Determination of D-Glucose in Corn Sirups By Use of Glucose Dehydrogenase ROY L. WHISTLER, LESLIE EIOLGEI,
AND
J . w'. HYLIN
Department of Biochemistry, Purdue Vnirersity, hfayette, Znd. There has heen need for a quantitative method for the determination of D-glucose in the presence of other sugars, and particularly for the determination of Dglucose in corn sirups. The present work shows that glucose dehydrogenase can be conveniently employed for the direct stoichionietric determination of D-gliicose i n a variet) of corn sirups. 111 the presence of oxygen the enzyme oxidizes D-glucose to D-gluconolactone, which is determined quantitatively hy titration with rtandard alkali. The enzyme ma) be obtained from a comniercial preparation. It is also proposed that the method may be generally applied for the specific qnantitative determination of D-glucose i n other sugars.
D
ETE:RRIIS.ATIOS of the aniount of n-glucose in :t mixture of other reducing sugars ip a frequent laborat'ory problem. I n practice, the wrii wet-milling industry requires an accurate knowledge of the 1)-glucose content' of corn sirups. The SichertBleyer riducing sugtr method ( 5 , 1 7 ) is widely used, but' the deternrin:ition suffers from lack of spccificit'y for D-glucose, from being nonstoichionictric, and from its requirement that all reaction conditions lie rigorously rontrollc?d for reproducibility, Fernient,:ition nii%hods (4,16) provide :L fairly reliable quantitative measure of u-glucose in :L niixturo of sug:irs, but the procedure is not well suited to routine aiialj Glucow tlchytlrogcnasc, :in enzJ-mr of wide occurrence, is very specific in catalyzing the osid:Ltion of glucose in the presence of oxygw to procluce u-gluconolact,one :ind hydrogen peroxide ( 2 , 6, 8, 10, 13, 1 8 ) . IIcmce with t.he aid of t,his enzyme a quantitative deteiininntion of n-glucose can l i evolved ~ which is both specific :tnd fncilc ( 1 , 11). Keiliii and 1X:irtrec (10) report. the :ict'ion of a mold glucose dehydrogmase on about fifty sugnrs, including maltose, which is oxidized a t most tu thc extent of only 0.2%. Of other sugars test,ed only four-winannose, u-xylose, 6-niono-o-methyl-nglucose, and ~,ti-di-o-methyl-o-glucosc-are oxidized to an estent of approximately 157,. The reaction is followed by manometric measurement of t,he oxygen consunled. Palmer (16) uses a similar procedure for the determinxtion of D-glucose in the hydrolysis products of fructans. Glucose tlehydrogenase can be prepared from culture filtrates of Aspergilliis rt i g e r and PenicilZimL notalum. The preparations are variously tennetl penatin (Is),notxtin (6), penicillin B (18), and glucose osidnvc (10). .As Hent'ley and Seuberger (9)demonstrate that the enzyiiir has true tlehydrogenase activity, the designation glucosr clehydrogenaw is preferred. Usually the enzjme is acconil):uiietl bl- at least two other enzymes, mutarotase (12 ) and catalnsc. the former catdyzes the mutarotation of a-n-glucose to p-D-glucose while the lat,ter catalyzes the decomposition of hj-drogcn peroxide to water and oxygen. Thus the series of ratctions niay be written as follows:
Table I .
D-GIIICOS Contents of Some Comniercial Corn Sirnps D-G1uoo.m Content, dehydrogenase method0
25.8 26.3 37.4 38.4 38.7
6.8 9.8 19.2 I6 0 18.7 20.2 36.8 RE. 1 :36.9 48.1
40.3 5.5. 8 38 9
59,s
65,3
5.4
8ichPi.t-Rleyer
Paper chromatography
ine t hodd
B'o
9.0 li.5 18 5
18. i 20.0 33.5 36.0 40.0 46.5
19.6 41.8 41 1
:o" ;
Based on dry weight. b DE determined by method of Lane and Eynon (f4). Average of three determinations, results of which were within = 0 . 5 % of glucose found. d Average of a t least two determinations, which were within f 3 7 of one another. a Average of a t least eight determinations. which were within =l.5% of one another ( 1 9). a
I n this 1:iboratory the method has been adapted t.o the measurement of o-glucose in corn sirups. After addition of the enzyme to the sirup, the oxidation of D-glucose proceeds to complet,ion in 4 hours a t 30" and the resulting acid may be quant'itatively titrated a t 50" with standard alkali. The higher temperature of alkali titration facilitates opening of the lact'one ring and thereby produces a clear end point by direct titration. General application of the method is now feasible with the recent availability of commercial enzymes. Although the I:ztt,er enzyme preparations contain amylase and maltase, these int,erfering substances may be reduced t.0 negligible proportions. The r)-glucose contents of a number of corn sirups differing in estcnts of hydrolysis are sliomn in Table I and coinparison is made with the wglucose contents as determined by t.he Sichert-Bleycr method. There is fair agreement between the two methods, considering the errors that can arise in the Sichert-Bleyer determination ( 7 ) . .knalysis of t.he corn siruDs ixtuer chromatog- bv" auantitat'ive _ '/z o? raphj- (19) agrees with the v n l u ~ s obt:iinerl by using glucose dehydrogen:iw. catalase 1
a-n-glucopyranose
1
1.
