Preparation And Assay Of Activities In Subcellular Samples

The lysis of cells has its consequences. For example, since most lytic procedures involve disrupting both the plasma membrane and the membranes surrounding internal organelles, opening a cell will result in a loss of boundaries that would otherwise segregate enzymes from degradation. Therefore, following the disruption of most cells, proteolytic activities from some locations such as lyso-somes gain access to areas from which they normally would have been excluded. Thus, precautions must be taken if problems arising from proteolytic activities are to be kept at a minimum or, better yet, prevented.

In the absence of any specific information about the nature of such activities, it is often best to mix a cocktail containing several or all of the available proteolytic inhibitors. These include such compounds as 1,10-phenanthroline, soybean trypsin inhibitor, leupeptin, benzamidine, antipain, aprotinin, phenyl-methanesulfanyl fluoride, and diisopropylfluorophosphate. As a precaution against any enzyme destruction, such a cocktail is best added to the buffer in which the cellular lysis will be carried out (Fig. 5.6).

Opening the cells can have other consequences. For example, many enzymes require cofactors for catalytic activity. If, as a result of the lysis of cells and organelles, the enzymes become separated from these cofactors, a loss

PROTEOLYTIC INHIBITORY COCKTAIL

PROTEOLYTIC INHIBITORY COCKTAIL

LYSIS

CELL SUSPENSION

LYSIS

CELL SUSPENSION

HOMOGENIZATION

CENTRIFUGE

Figure 5.6 Overview of steps involved in the preparation of a cell-free lysate. The cells are resuspended in a buffered solution at a specified cell density. To this suspension is added a "cocktail" containing several proteolytic inhibitors. The cells in the suspension are lysed (here by homogenization). Finally the lysate is subjected to a very low speed centrifugation such as 5000g for 10 minutes to remove unbroken cells.

of activity can result. Also, since the activity of most enzymes is concentration-dependent and since lysis usually results in the dilution of intracellular components, the activity of enzymes present at low intracellular concentrations can be lost. Both these problems are difficult to guard against.

In addition to the foregoing more general concerns are questions concerning the localization of an enzyme activity. The location of an enzyme can determine the type of cell lysis, since it could be more advantageous to lyse the cell completely or in such a manner that the organelles are left intact. For example, some lysis methods such as sonication completely disrupt mitochondria, nuclei, and Golgi systems. If an activity is localized in an organelle such as a mitochondrion, it would seem sensible to adopt a method that leaves these structures intact, to facilitate their separation from the rest of the cellular debris. Thus, for the isolation of mitochondrial enzymes, sonication is not the method of choice for cell lysis.

Methods commonly used for lysis involve such equipment as the French press, the sonicator, and the blender or homogenizer. In Figure 5.6, a homoge-nizer is represented in sequence with the other steps involved in the lysis process. The choice of the lysis method, while dependent on the localization of the enzyme, is also a function of the type of cell under consideration. For example, when lysing bacterial cells with rigid cell walls, the French press may be one of the few physical methods that works. Alternatively, for disrupting cells that have fragile cell membranes, homogenization as shown in Figure 5.6 is often adequate.

Regardless of the procedure finally adopted, it is wise to measure its success. One of the easiest techniques for accomplishing this assessment is a micro scopic examination of samples taken from the lysate after each step of the procedure. Such information is very useful, particularly when one is experimenting with either a new type of cell or a new method for lysis.

5.5 INITIAL PURIFICATION AND ASSAY OF ACTIVITIES IN CELL-FREE LYSATES

While it is possible and often necessary to assay enzymatic activities directly in the lysate, it is often helpful to remove any remaining unbroken cells and any nonbiological debris (e.g., sand grains or glass beads that might have been added to facilitate the breakage). Again, centrifugation at a low speed, such as 5000 rpm, for about 10 minutes should be sufficient.

