Abstract
This lab investigates the purification and assaying of lysozyme using size exclusion chromatography and Bradford protein assay. Micrococcus Lysodeikticus is employed in this lab as the substrate for lysozyme and its enzymatic activities are observed under various pH and substrate concentrations for determining the optimal conditions for lysozyme activity. The highest lysozyme activity was observed at a substrate concentration of 0.4 mg/mL and at an optimum phosphate buffer pH of 7. These optimal conditions were set as standard conditions for assaying the purified fractions for lysozyme activity and for the protein assay. A solution of egg white is used as the source for lysozyme and it is purified using size exclusion chromatography with column Sephadex G-50 that has a fractionation range from 1,500 to 30,000 daltons. Size exclusion chromatography provides purification of lysozyme, however about 64% of its initial yield is lost in the process of purification through the beads. The highest lysozyme activity was observed for fraction # 12 indicating a structural mass range of 15,000da-13,500da for lysozyme. The protein assay indicated a significant concentration of protein in the neighbouring fractions of purified lysozyme, however the exact concentration of lysozyme in albumen remain inconclusive.
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Introduction
Lysozyme is a catalytic enzyme that digests bacterial cell wall and is found in significant amounts in egg whites. Egg white, also known as albumen, is the cytoplasm of the unfertilized egg cell, which consists of approximately 40 different proteins dissolved in water (Lee-Huang et al. 1999). The main proteins in albumen are ovalbumin, ovotransferrin, ovomucoid, globulins, lysozyme, ovomucin, avidin, etc (Lee-Huang et al. 1999). Lysozyme comprises about 3.5 % of the total protein weight in albumen (Lee-Huang et al. 1999) and thus a solution of egg white was used as the source of lysozyme in this lab. The main objective of this lab is to purify lysozyme using size exclusion chromatography for examining its structural properties and concentration in albumen.
In this experiment lysozyme is extracted and purified from albumen using size exclusion chromatography. The stationary phase in the column consists of a porous cross linked gel matrix of Sephadex G-50 with a fractionation range of 1,500da to 30,000da. Separation and purification by size exclusion chromatography is based on molecular size i.e. larger molecules elute first with the mobile phase while the smaller molecules get trapped within the beads and elute last (Lodish et al. 2000). The different proteins present in albumen should elute at different fractions due to their varying molecular sizes leading to the purification of lysozyme which has a literature structural mass of 14,400da. It is hypothesized that since lysozyme is a small molecule in comparison to the fractionation range of the Sehadex G-50 coloumn, a significant amount of lysozyme is likely to be trapped or retarded by the resin beads resulting in a low yield.
Substrate binding is used as a powerful tool in this experiment where the enzymatic reactions are used for detecting the presence of lysozyme. The structural composition of lysozyme consists of 129 amino acid residues folded into a compact globular structure with a cleft for substrate binding (Berg et al. 2002). When a substrate binds to the cleft, it hydrolyzes the peptidoglycan polysaccharide found in many bacterial cell walls, resulting in the osmotic lyses of the cell (Berg et al. 2002). Gram positive bacteria are more susceptible to the effects lysozyme due to their peptidoglycan cell wall being exposed to the extracellular environment (Lee-Huang et al. 1999). However, gram negative bacteria are less vulnerable to the presence of lysozyme due to their thin layer of peptidoglycan shielded by the outer membrane of lipopolysaccharide (Lee-Huang et al. 1999). In this lab, the gram positive bacteria of Micrococcus Lysodeikticus is used as a substrate for detecting the enzymatic activities of lysozyme. The Bradford assay on the other hand is used to estimate the concentration of lysozyme with respect to other major proteins present in albumen.
Lysozyme has great research importance since it possesses the capability to lyse gram positive bacteria. Lysozyme, like most of the other biomolecules are not found in nature in its isolated form and this lab investigates one of the most simplest methods for extracting and purifying lysozyme from albumen.
