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«Chapter 11 Two Reliable and Inexpensive Lysozyme Assays for Teaching Enzymology and Microbiology John A. Snyder Department of Biology Furman ...»

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Chapter 11

Two Reliable and Inexpensive

Lysozyme Assays for Teaching

Enzymology and Microbiology

John A. Snyder

Department of Biology

Furman University

Greenville. South Carolina 296 13

Donald Fritsch

Department of Biology

Virginia Commonwealth University

Richmond, Virginia 23284

Dr. Snyder earned his BA in biology from Case Western Reserve

and his M A and PhD in zoology from the University of Michigan.

He has been at Furman University since 1971, and is now an Associate Professor teaching zoology, developmental biology and biological techniques. His research interests focus on antibacterial enzymes in legumes and the structure and function of lysozyme isozymes in amphibians. Part of this chapter was adapted from Snyder (198 1) by permission.

Donald Fritsch was President of A.B.L.E. from 1981 to 1983. He was Chairman of the Workshop Committee for 1979 and 1980. His BS and MS degrees were earned in 1973 and 1976 from the University of Missouri, where he was also Superviser of Introductory Biology Laboratories. He has been Coordinator of General Biology Laboratories a t Virginia Commonwealth University since 1976.

168 Lysozyme Assays Introduction A simple and reliable enzyme assay is a valuable teaching aid in an introductory biology course. Of even greater value is one that is also useful in upper-level courses and for research. We present two assays for the measurement of the antibacterial enzyme lysozyme. Each satisfies a number of criteria for being a good teaching assay at any level of undergraduate instruction.

The Ideal Assay The enzyme assay of choice would demonstrate both didactic and practical qualities. For the purpose of demonstrating principles, the enzyme to be displayed would have readily understandable functions and would occur in those organisms that immediately capture the interest of students. Any enzyme important in human medicine fits this description. Then, to broaden the level of interest, the chosen enzyme would have qualities that make it a subject of study in other areas, such as protein chemistry, genetics, molecular evolution, or microbiology.

A good assay for such an enzyme is one that would serve as an introduction to the proper use of scientific tools and methods. It would require that students measure out and mix chemicals, and gather data for analysis. It would provide to students realistic activity that accurately teaches the research experience. The assay would be one that scientists actually use and that students could use later in projects for advanced courses. The ideal assay would also be adaptable to many situations. One could easily modify it to demonstrate the importance of temperature, ionic environment, and other factors. It could be a model of a class of assays for several different enzymes.

Finally, the assay of choice would be easy to set up, reliable in its results, undemanding of manual dexterity by inexperienced workers, and inexpensive.

Lysozyme Assays Lysozyme is one of several enzymes that can hydrolyze components of the walls of certain bacteria. The cell lysis, or bursting, that usually follows is the basis for its bacteriocidal activity in vivo. Most assays for lysozyme are turbidimetric, measuring the clearing of a suspension of dead bacterial cells as their walls break down (Klass et al. 1977). Both of the assays described here are based on this model.

The first method is for a radial diffusion assay somewhat similar in principle to widely used antibiotic assays. In these, a test compound diffuses out from a filter paper disc placed on a nutrient agar surface seeded with bacteria, producing a visibly clear circle within which bacterial growth has been inhibited. As modified for lysozyme, non-nutritive agar contains an opaque suspension of dead bacteria of a species susceptible to cell wall disruption by lysozyme. Wells, punched in the agar sheet, are filled with solutions to be asLysozyme Assays sayed. Any bacteriolytic enzyme diffusing out of a well shows its presence by producing a ring that is cleared of intact cell walls. After a standard incubation period, the diameter of each cleared ring is proportional to the concentration of lysozyme in the solution that diffused out of the well. This assay has been variously labeled the “intra-agar lysis assay” (Nace et al, 1965) or the “lysoplate assay” (Osserman and Lawlor 1966).

Our second method of lysozyme detection involves spectrophotometric measurement of light transmission through a suspension of bacteria in buffer.

As lysozyme destroys cell walls the suspension clears, causing a gradual increase in measured “percent transmittance.” Although one can perform this assay using dead cells (as with the first assay), the method presented here involves harvesting and using living bacteria as the substrate.