The supernatant fraction obtained in this step can be centrifuged at 30,000g to produce a second supernatant fraction often referred to as an S-30 fraction. Centrifugation of this S-30 fraction at 100,000g will produce a third supernatant fraction called an S-100 fraction. Each centrifugation removes insoluble material of decreasing size or mass. For example, while the pellet from the S-30 fraction contains many large organelles, such as intact mitochondria, the pellet from the S-100 fraction contains ribosomes, the endoplasmic reticulum, and other smaller membranous structures.

Many enzymes are soluble and will be recovered in the S-100 fraction. In addition, their activity can be measured rather conveniently by adding a sample of the S-100 fraction directly to a reaction mixture. Of course, since the S-100 fraction will contain an excessive amount of extraneous protein, it will be necessary to terminate the reaction and filter the sample prior to injecting it onto the HPLC for analysis.

Note that while the S-100 fraction can be used in this form, it would be best to have it dialyzed to remove unwanted low molecular weight compounds before using it in an assay. In addition to dialysis, a simple salting out can be performed to remove some of the extraneous protein material. Ammonium sulfate is often added for this purpose to remove unwanted proteins or to precipitate the enzyme in question. Any ammonium sulfate should be removed before the sample is used in an assay, because the salt might affect activity. Again, dialysis can be used, or alternatively the sample can be passed through a gel filtration (G-25) column.

If it is necessary to purify the enzyme further, the next step should be one that has a high capacity, that is, one that can process large amounts of protein. Such steps, however, often are not very specific. For example, salting out is a technique of high capacity but low selectivity. This step should be followed by steps of decreasing capacity but greater selectivity. Such techniques include ion-exchange, gel filtration, or affinity chromatography and even HPLC itself. The choice should take into account what is known about the protein, its size and shape, its solubility, and even its substrate specificity. Also, the quantity of protein required should be considered. If only analytical amounts of the enzyme are needed, several methods including HPLC can be included.

5.6 HPLC FOR PURIFICATION OF ENZYMES: A BRIEF BACKGROUND

Early separations of proteins by HPLC relied on controlled-pore glass beads with a coating of 1% polyethylene glycol. In a later modification (3% polyethylene glycol coating on the beads), the technique was used for the separation of plasma proteins. The development of noncompressible ion-exchange supports has allowed various laboratories to separate the isoenzymes of lactate dehydrogenase. Refinement of this anion-exchange support to a microparticu-late size (40 /im) has enhanced the resolution of lactate dehydrogenase and creatine kinase isoenzymes as well as the purification of alkaline phosphatase.

Reversed-phase chromatography has also been used to separate proteins. However, the required use of alcoholic gradients or paired-ion reagents with the reversed-phase support should be avoided, to cancel the potential for inactivation of the enzymatic activities.

High pressure gel permeation chromatography (HPGPC) was developed to correspond to gel filtration, where large molecules are partially excluded from the porous coating of the support and thus are eluted before smaller molecules. A silica-based packing with an organochlorosilane chain containing other functional groups has been used to separate plasma proteins on the basis of their molecular size as well as by their ionic, polar, and hydrophobic interactions with the column packing and the mobile phase.

HPLC is useful as an analytical tool in several applications in addition to its role in the purification of an activity. For example, HPLC can be useful in the establishment of gradients. Instruments have been manufactured for use with HPLC that can control the flow and mixing of solvents and thereby generate gradients with a variety of concentrations and "shapes." In addition, since it is possible to carry out separations on the HPLC column in a comparatively short time, a number of these gradients can be applied to an analytical scale column, and the one best suited to the separation established fairly rapidly. Armed with this information, it is a relatively simple matter to carry over these gradient conditions to a non-HPLC ion-exchange column.

The speed with which it is possible to perform an analytical run makes HPLC useful for what might be called a pseudopreparative function. If rather modest amounts of a purified protein are required—for example, to carry out an analysis by polyacrylamide gel electrophoresis or even for antibody production—and if the HPLC column provides adequate separation and purification, it is often possible to produce enough material for such purposes by merely repeating the same run on an analytical column several times. By collecting the appropriate fraction, it is possible to generate sufficient material to advance to the next stage of the purification or to perform the experiments of interest. Again, it is the speed of the separation that makes the approach feasible.