Materials and Methods
A solution of egg white diluted to ¼ with 0.1 M phosphate buffer pH 7 and filtered though glass wool is used as the source for lysozyme (Laboratory Manual. 2007). The solution is put through size exclusion chromatography with G-50 Sephadex column (fractionation range of 1,500-30,000 da) to produce 24 test tubes of equal egg white fractionations of 0.75 mL (Laboratory Manual. 2007). Numerous assays are conducted with varying pH and micrococcus (substrate) concentration to determine the optimal conditions for the highest enzymatic activity of lysozyme. After the collected column fractions and prepared egg white solution were left in the lab for two weeks, the odd numbered test tubes were assayed for lysozyme activity and the even numbered test tubes were assayed for protein at optimal pH.
Refer to York University Department of Biology Laboratory Manual Summer 2008, SC/Biol 2020 Cell Biology and Biochemistry Pages 54-57 for a more detailed procedure of the lab. Also refer to the attached flow sheets for a thorough step by step procedure for this lab.
Results
The addition of micrococcus to a solution of lysozyme results in the rapid decrease in its optical density value due to its enzymatic reactions. Various assays are conducted in this lab to examine various properties of lysozyme. The substrate concentration assay indicated 0.4 mg/mL of micrococcus to be the optimal substrate concentration for lysozyme as it resulted in the highest enzymatic activity of 250 units. The pH assay on the other hand indicated pH 7 to be the ideal pH for the phosphate buffer as it resulted in the high lysozyme activity of 300 units. These observations led us to set 0.4 mg/mL micrococcus and buffer pH of 7 as standard conditions for assaying the size exclusion column fractionations for protein activity. When assaying the odd numbered fractionations for lysozyme activity, fraction # 15 reached the highest enzymatic activity of 900 units specifying the presence of concentrated lysozyme in that fraction of egg white. The neighbouring fractions (#14 and #16) showed significant protein concentrations of 4.4 mg/mL and 1.6 mg/mL when assayed however the highest protein concentration as observed in fraction # 12 which indicated a protein concentration outside the standard curve range. Upon dilution, the protein concentration of fraction # 12 was calculated to be 9mg/mL. Based on the results, a protein fold of 1.36 was calculated and the results showed a high lysozyme yield loss of about 64%.
Calculations:
Sample calculation of Micrococcus dilution:
Target: 3 ml of 0.4 mg/ml Micrococcus
C1V1 = C2V2
(10 mg/ml)(x ml) = (0.4 mg/ml)(3 ml)
X = 0.12 ml of Micrococcus
3 ml – 0.12 ml = 2.88 ml
Therefore, 0.12 ml of Micrococcus and 2.88 ml of Phosphate buffer will be required
Sample calculation of total protein in fraction # 15:
protein concentration of 0.52 mg/mL
Volume in fraction: 0.1 mL
Total protein = 0.52mg/mL X 0.1mL = 0.052 mg
Sample calculation of Total Enzyme Activity in fraction # 15
Activity = ΔOD x 1min/0.001
Activity = (0.4) x 1min/0.001
Activity = 400 units
Sample calculation of Specific Activity for fraction # 15:
Enzyme activity of column fraction 15 = 90 units
Total protein = 0.052 mg
Specific Activity = Enzyme Activity/Total protein
Specific Activity = 400 units/( 0.052 mg)
Specific Activity = 7692 units/mg protein
Sample calculation of initial specific activity of the egg white at 0.3 mg/ml substrate:
Specific Activity = Enzyme Activity/Total Protein
Specific Activity = 90 units/(4.5 mg/ml x 0.1 ml)
Specific Activity = 200 units/ mg protein
Calculation of Fold Purification:
Fold Purification = Specific Activity of fraction/Specific Activity of egg white
Fold Purification = (7692 units/ mg protein) / (200 units/ mg protein) = 38.46
Discussion
This lab experiment examines the purification and assaying of egg white lysozyme. Lysozyme is both a protein and an enzyme that catalyzes the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan (Lodish et al. 2000). It is found in abundant quantities in albumen (egg whites) where it protects the egg embryo from bacterial invasion. Several assays were conducted in this lab experiment and each assay demonstrated a significant property of lysozyme.
