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 at p H 5.6 to produce 1p M D-gluconic acid per minute at 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 at 4" for 15 minutes. Bfter centrifugation the ethyl alcohol concentration of the separated supernatant is raised to 61% and again held at 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 at 50" t o 60 ; otherwise the end point is ill defined because of the relatively slow hydrolysis of the lactone. Alternatively, at 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 at 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) at 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 at 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 at 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, L4MERICASCHEMICaL 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 L CHEY., . 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
