Designing a coupled assay system for aspartate aminotransferase

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0307-4412(94)00116-2 Designing a Coupled Assay System for Aspartate Aminotransferase PHILLIP M ARNOLD and GRAHAM R PARSLOW Russell Grimwade School o f Biochemistry University o f Melbourne Parkville 3052 Australia

Introduction This exercise places responsibility on the students to plan their own experiment rather than following an established procedure. In this experiment, second-year students work in groups of four to design an assay for aspartate aminotransferase (AST) from basic principles. Instructors provide minimal guidance, as the emphasis is on students solving the problem themselves, if necessary by trial and error. The AST reaction can be coupled to a second, indicator reaction catalysed by malate dehydrogenase (MDH), which allows AST activity to be monitored spectrophotometrically by measuring the oxidation of N A D H at 340 nm. L-aspartate + o~-oxoglutarate ~ L-glutamate + oxaloacetate oxaioacetate + N A D H ~ malate + NAD + + H + Prior to coming to class, students are required to read about a variety of dehydrogenase reactions (including MDH), and are asked which reactions could be coupled to the AST reaction. To aid students in their planning, they are provided with a limited list of reagents, and some basic information on the properties of AST, including the KM values for substrates.

Materials Fresh rat liver was obtained from laboratory animals. All chemicals were of reagent grade. Malate dehydrogenase was obtained from Boehringer Mannheim. Students were given the following list of reagent stocks to choose from when designing the assay: not all of the substrates are actually necessary or appropriate for assaying AST.

Substrates 300 150 150 150 3 3

mM mM mM mM mM mM

(c) Conducting the assay Once the students have worked out what volumes of buffer, substrates and tissue extract to put in their assay, they perform the assay by adding all the components except for N A D H and one of the AST substrates to a cuvette. The cuvette is pre-incubated at the chosen temperature for 2 minutes, then the cuvette is placed in a spectrophotometer and N A D H added. After observing the absorbance for about 1 minute, the reaction is started by addition of the final substrate (usually et-oxoglutarate). The assays shown in Figures 1 and 2 were conducted as follows: (i) an assay mixture containing 1.6 ml of 100 mM phosphate, pH 7.4, 1.0 ml of 300 mM aspartate, 0.1 ml of tissue extract and 0.1 ml of MDH (25 IU ml -t) was incubated at 25°C for 2 minutes. (ii) 0.1 mi of 3 mM NADH was added, and the absorbance at 340 nm was monitored. (iii) 0.1 ml of 150 mM et-oxoglutarate was added to start the reaction. When using undiluted rat liver extract, a discernable amount of N A D H oxidation takes place prior to the addition of exogenous ot-oxoglutarate or aspartate (Figure 1). This activity is due to the presence of AST, MDH, aspartate, a-oxoglutarate

Enzyme

aspartate oxaloacetate ot-oxoglutarate malate NAD ÷ NADH

0,6

.

25 IU ml -~ malate dehydrogenase

Buffers 100 100 100 100

mM mM mM mM

Tris, pH 8.5 sodium phosphate, pH 7.5 sodium acetate, pH 5.5 sodium acetate, pH 4.0

Spectrophotometric measurements were taken using a PerkinElmer Lambda 2 spectrophotometer attached to an Epson LX40(1 printer.

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Methods (a) Enzyme extraction Each group of four students homogenised approximately 1 g of rat liver in 5 ml of ice-cold 0.9% NaCI, using a chilled mortar and pestle. Cell debris was removed by a low-speed spin in a microfuge, and the supernatant stored on ice. (b) Designing the assay The laboratory manual tells the students that AST has KM (et-oxoglutarate)= 0.2 mM, and KM(aspartate) = 5 mM. Students are instructed to aim for an aspartate concentration of the order of 5-20 times K M or greater, so that substrate concentrations are saturating, and to BIOCHEMICAL

use a 10-15 fold excess of aspartate over et-oxoglutarate in their assay mixture to help drive the reaction to the right (AST is competitively inhibited by high et-oxoglutarate concentrations). Students then have to choose a concentration of NADH which will give a starting absorbance of less than 1.0. They can calculate this concentration using the Beer-Lambert equation and the molar extinction coefficients of NAD ÷ and NADH at 340 nm. The next thing to consider is how many units of MDH activity need to be present (MDH must be present in considerable excess over AST so that the rate of N A D H oxidation is not limited by the availability of MDH). Students calculate what change in absorbance would be caused by 1 unit of AST in a total assay volume of 3 ml. After doing their sums, they find that 0.1 unit of activity causes a change in absorbance of 0.21 per minute. Students are told to aim for a rate of change in absorbance of 0.05-0.3 min -~, and to use 10-30 fold excess of MDH over AST in the assay. If the rate of absorbance change is too high, the tissue extract should be diluted prior to adding it to the assay.

