled Products-The panose-coupling reaction was used to prepare a mixture of branched oligosaccharides of varying degrees of polymerization. This mixture was then separated by mass paper chromatography into fractions having the same degree of polymerization. A reaction mixture containing 1 gm. of panose, 1 gm. of a-schardinger dextrin, 45 units of B. macerans amylase, and a grain of thymol was incubated at 4 for 5 days. At this time 15 more units of B. macerans amylase were added, and incubation was continued at 4 for 12 days. At the end of this period the solution was filtered and concentrated, and the sirup was added to the top of a carbon-celite column containing 5 gm. of adsorbent. The column was washed with 4 ml. of 1 per cent ethanol. A paper chromatogram indicated that large amounts of the products of low molecular weight, including panose, had been washed off the column, which was then washed with 3 ml. of 25 per cent ethanol and 35 ml. of 4 per cent ethanol. A paper chromatogram indicated that the compounds of interest were contained in these fractions. The solution was concentrated in vacuo,and applied to sixteen paper chromatograms bystreaking the sirup across the paper with a fine bore pipette. The chromatograms were given ten ascents in the 3 : 4 : 6 solvent system. Narrow strips were cut from the edges and the middle of each chromatogram and developed with the copper-reducing spray. n this way the positions of the compounds on the remaining filter paper were determined and cut out,. The various fractions were extracted, and the resulting solutions concentrated to small volumes in vacua. Each fraction was chromatographed a second time for further purification. The concentration of each fraction was determined by a measurement of the optical rotation. somaltose-coupled Products-A second series of branched oligosaccharides was prepared by using isomaltose as a cosubstrate in a B. macerans amylase-coupling reaction. A reaction mixture containing 1.2 gm. of isomaltose, 1.2 gm. of a-dextrin, 75 units of 8. macerans amylase, and a grain of thymol was incubated at 4 for 12 days. The methods of separation and purification of the isomaltose-coupled oligosaccharides were the

3 R. SUMMER AND D. FRENCH 471 same as those used for the panose-coupled products. Since the isomaltose products had a lower molecular weight, the various fractions were sufficiently separated on the first chromatograms after eight ascents in the 3 : 4: 6 solvent system. Branched Heptasaccharide-A branched heptasaccharide, which has been shown to be a single compound having the structure1 o (6), was isolated from a salivary amylase digest of waxy maize starch. This compound was separated on paper by the multiple ascent technique as previously described. Xweet Corn Glycogen-Sweet corn glycogen was purified according to a method described by Hassid and McCready (7), and the resulting product was washed with absolute ethanol and stored in a vacuum desiccator. Ma&oheptaose-The maltoheptaose was prepared in this laboratory by acid hydrolysis of P-Schardinger dextrin (8). Flavaxole Derivatives--n certain instances flavazole derivatives of the reducing sugars were prepared and separated on paper as described by Nordin (6). The solvent system commonly used in this separation was water-saturated methylethyl ketone. Reducing Determinations-All the determinations of reducing sugars were made by the Nelson calorimetric method (9). A standard curve was made for maltose, and reducing values were calculated as maltose. Results The action of,8-amylase on fractions Bs, Bs, and By from the panosecoupled mixture was followed qualitatively by paper chromatography. After extensive degradation by P-amylase, a chromatogram was made of each of the three digests, a densitometer recording of which is found in Fig. 1. t is evident that there was a resistant fraction in each of the three groups. n Table are presented the possible isomers for the three panosecoupled fractions. R-enzyme was used to degrade each of the resistant fractions, and paper chromatography to identify the products of degradation. From these experiments it was concluded that fractions BGa, Be,, and probably B,= were the P-amylase-resistant fractions in each case. Whether or not Bse or Bea might also be resistant was not definitely settled. Similar experiments were carried out on the isomaltose-coupled fractions. O- signifies a glucose unit with its reducing group.... O-O... signifies 2 glucose units bonded with an or-l,4 linkage.... signifies 2 glucose units bonded with an a-l,6 linkage.... b

