L2. Isolation and partial characterization of human salivary amylase

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1 L2. Isolation and partial characterization of human salivary amylase Adapted from material developed by Martin Chapman at London South Bank University ( and protocols developed at Smith College, Northampton, MA and Bruce Reid at Kean University in Union, NJ Background Information Starch is a combination of two glucose polymers, amylose and amylopectin, in a typical ratio of 20:80. Amylose is an unbranched, single chain polymer of several hundred to several thousand glucose molecules linked by α-1,4-glycosidic bonds. Amylopectin is a branched polymer containing α-1,4-linked glucose chains joined with α-1,6 glucose branch points. The ratio of amylose to amylopectin and the degree of branching in the amylopectin varies in different plant species. Each amylopectin molecule contains a million or so residues, about 5% of which form the branch points. Single helix amylose behaves similarly to the cyclodextrins by possessing a relatively hydrophobic inner surface that holds a spiral of water molecules, which are relatively easily lost to be replaced by hydrophobic lipid or aroma molecules. It is also responsible for the characteristic binding of amylose to chains of charged iodine molecules (e.g. the polyiodides; chains of I 3 - and I 5 - forming structures such as I 9 3- and I Neutral I 2 molecules may give polyiodides in aqueous solution and there is no interaction with I 2 molecules except under strictly anhydrous conditions) where each turn of the helix holds about two iodine atoms and a blue color is produced due to donor-acceptor interaction between water and the electron deficient polyiodides. In this experiment you will use this property of iodine binding to analyze hydrolysis of three types of starch from tapioca, potato and maize by salivary amylase. The molecules are oriented radially in the starch granule and as the radius increases so does the number of branches required to fill up the space, with the consequent formation of concentric regions of alternating amorphous and crystalline structure. In the diagram below: A - shows the essential features of amylopectin. B - shows the organization of the amorphous and crystalline regions (or domains) of the structure generating the concentric layers that contribute to the growth rings that are visible by light microscopy. C - shows the orientation of the amylopectin molecules in a cross section of an idealized entire granule. D - shows the likely double helix structure taken up by neighboring chains and giving rise to the extensive degree of crystallinity in granule. There is some debate over the form of the crystalline structure but it appears most likely that it consists of parallel left-handed helices with six residues per turn. BL4820 Page 17 Biochem. Techniques

2 In order to make use of the carbon and energy stored in starch, hydrolytic enzymes, amylases, must first break down the polymer to smaller units that can be imported into cells and further modified. Several different enzymes are involved in breaking down starch. Amylopectinases, also known as debranching enzymes, hydrolyze the α-1,6-glucose linkages. Amylases, which hydrolyze the α-1,4 linkages are categorized into two classes according the extent of depolymerization. Bacterial α-amylases are typically liquifying enzymes, which cause clearing of starch gels by partial hydrolysis of the polymer. Products of degradation by this class are typically dextrins, short, α-1,6-linked glucose multimers. In contrast, fungal α-amylases are typically saccharifying enzymes. This class cleaves two glucose units (maltose) from the nonreducing end(s) of the polymer in a processive manner, resulting in complete destruction of the polysaccharide. Finally, amyloglucosidase or glucoamylase cleaves single glucose units from the nonreducing end(s). This enzyme can also cleave smaller dextrins and maltose into glucose. Many human secretions contain amylase isozymes (enzyme variants). The enzyme is found in saliva and pancreatic secretions, where it serves an obvious role in polysaccharide digestion. αamylase is also found in blood, sweat, tears, and genital mucus, possibly for anti-bacterial activity (2). α-amylase determination has been recognized as an important diagnostic tool for many BL4820 Page 18 Biochem. Techniques