The antibacterial activity of lysozyme immediately captures student interest, especially when demonstrated in their own body fluids. The chance manner in which Sir Alexander Fleming discovered its antiseptic property, followed by the recognition and careful analysis of its potential medical value, is a classical example of a prepared mind responding appropriately to a fortuitous event (Baumel et al. 1976). Medically oriented students will also be interested in lysozyme’s potential roles in cartilage formation (Kuettner et al.

1974) the immune mechanism (Kokoshis et al. 1978) and cancer therapy (Osserman et al. 1973). High lysozyme levels in human serum or urine are diagnostic of certain diseases, including leukemia (Pruzanski and Platts 1970).

A lysozyme assay also becomes a point of entry for discussion of protein structure, since chicken egg-white lysozyme was the first enzyme whose three-dimensional shape was accurately shown by X-ray crystallography (Blake et al.

1965). This, is turn, led to its extensive use as a reference protein in the field of molecular evolution (Wilson and Prager 1974).

Students can easily modify the assays described here in order to test the optimum pH of a particular lysozyme and to show the effects of various ions or of ambient temperature. The intra-agar assay is also the basis for determining the location of lysozyme isozymes in polyacrylamide gels after electrophoresis (Ostrovsky et al. 1976). Variations upon the theme of a radial diffusion assay exist to test for several other enzymes that catalyze the breakdown of opaque substrates (Davis 1977).

The intra-agar assay described here is a very reliable test. Since all standards and samples of unknown enzyme concentration can be tested simultaneously on a single plate, small measuring errors in the preparation of the plate do not significantly affect results. However, two cautions must be issued for both this assay and the spectrophotometric method. First, neither of them measures initial reaction rates. Therefore, an instructor cannot use them to teach enzyme kinetics. The spectrophotometric assay, if modified to operate at 37°, will run fast enough to obtain measurements in the first minute, but 170 Lysozyme Assays the complexity of the bacterial cell wall and the possibility of several reactions occurring simultaneously make evaluation of these measurements difficult. The second caution, applicable to both assays, is that a positive reaction is not sufficient proof of lysozyme’s presence. One must also investigate physical and chemical properties, such as molecular weight and isoelectric point, before assigning the name “lysozyme” to the enzyme in question (Jolles 1967).

The reagents and equipment needed for either assay are neither exotic nor expensive. Reagents can be stored for over a year. Although a student cannot obtain quantitative results with the intra-agar assay until several hours have elapsed, the time required to prepare plates and start the assay does not exceed one hour, exclusive of the time used in preparing the standards and other samples. An instructor may choose to provide samples ready for assay, or plates ready to receive samples, or to use a complete laboratory period to instruct students in obtaining, preparing, and analyzing their own samples.

Hen’s egg-white, human saliva, and human nasal secretion all produce lysis rings within five minutes after application. (See Appendix A.) A bonus associated with the assay is that students can take home with them a valuable reminder of their experiment-the developed plate.

The spectrophotometric assay, while requiring a more sophisticated piece of equipment, the spectrophotometer, does provide the student with results within minutes. As required by the instructor or time constraints per lab period, students may do none or all of the simple set-up procedures.

Instructors’ Materials The following instructions are for single 100 X 15 mm petri dish sufficient for the assay of up to 30 samples. One can easily prepare up to four such plates in a single batch by appropriate multiplication of reagent quantities.

Many more are possible if smaller petri dishes are used.

Place 6.0 g of agar cubes and 6.

0 ml of phosphate-NaCl buffer into a 125 ml erlenmeyer flask. Seal the flask with aluminum foil, attach a test tube holder to its neck, and place it in a boiling water bath to melt the agar.

Swirl occasionally.

While waiting for complete melting of the agar, weigh out 15 mg of dried Micrococcus cells and place them in a grinding device such as a glass tissue grinder or a very small mortar and pestle. Add 0.5 ml of buffer and mix thoroughly to suspend the cells. Transfer the suspension to a 25 ml graduated cylinder.

When the agar cubes are thoroughly melted, pour the resultant liquid into the graduated cylinder, up to a total volume of 11 ml. Mix thoroughly to produce a uniformly opaque suspension of Micrococcus.