EDUCATION

23(1) 1995

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Time (min.utes) Figure 1 A S T assay using undiluted rat liver extract. Rate of reaction is too high to be sure that MDH is present in excess over AST

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+ ~-0x0glutarate

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AST. Explain how pH could influence the enzymes involved in the assay system.

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Conclusion Students benefit from discussing an experimental problem amongst themselves, identifying problems and sharing ideas. At the completion of the class, students have developed an understanding the factors influencing the activity of an enzyme. This approach is more challenging to students than following a written protocol without any critical evaluation of the procedure, and many students expressed satisfaction at having devised something of their own which actually worked. The exercise can be completed within three hours, and requires only basic chemicals and standard laboratory equipment.

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0307-4412(94)00104-9

(minutes)

Figure 2 A S T assay usmg rat liver extract diluted 1/10 with O.9% NaCl

and oxaloacetate in the liver extract itself, When the liver extract is diluted by 1/10, this effect disappears (Figure 2). Students are asked to explain the activity observed with undiluted extract prior to addition of o~-oxoglutarate, and to design experiments to test their hypothesis. Students will find that reaction mixtures containing only buffer, liver extract and N A D H (ie no exogenous o~-oxoglutarate, aspartate or MDH) also give rise to N A D H oxidation, indicating that the undiluted tissue extract contains sufficient endogenous substrate to maintain weak activity.

A Rapid and Inexpensive Procedure for the Determination of Proteolytic Activity S CASTRO and A M B CANTERA Cdtedra de Bioquimica Facultad de Quimica Gral Flores 2124, CP 1157 and Laboratorio de Bioquimica lnstituto de Quimica Facultad de Ciencias Montevideo, Uruguay

Introduction (d) Varying the assay conditions

When students have devised a coupled assay procedure that works, they can investigate the effect of altering some of the variables in the system: (i) measure AST activity in the presence of 5 mM, 25 mM and 50 mM aspartate (ii) replace the buffer selected with each of the other three buffers provided and examine the effect of buffer pH on the measured AST activity (iii) examine the effect of temperature on the measured AST activity by preincubation of the assay mixture at 25°C, 30°C and 37°C.

Questions Each student is required to answer the following questions in their laboratory report (1) Describe the assay procedure you devised for assaying AST activity, giving details of what you put in the assay mixture. Include the initial concentration of each substrate in the assay mixture. (2) Work out how many p~moles of aspartate, ~-oxoglutarate and N A D H were present initially in your assay mixture (note that 1 mM = 1 p.mole per ml). How long would it take for all the N A D H in your assay system to be used up if the added tissue extract contained 0.5 IU of AST activity? (3) Using your absorbance versus time plots, calculate the observed AST activity in: (a) IU per ml of rat liver extract; ( b ) IU per g of rat liver tissue. Only NADH absorbs light at 340 nm with a molar extinction coefficient of 6220 M -t cm -I. (4) Why is it important to define the reaction conditions used to measure the activity of an enzyme? Briefly state the effect of buffer pH and reaction temperature on the measured activity of BIOCHEMICAL

EDUCATION

23(1) 1995

Work on enzymes involves, in most cases, partial purification and determination of activity. These are important for basic and applied research as well as for industrial production and teaching purposes. Proteases or proteinases are important enzymes from an economic viewpoint. They are used as coagulating agents in cheese manufacture, in the detergent industry, in the tenderizing of meat, in the chill-proofing of beer, the production of protein hydrolyzates, the modification of gluten, and the prevention of gelatinization of fish solubles. I Thus, determination of proteolytic activity is very important on the industrial scale and it is important for students to learn about it. Proteolytic enzymes are ubiquitous in biological tissues, fluids, and bacteria, but the ease of their isolation varies considerably: bacteria are an appropriate source of proteases in the laboratory. Bacillus subtilis is easily maintained and grown in different media, eg SP 2 and LB 3, and strains are available that produce plentiful exoproteases 4"5 which are used in industrial and commercial processes. Several methods are used to determine proteolytic activity, eg hydrolysis of casein, 6 hemoglobin7 or azocasein. 8 The casein hydrolysis method is not expensive, but casein is difficult to dissolve and the pH must be controlled, because the substrate precipitates at pH 4. The hemoglobin hydrolysis method is relatively expensive, and the substrate must be denatured and standard curves prepared. The azocasein hydrolysis method avoids this but again is relatively expensive. Our proposal here is to use skim milk as a substrate. The method is inexpensive, rapid, involves little equipment, and can be used in teaching courses as well as in industrial training courses. Essentially all that is required is skim milk and a water bath!