3 years (4, 6, 7), because elevated levels of the enzyme are associated with liver and pancreatic disorders, as well as other diseases. Human saliva contains a mixture of isozymes that cleave α- 1,4 and the α-1,6 glucosidic linkages at a relative rate of 1:20, resulting in the splitting off of simple glucose units into the solution. L2.1 Purification of human salivary α-amylase SAFETY NOTE: Procedures using human tissue or body fluids have a potential risk of releasing infectious agents. A federally-defined ranking for laboratory practices defines four levels of potential risk, called Biosafety Levels, with Biosafety Level 1 (BSL-1) being the lowest risk and Biosafety Level 4 being the highest. Procedures using material obtained from apparently healthy individuals are rated Biosafety Level 2. The requirements for BSL-2 practice will be described in lab, and must be followed for any procedure using human-derived materials! In the early 1960s, purifying salivary α-amylase required a starting volume of 1-2 liters of saliva. In this lab, a microscale method for isolating a purified enzyme is described. Indeed, frequently only small samples of enzyme-containing material are available for protein purification, which has encouraged the development of microscale procedures. This method is based on the highly specific binding, but low catalytic activity, of the enzyme with glycogen at 4 ºC. Once the enzyme is bound to this substrate, the resulting complex is precipitated by the addition of ethanol (3). The enzyme, essentially free of other proteins, is thus obtained in a single purification step. Glycogen and its hydrolysis products still bound to the enzyme can be subsequently removed by allowing the solution to warm to room temperature. This step is required to study α-amylase activity or to permit crystallization of the enzyme. In your notebook, prepare a flowchart of the purification steps described below. Identify the fractions produced at each step and indicate which fractions will contain the amylase. For each step of the purification, collect an appropriate (>100 µl) sample to use later to assay for protein concentration and amylase activity (see table below). Store the well-labeled samples at 4 C. The points in the purification where you should save a sample to assay for amylase activity and total protein concentration are indicated in bold. 1. Collect saliva in two microcentrifuge tubes for a total volume of approx ml. Balance the tubes and centrifuge the saliva at 8,000 g for 5 minutes. 2. From each tube, transfer 1.0 ml of clear supernatant to a 2 ml microcentrifuge tube. Save the remaining saliva. All subsequent samples should be kept on ice and the operations carried out at 0-4 C. 3. Calculate the volume of 95% ethanol to add to 1.0 ml of supernatant to reach a final concentration of 40% ethanol. To each sample add this volume of ice-cold 95% ethanol, mixing after each drop. 4. After a final, thorough mixing, centrifuge the mixture at 10,000 g for 10 minutes. 5. Remove 1.0 ml of the supernatant from each tube, (containing about 50 Somogyi Units of amylase) and place in separate microcentrifuge tubes kept on ice. Allow the supernatants to cool to 0 º. Save the remaining solutions and the pellets. 6. To each tube of supernatant, add the following ice-cold reagents in the order shown, mixing after each addition; 0.06 ml 0.2 M phosphate buffer, ph ml glycogen reagent (1 mg glycogen) 0.08 ml 95% ethanol. BL4820 Page 19 Biochem. Techniques

4 7. Mix each suspension well and let stand on ice for 5 minutes before centrifuging at 5000 g for 3 minutes. 8. The precipitate, (which contains most of the amylase) should appear as a very small white patch on the side of the microcentrifuge tube. Carefully decant the supernatant. Save supernatant. 9. Combine the pellets in one tube by resuspending in 1.0 ml of the following ice-cold solution. (Mix well before use.) 0.75 ml H 2 O ml 0.2 M phosphate buffer, ph 8.0, 0.6 ml 95% ethanol 10. Centrifuge at 5000 g for 3 minutes. (Remember to balance the centrifuge.) 11. Carefully decant the supernatant. Save the supernatant. Resuspend the precipitate in 0.4 ml of 0.2 M phosphate buffer, ph 8.0. L2.2 α-amylase ACTIVITY ASSAY The procedure described is essentially that of Somogyi (5), who first quantified amylase activity by measuring the time required to hydrolyze starch, in a carefully standardized substrate solution. A simple assay to measure this time takes advantage of differently colored products generated by the reaction between iodine and the saccharides depending of their degree of degradation: α-amylase α-amylase Starch (polysaccharide) Oligosaccharides Glucose + Maltose (mono/disaccharides) (Blue) (Red) (Yellow) After mixing the amylase samples with a standardized starch solution, the reaction is monitored by removing portions of the mixture at timed intervals and adding these to aliquots of an iodine solution. As long as starch is present, a blue-purplish color will develop. As the incubation proceeds, the color will change from blue to blue-purple, to red-purple and then to reddish-brown. If the solution remains yellow, all the starch has been hydrolyzed to glucose and maltose and the assay must be repeated. The reaction is considered to have reached its endpoint when samples produce a reddish-brown color with iodine. The time required to reach the endpoint is a function of α-amylase activity expressed in Somogyi units (one Somogyi Unit is defined as the amount of amylase required to produce the equivalent of 1 mg of glucose in free aldehyde groups in 30 minutes at 40 C. (Somogyi Units/dL may be converted to International Units (µmol minute -1 L -1 ) by multiplying by 1.85.) The best estimate of amylase activity can be made using samples diluted to around 3-6 Somogyi Units/dL, so that the assay reaches the endpoint after 3-6 minutes. In the first part of the lab you will use a dilute, unfractionated saliva sample to test three different starches from tapioca, potato, and corn. This will acclimate you to the assay. When you have good timepoints and endpoints for those samples, choose one of the starch solutions to use as a substrate to evaluate your purification scheme. You will also need to perform protein assays on your fractions so budget your time/personnel appropriately. BL4820 Page 20 Biochem. Techniques