Lysozyme Assays

4. Immediately remove 10 ml of the suspension with a serological or mohr pipet. (Careful! Use a safety pipet filler to avoid burning your mouth.) The Intra-Agar Lysozyme Assay Materials

1. Agar (such as Difco "Bacto Agar") rehydrated in distilled water at 4% concentration (w/v) to yield 500 ml. Cut the gel into cubes of approximately

0.5 cm and wash them in running tap water for several days to remove colored impurities. Soak washed cubes for at least 24 hours in several changes of distilled water. Store cubes indefinitely at 5" to 10°, covered with distilled water.

This amount is sufficient for preparing approximately 80 of the plates described below.

2. Buffer, pH 6.6, 0.067 M. Optimum pH varies according to the source of the lysozyme being tested, but this buffer is good for most animal tissues.

Na2HPO4 3.56 g KH2PO4 5.65 g H2O to make 1000 ml Add 22.67 g NaCl to this buffer, since these ions enhance the lytic activity of many animal lysozymes (Metcalf and Deibel 1969).

3. Bacterial cells. Obtain this material in dried form from a biochemical supply company, ordering it as Micrococcus lysodeikticus (M. luteus). One gram, suitable for over 50 assays, costs only a few dollars. Store below 0" indefinitely.

4. Lysozyme standard, purified from chicken egg-white. Obtain it from a biochemical supply company in lyophilized form. One gram costs a few dollars and is sufficient for over 1000 assays. For use, make a series of 2 ml solutions in distilled water, ranging from 0.1 to 10.0 mg/ml. One can store powder or solutions below 0" indefinitely with little loss of enzymatic activity. Transfer this volume to a 100 X 15 mm petri dish placed on a perfectly horizontal surface, to form a uniform layer over the bottom of the plate. Cover, and allow the agar to gel undisturbed for about 20 minutes.

5. With either a gel puncher (such as is used for immunodiffusion assays) or a cork borer of 4 mm or smaller diameter, punch the desired number of sample wells into the agar. Remove agar plugs by suction through a Pasteur pipet connected to a water aspirator or other weak vacuum source.

6. Using a 22 to 26 gauge needle attached to a 1 ml syringe, fill wells with standards or test solutions. You can use the same needle and syringe for all samples if you rinse them between samples by drawing up and squirting out water five to ten times. Fill each well exactly to its top.

172 Lysozyme Assays

7. Cover the plate, seal it in clear plastic wrap to avoid dessication, and place it in an incubator set between 37" and 45". For full development at 45°, incubate at least 3 hours. At 37°, increase total incubation time to 10 hours.

Lysis activity also occurs visibly at room temperature, but at least 24 hours must be allowed for full development. A solution very rich in lysozyme will produce an observable ring of lysis at the well margin within 15 minutes, even at room temperature.

8. To obtain quantitative results, measure lysis diameters made by standards and "unknowns", using vernier calipers or a ruler graduated in millimeters. For best visibility during measuring, hold the plate toward a light source and provide a darkfield background (see Fig. 11.1). A bacterial colony counter is ideal. To construct a standard curve, plot the concentrations of chicken eggwhite lysozyme standards vs. their lysis diameters. Use three-cycle semilog graph paper, placing concentrations on the logarithmic axis.

Figure 11.1.

An intra-agar lysis assay plate after 5 hour incubation at 37". Upper

left: purified chicken egg-white lysozyme, 1.0 mg/ml. Upper right: chicken eggwhite, whole. Lower left: human saliva. Lower right: human nasal secretion. Center:

human fingernail clipping. Bottom: distilled water (control).

Lysozyme Assays

9. To stop further lysis, cover the agar surface with a 0.1 N NaOH solution for at least 15 minutes. This treatment may also make the border of the lysis zone around each well more distinct.

The Spectrophotometric Assay for Lysozyme Medium preparation Materials: 1 liter flask Aluminum foil-to cover flask 20 100 X 15 mm petri dishes Agar-7.5 grams Nutrient broth-4 grams 500 ml of distilled water

Prepare nutrient agar petri dishes as follows:

1. Place 7.5 grams of agar and 4 grams of nutrient broth in a 1 liter flask.

2. To this flask add 500 ml of distilled water and swirl the contents in the flask for a few seconds.

3. Cover the mouth of the flask with aluminum foil or an inverted beaker.

4. Autoclave at 15 to 20 psi for 20 minutes.

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