4 472 &~MYLASE ON OLGOSACCHARDES After extensive degradation by,&amylase, resistant fractions Bq, Bs, and Be remained, which were separated from the degradation products by paper chromatography. The possible isomers for the various isomaltosecoupled fractions are found in Table. Flavazole derivatives were made from both the raw B, group and the,&amylase-resistant Bh fraction. The two Bq derivatives were chromatographed in water-saturated methylethyl ketone. The raw B, flavazole fraction indicated at least two components, with possibly a third, and the /?-amylase-resistant B* flavazole indicated only one component. Each of these flavazole fractions was oxidized for 3 minutes at room temperature with potassium periodate. The raw B, flavazole fraction produced flavazole aldehyde, while the p- amylase-resistant Bq flavazole did not. These experiments indicated that DSTANCE FROM ORGN FG. 1. Densitometer tracings of paper chromatograms of p-amylase digest of panose-coupled products. Symbols B7, Bg, B 5, and B, indicate positions of branched heptasaccharide, branched hexasaccharide, branched pentasaccharide, and panose. Gz indicates maltose. fraction Bda was probably the only resistant member of the B, group. BE fraction was digested with large amounts of salivary amylase, and the products were converted to flavazole derivatives. A chromatogram of the flavazole mixture indicated two Ba flavazoles. Therefore, the resistant Bg fraction contained both Bsa and Bsc. The p- amylase-resistant B6 fraction was digested with R-enzyme, and the products were identified by paper chromatography. The BG-resistant fraction contained only Bs,. The action of /?-amylase on the branched heptasaccharide obtained from salivary breakdown of amylopectin was followed by paper chromatography.,&amylase apparently had no action on this compound even after an extended degradation period. The rate of,&amylase act,ion on these various branched fractions, sweet corn glycogen and maltoheptaose, was determined by 6he Kelson method for reducing sugars, Each reaction mixture was made up in a 25 ml. volumetric flask containing 2 ml. of buffer (ph 4.82,.2 M acetate), a known amount of substrate, and a known amount of enzyme. The volumetric

5 R. SUMMER AND D. FRENCH 4i3 Panose-Coupled TABLE Oligosaccharides Eraction BE Fraction Be Fraction B? a o- o / b o- c d e o Fraction C)-O-O- O-O B4 o- o o-- o TABLE somaltose-coupled _- Fraction o- O-b Oligosaccharides Bs o o- o- o Fraction Bs o- o o- o-6 o-! o

6 474 P-AMYLASE ON OLGOSACCHARDES flask was then made up to the mark with distilled water, and the reaction mixture was incubated at 35. Duplicate 1 ml. aliquots were withdrawn periodically and analyzed for reducing power. The initial rates for the panose products are plotted in Fig. 2, for the isomaltose products in Fig. 3, and for glycogen and maltoheptaose in Fig. 4. ENZYME UNTS X TME MN. MG. SUBSTRATE FG. 2. Production of maltose by action of (3-amylase on panose-coupled products ENZYME UNTS X TME MN. MG. SUBSTRATE FG. 3 FG. 4 FG. 3. Production of maltose by action of 8-amylase on isomaltose-coupled products. FG. 4. Production of maltose by action of fi-amylase on maltoheptaose (GT) and sweet corn glycogen. DSCUSSON The qualitative study of the action of P-amylase on the various oligosaccharides, analogous to the branch points in amylopectin or glycogen, led to the conclusions summarized in Table. t is apparent that one chain exerts an influence on the other insofar as the action of P-amylase is concerned. The presence of 1 glucose unit on the opposite chain appears to make a resistant linkage out of one which was formerly non-resistant. This is not surprising, for the enzyme itself has a molecular weight of 152, f 1 per cent (1). On examination of a Fischer-Hirschfelder- Taylor model of the particular oligosaccharides involved, it seemed reason-

