PURIFICATION OF SALIVARY a -AMYLASE

I- BACKGROUND AND PURPOSE a-Amylase (a-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) is an enzyme that degrades starch, first to oligosaccharides and then in turn to maltose and glucose, by hydrolyzing  a -1,4-glucan bonds.  In digestion, the role of a -amylase is primarily the first reaction of this process, generating oligosaccharides that are then hydrolyzed by other enzymes.
 

	a-Amylase		a-Amylase
Starch	--->	Oligosaccharides	--->	Maltose + Glucose

In vitro, a-amylase is also able to hydrolyze the  a-1,4 linkages in glycogen, but has no activity on the  a-1,6 linkages responsible for the more highly branched structure of glycogen.  These branched structures also reduce the activity of a-amylase toward glycogen by limiting the accessibility of the target  a -1,4-glucan bonds

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The enzyme is found in saliva and pancreatic secretions, where it serves an obvious role in polysaccharide digestion.  More surprisingly,   a-amylase is also found in blood, sweat and tears, possibly for anti-bacterial activity (2).   a -Amylase determination has been recognized as an important diagnostic tool for many years (4, 6, 7), because elevated levels of the enzyme are associated with liver and pancreatic disorders, as well as other diseases.
 Enzyme Purification
In the early '60s, purifying salivary a-amylase required a starting volume of 1 - 2 liters of saliva.  That is a lot of spit!  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   a-amylase activity or to permit crystallization of the enzyme.
The data shown in Table I demonstrate the efficiency of the method for purification of  a-amylases from various sources.
 

Table 1: Specific Activity of  Amylase During Purification
	Spec. Activitya of Extract in  40% Ethanol	Spec. Activitya washed enzyme- glycogen complex	Spec. Activitya of pure enzyme b	Yield of enzyme (%) from glycogen ppt.
Pancreatin	45	1200	1260	95%
Human Saliva	500	2400	1960	75%
Rat parotid gland	800	2700	2500	95%

_____________________________________________________________________________ ^aSpecific activity is expressed in Somogyi Units/mg protein. "Somogyi Units" are defined below.
^bValues obtained from the literature
Enzyme 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:
 

	a-Amylase		a-Amylase
Starch (polysaccharide)	--->	Oligosaccharides	--->	Glucose + Maltose  (mono/disaccharides)
[Blue with iodine]		[Red with iodine]		[Yellow with iodine]

After mixing the amylase-containing 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 a-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.)
[Salivary Amylase] (Somogyi Units/dL)   =          Temperature  Factor           
                                                                           Endpoint time [min]
 The temperature factors for the incubation temperatures are:
 

Incubation Temperature (°C)	Temperature Factor
37	18
40	16

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.

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II- PROTOCOLS 1.  a-AMYLASE PURIFICATION
 Reagents

Ethanol (95%) at 0 °C Glycogen reagent - A 2% solution (20 mg/mL) of oyster glycogen (Sigma, Type II) in distilled water.  The solution should be centrifuged at 11,000 g for 10 minutes before use and stored at 4 °C. 0.2 M sodium phosphate buffer, pH 8.0

 Procedure

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 being the lowest risk and Biosafety Level 4 being the highest.  Procedures using  material obtained from apparently healthy individuals are rated Biosafety Level 2 (BSL-2).  The requirements for BSL-2 practice will be described in lab, and must be followed for any procedure using human-derived materials!

In your notebook, prepare a flowchart of the purification steps described below.  Identify the fractions produced at each step and indicate which fractions will (we hope) 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. 3 - 3.5 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 8.0 0.05 mL glycogen reagent (1 mg glycogen) 0.08 mL 95% ethanol.

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 H₂O
0.072 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.

12. Resuspend the precipitate in 0.4 mL of 0.02 M phosphate buffer, pH 8.0.
 

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)

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2- TOTAL PROTEIN ASSAY  - see also  Bradford Protein Assay
Dye stock - Coomassie Blue G (C.I.# 42655) (100 mg) dissolved in 50 mL of methanol. (If turbid, the solution is treated with Norit (100 mg) and filtered through a glass-fiber filter.)  The solution is added to 100 mL of 85% H₃PO₄, and diluted to 200 mL with water.  The solution should be dark red, and have a pH of -0.01. The final reagent concentrations are 0.5 mg/mL Coomassie Blue G, 25% methanol, and 42.5% H ₃PO₄.  The solution is stable indefinitely in a dark bottle at 4 °C.
Assay reagent - The assay reagent is prepared by diluting 1 volume of the dye stock with 4 volumes of distilled H₂O.  The solution should appear brown, and have a pH of 1.1.  It is stable for weeks in a dark bottle at 4 °C.
Protein Standards - Prepare six protein standards (1 mL each) containing 0, 250, 500, 1000, 1500 and 2000 µg/mL bovine serum albumine (BSA), prepared in the same buffer as the samples to be assayed.
Assay Procedure - Set the spectrophotometer to measure a single wavelength, 595 nm, and an absorbance range of 0 to 2 Absorbance units.  Use the same 4 mL plastic cuvette for all measurements.
Blank the spectrophotometer, using distilled water.  Starting with the lowest protein concentration and working up,. assay each standard and the test samples by mixing:
     2.0 mL Assay reagent
     0.04 mL of protein solution
Record the absorbance of the protein standards and samples at 595 nm.   Shake out remaining drops between samples.
Prepare a graph of Absorbance at 595 nm vs [Protein] for the protein standards, and use this standard curve to determine the protein concentration of your test samples.
If the absorbance of a test sample falls above the linear range of the standards, dilute the sample and repeat the determination.

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3- a-AMYLASE ACTIVITY ASSAY   Reagents

  Starch substrate - Soluble starch, 0.75 g/L in 20 mM Tris-phosphate buffer, 10 mM NaCl, 1 mM NaF, 0.1 mM NaN₃, 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).   Dilutent solution - 0.5% NaCl solution

 Equipment

7 test tubes, [3 mL, 10 x 75 mm] and test tube rack syringe, 1 mL, with needle Micropipettor (1 mL, adjustable) Clock or timer Heat block or water bath, 37°C or 40 °C

 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. Using a needle and syringe, transfer 1.5 mL of starch solution to the remaining test tube. Place the test tube in a heat block at 37 °C and allow to incubate for 2 minutes. Add 0.1 mL of diluted saliva (Note 1) to the 1.5 mL of starch substrate in the heat block. 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 ). 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 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 (starch-iodine 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 fluids, such as whole blood, bile, or serum that is highly icteric or hyperlipemic (turbid)
 

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 REFERENCE 1. Anonymous. 1985. 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 AM, Laqua H. 2000. "Quality of salivary tears following autologous submandibular gland transplantation for severe dry eye."  Graefes Arch Clin Exp Ophthalmol.238: 45-52.
3. Schramm, M. and Loyter, A. 1966. "Purification of a -Amylase by Precipitation of Amylase-Glycogen Complexes" Methods in Enzymology . 9:533 - 537.
4. Searcy, R.L, Wilding, P. and Berk, J.E. 1967. "An appraisal of methods for serum amylase determination" Clin Chim Acta15:189.
5. Somogyi, M. 1960 "Modification of two methods for the assay of amylase." Clin Chem23.
6. Henry, R.J. and Chiamori, N. 1960. "Study of the saccharogenic method for the determination of serum and urine amylase" Clin Chem 5: 434.
7. Young, D.S., Pestaner, L.C. and Gibberman, V. 1975. "Effects of drugs on clinical laboratory tests" Clin Chem 21:10