mutarotase
8-u-glucopyranose I0
2
glucose n-glucono-&lactone dehydrogenase ~~
+
I + H,Oz
n-gluconic acid As is apparent, the reaction m a y be followed stoichiometrically nleasurelllents (11 ) or alkali tit,rat,ion ( 2 , 6) of the acid formed.
I,\.,,ither
%a
GlilCOSe
Dextrose Equivalent 6 17.1
.
ENZY>IE PREPARATION
Procedure A. A conimercial enzyme .wlution which contains approximately 27, of D-glucose is dialyzed in cellophane tubing against distilled r a t e r at 2" for 24 hours. (Deoxygenaw from Takamine Laboratory, Inc., Clifton, K. %J,, was used. The new enzyme Preparation, Glucatase, of Chas. 1'fi.w Co., Inc., Brooklyn 6,N. Y., may also be suitable, but because of' its newness, comp1et.eresults are not available in this laboratory. ). The dialyzate retains 99% of the original dehydrogenase activit,?,,
1215
ANALYTICAL CHEMISTRY
1216
but shows considerable catalase, maltase, and amylase activity. The last two enzymes are inactivated by holding the solution, buffered to pH 11 with glycine-sodium hydroxide (9),a t 2" for 15 hours. -4fter the pH has been adjusted to '7.0 with acetic acid, the solution (20 ml.) is mixed with 5 drops of saturated tannic acid solution and allowed to stand a t 2' for half an hour to produce a tannic acid complex (3, 6). This is centrifuged and washed once with water and twice with acetone to remove tannic acid. The residue is extracted with water (20 ml.) and the extract is clarified by centrifugation. The preparation shows 20% of the dehydrogenase activity of the original sample, and when allowed to act on standard D-glucose solutions brings about 100 =t270 conversion to D-gluconic acid in the presence of either maltose or starch dextrins. The enzyme solution had an activity of 4.0 units per ml. Unit activity is that amount of enzymic material that reacts with an excess of 0.1 Jf D-glucose solution a t p H 5.6 to produce 1p M D-gluconic acid per minute a t 30'. If either the alkali treatment or the tannic acid procedure is omitted, the product shows amylase and maltase activity. Procedure B. X dry, powdered enzyme preparation from the same commercial source is stirred with 10 portions (v./w.) of distilled water for 5 minutes and then centrifuged. The supernatant is treated with ethyl alcohol to a concentration of 54% by volume and held a t 4" for 15 minutes. Bfter centrifugation the ethyl alcohol concentration of the separated supernatant is raised to 61% and again held a t 4' for 15 minutes. The suspension is centrifuged and the precipitate dissolved in a volume of distilled water double that of the original extract. This solution is dialyzed against water containing 0.5% calcium acetate for 15 hours. Any insoluble matter formed is removed by centrifugation and discarded. The enzyme solution had an activity of 12.4 units per ml. Procedure A applied to the dry enzyme preparation does not yield a product free of amylases.
Table 11. Rates of Oxidation of Varying Amounts of D-Glucose Time, Hours 1 L
4 6 8 10
2
3
4
97 98 94.5 94 92
99 99 5 96 95 5 94.5
Oxidation, 82 82 79
io 65
94 94 90.5 89,s 87
R
Enzyme solution prepared in the above manner produces theoretical ( f 2 % ) oxidation of D-glucose in the presence or absence of maltose and dialyzed soluble starch. DETERMINATIOIV OF D-GLUCOSE
A solution ( 5 ml.) containing approximately 20 mg. of n-glucose is mixed with the enzyme solution ( 1 ml.) at 30' and oxygen is passed through a small orifice into the mixture for 4 hours. The pH changes from about 6.8 to 3.5. D-Gluconolactone produced in the reaction is determined by titration with 0.01 N sodium hydroxide solution (carbonate-free) using phen:lphthalein as indicator. Titration is conducted a t 50" t o 60 ; otherwise the end point is ill defined because of the relatively slow hydrolysis of the lactone. Alternatively, a t the end of enzyme action an excess of standard alkali may be added to saponify the lactone and excess alkali titrated with standard acid. For each new enzyme preparation a blank determination must be made on a solution containing enzyme only. The blank was usually 0.02 to 0.03 ml. of 0.01 -V sodium hydroxide solution. The rate a t which various concentrations of D-glucose in water ( 5 ml.) are oxidized by the above conditions using an enzyme solution (1 ml.) with an activity of 4 units per ml. is shown in Table 11. Thus concentrations of 2 to 4 mg. per ml. of D-glucose are optimum. The rate of oxidation of D-glucose (20-mg. samples as above) a t various temperatures is shown in Table 111. These results indicate that the optimum temperature for the reaction is in the neighborhood of 50'. For convenience the analyses reported here were performed a t room temperature (30'). I n the presence of buffer a t p H 5.6 (the optimum p H of the enzyme) the oxidation of D-glucose is greatly accelerated (6). Thus, if the reaction mixture is maintained a t pH 5.6 by inter-
Table 111. Variation in Rate of Oxidation of D - G l u c o s e with T e m p e r a t u r e Time, hlin. Temp.,
C.