Finally, HPLC can be used as an analytical method to monitor the efficiency of different purification methods. For example, imagine that gel filtration chromatography has just been carried out on a sample of an S-100 fraction. Several peaks are observed. A question usually asked at this point relates to the homogeneity of each of the peaks. HPLC is ideal to answer this question, since it allows each peak to be analyzed in just a few minutes. Also, different columns can be used for the analysis, and therefore the homogeneity of any peak can be verified under a variety of conditions.

As another example, imagine that an S-100 fraction has been prepared, ammonium sulfate has been added, and the fraction that precipitates between 30 and 50% has been obtained. This sample is subjected to affinity and ionexchange chromatography. Have these procedures been successful in removing extraneous proteins? While it is possible to answer this question by a determination of total protein content, it may be more informative to analyze each of the samples for its constituent proteins.

The speed of the HPLC analysis makes such a determination possible. Figure 5.7 represents the analysis of samples after each of four stages of a typical purification. The analysis was made using gel filtration HPLC, and as shown this could be accomplished in 20 minutes.

Comparison of the profiles reveals that proteins present in the sample prepared by ammonium sulfate fractionation (Fig. 5.7/1) were removed following affinity chromatography (Fig. 5.1 B) and ion-exchange chromatography (Fig. 5.7C). The profile of the activity following ion-exchange HPLC (Fig. 5.7D) shows considerably less protein than was originally present.

5.7 STRATEGY FOR USE OF HPLC IN THE PURIFICATION OF ACTIVITIES

HPLC has received a great deal of attention as a method for the purification of enzymes, and the results of such studies are rapidly pervading the literature. The reader should consult recent reviews for information concerning the enzyme of interest.

Consistent with our purpose, this volume presents the general principles of how to begin the purification procedure using HPLC. The experience gained over the years, in the purification of enzymes has led to the general approach for purification detailed in this section.

To obtain the separation needed for purification, a few initial runs are carried out to verify solubility and to get some idea of the complexity of the sample. Separation of the proteins can require modification of several variables, including the concentration range of the salt used for the gradient as well as the salt itself. For example, the range of salt concentrations used in the gradient can have a significant effect on the elution profile of a series of

10 15 20 Retention time (min)

Figure 5.7 The use of HPLC to monitor enzyme purification. These profiles were obtained by gel filtration chromatography during the purification of the enzyme sAMP synthetase. The column was a TSK-250 (BioSil, 7.5 mm X 30 cm), and the mobile phase was 0.1 M potassium phosphate (pH 6.0). The column was monitored at 280 nm. Profiles obtained after (/4) 30 to 50% ammonium sulfate precipitation, (fl) affinity chromatography, (C) ion-exchange chromatography on DE-52, and (D) HPLC ion-exchange chromatography on AX-300.

MW x 1000

MW x 1000

10 15 20 Retention time (min)

5 10 15

Retention time (min)

Figure 5.7 The use of HPLC to monitor enzyme purification. These profiles were obtained by gel filtration chromatography during the purification of the enzyme sAMP synthetase. The column was a TSK-250 (BioSil, 7.5 mm X 30 cm), and the mobile phase was 0.1 M potassium phosphate (pH 6.0). The column was monitored at 280 nm. Profiles obtained after (/4) 30 to 50% ammonium sulfate precipitation, (fl) affinity chromatography, (C) ion-exchange chromatography on DE-52, and (D) HPLC ion-exchange chromatography on AX-300.

proteins (Fig. 5.8/4,B). The pH might also be changed, and the effect of such a change on the separation can be seen by comparing the chromatograms of Figure 5.8(A,B vs. C,D). During this initial phase, when the purpose is merely to establish conditions for the separation, it is usually not necessary to collect fractions or to assay enzymatic activity. Throughout this phase of the work, the separations can be monitored at 280 or 230 nm in the absence of aromatic amino acids in the protein.