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Enzymes have optimal conditions at which it functions most effectively and it is important that enzymes are studied under optimal conditions for the most accurate results and observations. The substrate concentration assay and pH assay were employed in this experiment to determine the optimal conditions for lysozyme since its enzymatic activity varies with substrate concentration and buffer pH. Appropriate substrate concentration is significant for an enzymatic reaction because a high substrate concentration might outnumber the available active sites on lysozyme while a low substrate concentration will leave vacant binding sites on the lysozyme. This lab proved 0.4 mg/mL of Micrococcus to be the optimal substrate concentration for lysozyme with a high enzymatic activity of 250 units. The determination of the optimal pH at which the substrate-enzyme binding is carried out most efficiently is another important aspect of an enzymatic reaction. An environment too acidic or basic could cause hindrance for the substrate-enzyme binding and thus result in low lysozyme activity. The phosphate buffer pH assay in this lab proved pH 7 to be the optimal pH at which the enzymatic activities of lysozyme are carried out most efficiently with an enzymatic activity of 300 units.
Size exclusion chromatography is used in this experiment for extracting and purifying lysozyme from the mixture of approximately 40 proteins that is present in albumen. Other proteins that are present in significant quantities in egg whites are ovalbumin (66 kDa), ovotransferrin (77.8 kDa), ovomucoid (28 kDa), ovomucin, avidin (18kDa), etc. however none of these proteins share the same molecular mass as lysozyme which has a literature molecular weight of 14.5 kDa (Lee-Huang et al. 1999). This unique mass distinction between the proteins present in albumen allows size exclusion chromatography to be an effective method in purifying lysozyme as its separation method is solely based on molecular mass difference.
Specific selection of resin for the column is another important factor in protein purification as the porosity and fractionation range of the column should be focused on the protein being purified. This experiment uses the Sephadex G-50 column for protein purification. The stationary phase of Sephadex G-50, has a bead matrix with a well defined pore size for separating proteins within the fractionation range of 1,500da – 30,000da. Sephadex G-50 is well suited for lysozyme purification because lysozyme has a literature molecular mass of 14,400da (Lee-Huang et al. 1999) which falls midway between the column fractionation range.
Purification of lysozyme however would come with the cost of obtaining low lysozyme yields. During purification, the larger molecules elute first while the smaller molecules like lysozyme travel through the beads and elute last. This affects the yield of lysozyme as some of its initial mass gets trapped within the gel matrix beads during separation while another small fraction of the initial yield is lost due to the retardation of the lysozyme during the in and out diffusion of the protein from the beads in the matrix (Laboratory Manual. 2007). Thus for a method like size exclusion chromatography, I would predict very low yields. The prediction was proved to be true as the data collected from the experiment demonstrated a low yield of 36%. Thus even though the method of size exclusion chromatography is effective in purifying lysozyme, it comes with the disadvantage of low yield.
Micrococcus, the substrate for lysozyme in this experiment, is a gram positive bacteria with an exposed peptidoglycan cell wall (Lee-Huang et al. 1999). Like any other enzymes, lysozyme is very specific about its substrate and the ability of lysozyme to bind to micrococcus and lyse the cell allows us to follow its enzymatic activity through the decrease in optical density detected on the spectrometer. Lysozyme activity assay demonstrated a peak for the highest enzymatic activity at fraction # 15. The peak represents the single protein species of lysozyme because micrococcus can only be digested by lysozyme and no other albumen proteins. Therefore all activities observed for lysozyme assay is due to the presence of lysozyme binding and hydrolyzing the peptidoglycan in the cellular walls of micrococcus. Micrococcus is thus the ideal substrate for this experiment however it also poses some disadvantages as well due to its biohazardous nature. Micrococcus needs to be handled with extra caution and is to be discarded of appropriately.
The lysozyme assay showed the highest specific activity of 900 units in fraction # 15. This indicated that fraction # 15 contained purified lysozyme. Considering the fractionation range of 1,500 – 30,000 da of the chromatography, and the elusion of a fixed volume into 24 separate test tubes, an estimation of the molecular weight range for fraction # 15 could be made around 15,000 da – 13,500 da, a range that covers the literature molecular mass of lysozyme, 14,400 da. Thus, our detection for the fraction of egg white containing purified lysozyme was pretty accurate.