30 40 50 55 60
30
60
65 77.5 83 91 69
83 92 94.5 92 74.5
90
120
150
180
94 96 97 93 71.5
96.5 97.5 98 93.5 75.5
97.5 98.5 98.9 93.8
98.5 99 99 94
Oxidation, %
mittent titration with sodium hydroxide solution, oxidation is complete in 2 hours a t 30'. The reaction may be conveniently performed with an automatic titrator. DETERMINATION OF D-GLUCOSE IN CORN SIRUP
Each sirup is diluted to a volume such that the D-glUCOSe concentration is approximately 0.4%. Aliquots ( 5 ml.) of the sirup solutions are mixed with enzyme solutions ( I ml.) and oxygen is bubbled through the mixtures a t 30' for 4 hours. The reaction mixtures are then titrated a t 50' to 60' with 0.01 N sodium hydroxide solution using phenolphthalein as indicator. Results are given in Table I. LITERATURE CITED
(1) Beck, W. S., Federation Proc., 11, 184 (1952). (2) Bentley, R., and Neuberger, A , , Biochem. J., 45, 584 (1949). (3) British Drug Houses, Ltd., and Skrimshire, G. E. H., Brit.
Patent 561,175 (1944). (4) Bryant, A. P., and Jones, R. C., Ind. Eng. Chem., 25,98 (1932). (5) Cantor, S.M., and Smith, R. J., Division of Sugar Chemistry and Technology, 100th AIeeting, L4MERICAS CHEMICaL soCIETY, Detroit, Mich., 1940. (6) Coulthard, C. E . , Rlichaelis, R., Short, W. F., Sykes, G., Skrimshire, G. E. H., Standfast, A. F. B.. Birkinshaw. J. H.. and Raistrick, H., A-nture, 150, 634 (1942); Biochem. J . , 39, 24 (1945). (7) Fetzer, W. R., A ~ A LCHEY., . 24, 1129 (1952). (8) Franke, W., and Deffner, AI., Ann., 541, 117 (1939). (9) International Critical Tables, Vol. I, p. 81, New York, LlcGrawHill Book Co., 1926. (10) Keilin, D., and Hartree, E. F., Biochem. J . , 42, 221 (1948); 50,331 (1952). (11) Ibid., 42,230 (1948). 112) Ibid.. 50.341 11952). (13j Kocholaty, I?., J . Bact., 44,143 (1942): 46,313 (1943); Science, 97,186 (1943). (14) Lane, J. H., and Eynon, L., J . Soc. Chem. Ind., 42, 32T, 143T, 463T (1923); 44, l5OT (1925); 46, 434T (1927); 50, 85T (1931) (15) McLachlan, T., Analyst, 53, 583 (1928). (16) Palmer, A , , Biochem. J . , 48,389 (1951). (17) Sichert, K., and Bleyer, B., Z . anal. Chem., 107, 328 (1936). (18) Van Bruggen, J. T., Reithel, F. J., Cain, C. K., Katzman, P. 9.,
.
Doisy, E. A . , Rluir, R. D., Roberts, E. C., Gaby, W. L., Homan, D. M., and Jones, L. R., J . Biot. Chem., 148, 365 (1943); 147,47 (1943). (19) Whistler, R. L., and Hickson, J. L., manuscript in prepa-
ration. RECEIVED for review December 26, 1952. Accepted M a y 29, 1953. Journal Paper KO.676 of the Purdue Agricultural Experiment Station,
Fluorometric Determination of Aluminum-Correction Attention has been called to an error in the historical part of the article, "Fluorometric Determination of Aluminum" [Goon, Petley, ?rlc?vIullen,and Wiberley, AKAL.CHEM.,25, 608 (1953)]. Two sentences beginning with line 3 are erroneous and should be replaced by the following: "Davydov and Devekki ( 1 ) using quercetin, an isomer of morin, developed a fluorescent method for the quantitative determination of aluminum." Actually Davydov and Devekki were unsuccessful in attempting to use pontachrome blue black R as a reagent for aluminum, and not being able to obtain morin used by White and Lowe, developed a method using its isomer, quercetin. EDWARD GOON