When conditions for the separation have been attained, the enzyme should be located. Another run should be carried out for this purpose, the fractions collected, and the activity of each fraction determined. Figure 5.9 illustrates the results of such an analysis with the absorbance profile obtained at

5.7 STRATEGY FOR USE OF HPLC IN THE PURIFICATION OF ACTIVITIES 109 5

5.7 STRATEGY FOR USE OF HPLC IN THE PURIFICATION OF ACTIVITIES 109 5

Figure 5.8 Effects of ionic strength and pH on elution profiles obtained by gel filtration HPLC on five proteins: 1, thyroglobulin, 670,000; 2, gamma globulin, 158,000; 3, ovalbumin, 44,000; 4, myoglobulin, 17,000; 5, vitamin D-12, 1350. The column was eluted with sodium acetate (pH 6.8) (,4) at 50 mM and (B) at 100 mM. The column was also eluted with (C) 100 mM sodium acetate (pH 6.0) and (D) 150 mM sodium acetate (pH 6.0).

Figure 5.8 Effects of ionic strength and pH on elution profiles obtained by gel filtration HPLC on five proteins: 1, thyroglobulin, 670,000; 2, gamma globulin, 158,000; 3, ovalbumin, 44,000; 4, myoglobulin, 17,000; 5, vitamin D-12, 1350. The column was eluted with sodium acetate (pH 6.8) (,4) at 50 mM and (B) at 100 mM. The column was also eluted with (C) 100 mM sodium acetate (pH 6.0) and (D) 150 mM sodium acetate (pH 6.0).

280 nm of the sample as fractionated on an ion-exchange column. The fractions collected were assayed for three different enzymes (Fig. 5.9/1); the results are shown in Figure 5.9B-D. Three activities are present, and each activity has been separated into two components. Additional runs may be necessary to obtain sufficient material for subsequent purification. If an HPLC step is introduced early in the purification, several runs will be required to obtain enough sample for additional purifications.

To illustrate the rapidity of HPLC, particularly in comparison with the more conventional techniques, the same sample was separated by conventional ion-exchange chromatography. Figure 5.10 compares the two procedures. These data show that where 14 hours was required for the traditional method, only about 45 minutes is required with HPLC. Therefore, the total time needed to carry out this purification, not counting the time for the enzyme assay, could be as short as 3 to 4 hours. If necessary, the chromatography step could be completely automated. Finally, since each run will use only a fraction of the total volume of the starting material, the entire procedure will be economical.

Figure 5.9 Enzymatic activities of fractions following HPLC chromatography. A partially purified preparation was fractionated by ion-exchange HPLC (AX-300) with a mobile phase of 0.1 M potassium phosphate. Proteins were eluted with a gradient of sodium acetate. Column eluent was monitored at 280 nm. Fractions were collected, and each fraction was assayed for three different activities.

Figure 5.9 Enzymatic activities of fractions following HPLC chromatography. A partially purified preparation was fractionated by ion-exchange HPLC (AX-300) with a mobile phase of 0.1 M potassium phosphate. Proteins were eluted with a gradient of sodium acetate. Column eluent was monitored at 280 nm. Fractions were collected, and each fraction was assayed for three different activities.

Time (min)

Time (min)