5 You should create data for the following table: Step Vol. (ml) [Protein] (mg/ml) Total Protein (mg) Amylase Activity (SU/mL) Total Activity (SU) Specific Activity (SU/mg) Overall Yield (%) Centrifuged saliva (#2) 100 Supnt+EtOH & centrifug.(#5) Supnt+glycog & centrifuge. (#8) Wash soln. (#11) Resuspd. ppt. (#12) Reagents Starch substrate - Soluble starch, 0.75 g/l in 20 mm Tris-phosphate buffer, 10 mm NaCl, 1 mm NaF, 0.1 mm NaN 3, ph 7.0. (SHAKE WELL BEFORE USE.) The solution is slightly opalescent, and on standing threads or white sediment may appear due to retrograded starch. This does not interfere with the assay. Store at room temperature (18-26 C). Do not refrigerate or freeze. The starch solution is suitable for use as long as this reagent and iodine solution produce blue color. Iodine solution - 0.2% iodine, containing 0.2 mm potassium iodide and buffer. Dilute as necessary to give a blue color when mixed with the starch. Store tightly capped at room temperature (18-26 C). Diluent solution - 0.5% NaCl solution Assay Procedure 1. Pipet 0.8 ml of iodine solution into each of 6 test tubes [100 x 75 mm]. Keep tubes at room temperature. 2. Preparation of the reaction mixture: Swab the cap of the starch substrate bottle with ethanol. Transfer 1.5 ml of starch solution to the remaining test tube. Place the test tube in a water bath at 37 C and allow incubate for 2 minutes. Add 0.1 ml of diluted saliva (Note 1) or undiluted fraction to the 1.5 ml of starch Mix and record the starting time. 3. After 2 minutes, remove 0.20 ml of saliva-starch mixture from the test tube, leaving the remainder in the heat block. Add this aliquot to one tube of iodine solution. Mix and record the time and color (Note 2). BL4820 Page 21 Biochem. Techniques

6 4. After another 1 or 2 minutes withdraw a second 0.20 ml aliquot from the saliva-starch mixture, and add the aliquot to a second tube of iodine solution. Mix the solution and record the time and color. 5. If the blue color persists, continue the incubation with sampling at regular intervals until the reddish-brown endpoint is observed. 6. Record the total elapsed time required to reach the endpoint. NOTES: 1. An assay of normal saliva diluted 100-fold will usually reach the endpoint in approximately 4 minutes. [Use a series dilution; 1 part saliva to 9 parts 0.5% NaCl solution, then 1 part diluted saliva to 9 parts 0.5% NaCl solution.] 2. If the amylase activity of a solution is high enough to go past the endpoint in <1 minute (starchiodine is yellow), the amylase concentration of the sample is too high to be assayed directly with this method. To overcome this, the solution should be diluted at least 5-fold and the assay repeated. 3. Occasionally, a tube will be seen to revert from reddish-brown to a purplish color upon standing, but this does not alter the recorded endpoint. 4. The method cannot be applied to colored and/or turbid fluids, such as whole blood, bile, or serum that is highly icteric or hyperlipemic. L3.3 Total Protein Determination To create a purification table you should also determine the total protein in the various fractions. Use the BCA test and provided BSA standards as in Lab 1. References 1. Anonymous Procedure 700 "Amylase: Visual, Colorimetric Determination in Serum or Urine". Sigma Chemical Company, Sigma Diagnostics. 2. Geerling, G., Honnicke, K., Schroder, C., Framme, C., Sieg, P., Lauer, I., Pagel, H., Kirschstein, M., Seyfarth, M., Marx, A.M., Laqua, H "Quality of salivary tears following autologous submandibular gland transplantation for severe dry eye." Graefes Arch. Clin. Exp. Ophthalmol. 238: Schramm, M. and Loyter, A "Purification of a -Amylase by Precipitation of Amylase- Glycogen Complexes" Meth. Enz. 9: Searcy, R.L., Wilding, P. and Berk, J.E "An appraisal of methods for serum amylase determination" Clin. Chim. Acta15: Somogyi, M "Modification of two methods for the assay of amylase." Clin. Chem Henry, R.J. and Chiamori, N "Study of the saccharogenic method for the determination of serum and urine amylase" Clin. Chem. 5: Young, D.S., Pestaner, L.C. and Gibberman, V "Effects of drugs on clinical laboratory tests" Clin. Chem. 21:10 BL4820 Page 22 Biochem. Techniques