7 1:. SUMMER A1VD D. FREXCH 475 able that an extra glucose unit might well have prevented the approach or attachment of the bulky enzyme. t should be pointed out that the model substrates of low molecular TABLE Action of fi-amylase on Branched Oligosaccharides Non-resistant arrangements Resistant arrangements o-- --* O-O--o--c)- o- TABLE V Relative Rates on Glycogen, Maltoheptaose, and Branched Oligosaccharides / Substrate Relative rate o o- o- / d * The end-group arrangements listed in Table were arrived at by an examination of the action of fl-amylase on all the various branched oligosaccharides. n certain cases (arrangement d, non-resistant; arrangement c, resistant), these conclusions were not supported by direct evidence. However, the questionable end-groups do fit the over-all picture. Maltoheptaose*... Sweet corn glycogent.. Panose-coupled fraction BT.. L Bg.. Bg.. somaltose-coupled fraction Be L Bb B * For maltoheptaose, a conversion to moles of maltose per mole of substrate was made. t n the case of sweet corn glycogen, the results were converted to moles of maltose per exterior chain.

8 476 P-AMYLASE oiv OLGOSACCHARDES weight will not necessarily act the same way as the naturally occurring products of high molecular weight. n the model substrates, only two chains per molecule were available, but even in this relatively simple situation one chain appeared to affect the susceptibility to enzyme attack of the other. Amylopectin probably has 5 or more end-groups per molecule, depending upon the source (l), while glycogen might have as many as several thousand per molecule (11). This relatively large number of endgroups might exaggerate the effect noticed in the low molecular weight compounds. Furthermore, it would be helpful to have a model substrate of a doubly branched oligosaccharide a.vailable for study. t does seem reasonable, however, that, if there is an absolute limit to the action of /3- amylase, the end-groups found in this study would closely approximate the situation in the glycogen or amylopectin molecule. The relative rates of P-amylase on the various substrates, determined by measuring the slope of the curves in Figs. 2, 3, and 4 and based upon the rate of 1. for maltoheptaose, are found in Table V. They indicate that the rate slows considerably as it approaches a branch point. t seems reasonable that this change of rate might be even more drastic in the bulky natural molecules of glycogen and amylopectin. SUMMARY 1. The Bacillus macerans amylase-coupling reaction has been used to prepare two homologous series of singly branched oligosaccharides. Panose was used as a cosubstrate in one series, while isomaltose was used for the other. The products of these reactions were separated into isomeric fractions by multiple ascent paper chromatography. 2. The action of /?-amylase has been studied on these individual fractions, and the structural arrangements resistant to the action were ascertained. 3. The branched heptasaccharide from the salivary amylase hydrolysis of amylopectin was apparently completely resistant to P-amylase. 4. The rate of,!?-amylase on these various fractions, as well as on sweet corn glycogen and maltoheptaose, was determined. t was found that the rate slows considerably as the enzyme approaches the branch points, and the attainment of an absolute limit dextrin from a high molecular weight molecule such as amylopectin might be impractical, if not impossible. BBLOGRAPHY 1. Meyer, K. H., Experientia, 8,45 (1952). 2. Peat, S., Whelan, W. J., and Thomas, G. T., J. Chem. Sot., 4546 (1952). 3. Norberg, E., Thesis, owa State College (1949). 4. Hobson, P. N., Whelan, W. J., and Peat, S., J. Chem. Sot., 1451 (1951). 5. Wild, G. M., Thesis, owa State College (1953).

9 R. SUMMER AND D. FRENCH Nordin, P., Thesis, owa State College (1953). 7. Hassid, W. Z., and McCready, R. M., J. Am. Chem. Sot., 63, 1632 (1941). 8. French, D., Levine, M. L., and Pazur, J. H., J. Am. Chem. Sot., 71,356 (1949). 9. Nelson, N., J. Biol. Chews., 153,375 (1944). 1. Englard, S., and Singer, T. P., J. Biol. Chem., 187, 213 (195). 11. Greenwood, C. T., Advances in Curbohydrate Chews., 7,289 (1952).

10 ACTON OF β-amylase ON BRANCHED OLGOSACCHARDES Russ Summer and Dexter French J. Biol. Chem. 1956, 222: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites references, of which can be accessed free at tml#ref-list-1