Isolation of Yeast Invertase by Paul Melius Auburn University Auburn, Alabama 36830
Sephadex Gel Chromatography
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A biochemistry laboratory experiment
W e have needed an experiment to illustrate ~ r o t e i nand enzvme isolation and ~urification. which w& suitable for our undergraduate diochemistj laboratory. Because of the large number of students we have, most classical procedures were undesirable especially those utilizing large amounts of organic solvents or long, difficult procedures. An easily available source of enzyme was extremely desirable. We were also interested in a simple, economic enzyme assay procedure. Yeast invertase (6-fructo furanosidase) (1, d) appears to be an ideal system. The procedure for enzyme assay using sucrose as the substrate and 3,5dinitrosalicylate to detect the glucose and fructose formed is simple and fast. The procedure does require a calorimeter to measure light absorption a t 540 nm. The published enzyme purification procedures are not suitable for a 3-hr laboratory period, and they are difficult for students to perform. This problem was solved by subjecting yeast extracts to gel filtration on short Sephadex gel columns. These columns were simply prepared from 50-ml g.s. burets of the type usually used in quantitative analysis laboratories. A glass wool plug support for the Sephadex gel was used. The Sephadex was treated according to the manufacturer's directions' and poured into the columns to about the 20-30 ml mark. The gel was rinsed with 0.10 M , pH 4.8, acetate buffer and only a ml of buffer left on top of Sephadex bed. In this way the student is able to begin the experiment immediately at the beginning of the laboratory period when the columns have been prepared before the lab period. We have used two yeast preparations, one we prepared from wet Baker's Yeast, which we air-dried and ground in a hall mill and the other, Fleischmann's Dried Yeast which can be obtained a t most grocery stores. The Fleischmann's Yeast has given the best results, because the columns can be reused without extensive cleanup. About 3 4 ml of 0.1 M NaHCOa was used per gram of dry yeast and a smooth mixture was prepared and incubated a t 3540°C for a day. The mixture was centrifuged at 15,000 X G and the supernatant reserved as the yeast extract. This work was supported by s. grant from the Division of Chemical Education4uPont Small Grants Program. Karen Franklin provided assistance in performing the enzyme assays and protein analysis. 'Pharmacia Fine Chemicals, Inc., Pisoataway, New Market, N J
*The DNS reagent was prepared by dissolving 10 g of 3,5dinitosalicylie acid (1802 Eastmm Kodak) in 400 ml of 1 N NaOH with warming and good stirring. This solution raas added to 500 ml of water containing 300 g of dissolved sodium potassium tartrate tetrahydrate (Baker Analyzed) with adequate stirring. The solution was then made up to a findvolume of 1 1.
When 2 4 ml of the yeast extract was placed on the column and allowed to percolate into the Sephadex followed by elution with the 0.1 M acetate buffer, it has required about 11/r21/2 hr to elute the enzyme and the hulk of protein which trails the enzyme. The trailing inert protein has a yellow color, so this allows the student to predict approximately when the enzyme has eluted from the column. We have recommended collection of 1-3 ml fractions, and the enzyme assays can be performed when the first 10 or so fractions have been collected. All the procedures can be carried out at room temperature. In this way the student obtains some definite results during the first laboratory period, and can test for any changes of enzyme activity with time and conditions of storage (040°C). In order to measure the amount of enzyme activity, an appropriate quantity of enzyme, 0.1 ml of 1:10, 1: 100-1:1000 dilution was incubated with 300 pmoles of sucrose and 0.05 M acetate buffer to give a pH of 4.8. After about 1-10 min incubation 1 ml of 3,5-dinitrosalicylate reagent2 (DNS) was added to stop the reaction and to react stoichiometrically with the glucose and fructose formed in the enzymatic hydrolysis reaction. The reaction mixture was diluted with about 20 ml of water and the absorbance measured in the Bausch and Lomh Spectronic Colorimeter at 540 nm. The protein analysis were performed by the Miller (3) procedure, as the enzyme-containing fractions obtained from the Sephadex column were very dilute in protein. Usually the students carry out the protein analysis during the following laboratory period, particularly when the flow rate from their column is unusually slow. Thus, with the enzyme activity and protein concentration measured for the initial yeast extract and the twymr ohrxinrd by tllc Scphndrx chn~mntography,t l ~ r swccilic activities can be cn1rul:rted and comw:md. Thr activity of the enzyme can be expressed as units which are equal to pmoles of sucrose hydrolyzed per minute by comparing the enzyme reactions to a standard curve of concentration of an equimolar solution of glucose plus fructose. The specificactivities are expressed as pmoles of sucrose hydrolyzed per minute (units) per milligram of protein (4). During subsequent laboratory periods our students have determined the effects of temperature, pH, and substrate coucentration on the enzyme activity. With this information they can calculate the maximum velocity and R'lichaelis-Menton constant for the enzyme catalyzed reaction. Neumann and Lampen (6) reported a purified yeast invertase with a specific activity of 2700-3000 measured at 30°C. They found the molecular weight to be 270,000 + 11,000 daltons Volume 48, Number 1 1, November 1971
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and the enzyme was eluted with the void volume when passed through Sephadex G-200, which conforms with the results we have obtained. This enzyme has been known since 1860 by Berthelot (6) and has played an interesting role in the development of modern enzyme chemistry. Thus the ease and simplicity of working with this enzyme system plus its historical and biological significance in fermentation of sucrose make it ideal for the undergraduate biochemistry laboratory.
766
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Journal of Chemical Educalion
Literature Cited (1) C L A R J. ~ . M., "Experimental Bioohsmiatry." W. H. Freemen and Co.,
SanFrancisco. 1964,p. 101.
8.. Fmmom, D. S., a m SOHRAMY, M., "Method. of Ensymology."Vol. 1, AcademioPresa. New York. 19611,p. 251.
(2) Hmmam,
(3) M m a e . G.L.,Anal. Chm..81,984 (1959).
(4) "Report of the Commission on Enaymea of the International Union of Bioohemistry," Pergamon Preaa. 1961,p. 10. . P. AN= Laar~sr. J. 0..Biochemislry, 6,468 (1867). (5) N a a ~ m N N. AND MTRBACR, K., "TheEnaymes," Academic (6) Bown, P. D.,Lamr, H.. Press, 1960,chapter 22,p. 379.