The Bradford protein assay was used in this lab to examine the protein concentration of lysozyme with reference to the other proteins present in albumen. Theoretically lysozyme comprises about 3.5% of the total protein mass in albumen (Cançado et al. 2007). Even though it is a significant amount, there are other proteins comprising a higher concentration in albumen. The odd fractionations closest to #15 show fair concentrations of protein with # 14 showing a concentration of 4.4 mg/mL and # 15 showing 1.6 mg/mL. In order to get an accurate reading for the lysozyme protein concentration, fraction # 15 would have to be directly assayed instead of its neighbouring fractions. Thus the actual lyoszyme concentration in albumen remains inconclusive. The highest protein peak was observed for fraction # 12 with a protein concentration that went beyond the standard curve range. Upon dilution, the protein concentration of # 12 was determined to be approximately 9 mg/mL. This indicates that a protein that eluted in fraction # 12 is the most concentrated in albumen.
Specific activity is defined as the enzyme activity over total amount of protein (Laboratory Manual. 2007). The specific activity of 900 units/mg protein for fraction 15 and the specific activity of the initial egg white solution of 666.66 a fold purification of 1.35(Refer to calculations). The yield of lysozyme after purification was fairly low indicating a high percent of loss. This loss was due to a portion of lysozyme being trapped within the beads of the matrix during purification and another small portion being distorted during in and out diffusion of lysozyme (Laboratory Manual. 2007). The 64% loss in enzyme yield was worth the increase in purity because purifying the enzyme enabled us to estimate its structural mass and protein concentration in albumen.
Sources of error in this lab were tried to be kept at its minimal level however there could still be some errors that might have deviated the results slightly. Lysozyme activity was measured by detecting the difference in optical density on a spectrophotometer in a time period of 1 minute. Incorrect readings of the optical density caused by fingerprints/other residue on the test tube surface or reading the incorrect absorbance at an earlier or later time period could have been a major source of error as this lab is dependent on the accuracy of the optical density readings. Other sources of error could be dilution errors as some of the dilution require very minute amounts and pipetting the small amounts with the pipette provided for this lab accurately is very challenging.
New researches in the field of biochemistry help expand our knowledge about cell and molecular processes and thus research interest in the unique enzyme of lysozyme is of no exception. It was previously believed that lysozyme was used primarily as a constitutive defense against bacterial pathogens but recent research indicate that in certain species in the animal kingdom the structure of lysozyme is different and the structural difference enables lysozyme to incorporate other useful functions such as digesting bacteria for nutrition (Cançado, et al., 2007). Research has also shed light on urinary lysozyme C showing that a combination of urinary lysozyme C with certain RNases can be used to combat HIV-1 (Lodish et al. 2000). Other researches on lysozyme conducted by Lee-Huang et al. found that lysozyme from chicken egg white, human milk and human neutrophils combined with RNase A from bovine pancreas display activity against HIV-1 (Lee-Huang, et al., 1999). These are significant discoveries that not only broaden our knowledge in biochemistry but also define possible cures for HIV in the future.
Conclusion
Lysozyme is a widely distributed enzyme in the animal kingdom that lyse bacterial cells to protect organisms from bacterial invasion and this lab demonstrated some of the important characteristics of this unique enzyme. Micrococcus proved to be an ideal substrate for observing lysozyme activity due to its gram positive nature illustrated by its exposed peptidoglycan cell wall, the cleavage target of lysozyme. The substrate concentration assay and pH assay demonstrated how lysozyme is at its peak enzymatic activity at the optimal substrate concentration of 0.4 mg/mL and at a buffer pH of 7. Even though the exact protein concentration of lysozyme in albumen remains inconclusive, the neighbouring fractions assayed for protein concentration provided an estimate that lysozyme is present in significant amounts in egg white in comparison to the other albumen proteins. Even though lysozyme was successfully purified using size exclusion chromatography with Sephadex G-50 column, its purification resulted in the loss of lysozyme yield. The column beads trapped and retarded about 64% of the total lysozyme resulting in low yields.
Overall this was lab was well engineered to demonstrate how size exclusion chromatography can be used for purification based on molecular mass and how the unique activities of a certain enzyme with its specific substrate can be used to determine the purified fraction that contain the certain enzyme.
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