Figure 5.10 Ion-exchange chromatography of a detergent-solubilized membrane fraction. (A) Approximately 2 mg of protein from the fraction was injected onto an AX-300 anion-exchange column (250 mm X 4.1 mm) that was equilibrated with a 20 mM sodium acetate buffer (pH 6.3) containing 0.1 mM Z-314. A 20-minute linear salt gradient of 20 mM to 2 M sodium acetate (pH 6.3) with 0.1 mM Z-314 (top) was used to elute proteins. The protein profile is shown in the bottom panel. The flow rate was 1 mL/min, and the absorbance was monitored at 280 nm. Fractions of 1 mL were collected and immediately put on ice. For the amount of ATP pyrophosphohydrolase activity contained in each column fraction, the following assay was performed. To 75 /xL of each column fraction was added a reaction mixture such that 0.4 mM ATP, 0.4 mM MnCl2, and 50 mM Tris-HCl (pH 7.4) were in 100 fiL final volume. The reactions were run at 31°C for 2 hours and terminated at 155°C for 1 minute. An analysis of the reaction components was done by reversed-phase HPLC using /xBondapak C)8 column. The mobile phase was 65 mM KH2PO4 (pH 3.6), 1 mM tetrabutylammonium phosphate, and 2% acetonitrile. Assay tubes samples of 20 piL were analyzed, and the amount of AMP formed by the pyrophosphohydrolase in each column fraction is expressed as nanomoles of AMP per minute microliter. (B) Approximately 60 mg of protein from a detergent-solubilized membrane preparation was applied to a DE-52 column (30 cm X 2.5 cm) that had been equilibrated with a 20 m M sodium acetate buffer (pH 6.3) with 0.1 mM Z-314. After washing with 200 mL of buffer, a linear salt gradient (top) of 20 mM to 2 M sodium acetate in 2 L (total volume) was used to elute the proteins. Fractions of 15 mL were collected at 4°C, and absorbance was monitored at 280 nm. The protein profile is shown in the bottom panel. For the amount of ATP pyrophosphohydrolase activity contained in each column fraction, the enzymatic assay as described above was performed.

5.8 PROBLEMS RELATED TO THE ASSAY OF ACTIVITIES FOLLOWING THEIR PURIFICATION BY HPLC

While most of the problems in the assay of an activity purified by HPLC are expected and typical of chromatographic work with enzymes, the introduction of this technique into the purification scheme may lead to problems if the fractions obtained from the HPLC purification step are to be measured for enzymatic activity. For example, the salt in each fraction may inhibit any enzymatic activities it contains. Moreover, when ion-exchange HPLC is used the salt concentration will vary in the fractions. Thus it is prudent to study the effects of salt, at the concentration used for elution, on enzyme activity before the chromatography. If the salt is found to be detrimental, it will have to be eliminated or at least reduced in concentration before the chromatography. Removing the salt by dialysis may not be the appropriate way to proceed, however, since the inactivation of enzyme activities is not always reversible.

In addition, the detergents often added to enhance the solubility of proteins can cause problems: They can inhibit activities directly or, if they are removed, the resulting loss of solubility can cause precipitation. However, when HPLC is used for the purification, more often than not the protein concentration of the sample will be low, and the concentration of protein in each of the fractions will be lower still. Therefore, the formation of a precipitate may not be visible, and monitoring the sample at 320 nm, a wavelength useful in monitoring any light scattering, may be the only reliable method of detecting precipitates. As a precaution, it is good practice to dialyze the sample against the mobile phase that is to be used for the elution. When a gradient is used, dialysis against the starting and ending buffers should be carried out as well.

Many detergents, such as Triton X-100, absorb in the ultraviolet range and will therefore interfere with the detection of proteins at 280 nm. The use of nonabsorbing detergents will eliminate this problem. Detergents can also interfere with the operation of ion-exchange columns, and a reduction in detergent concentration may be required for the correct performance of the ion-exchange packing materials.

For example, we found that a 10% zwitterionic detergent was required for complete solubilization of the membrane fraction. However, we also found that the presence of detergent blocked the retention of some proteins on the ion-exchange column and, further, that dialysis of the proteins to remove the detergent resulted in the prompt precipitation of the protein. The problem was solved by trial and error. The protein was dialyzed against detergent solutions of various concentrations until a concentration low enough to permit ion-exchange chromatography but high enough for solubility was found.

Again, it is emphasized that because salt solutions often act to precipitate detergents, as in the precipitation of sodium dodecyl sulfate by potassium, it is necessary to check the solubility of the protein solutions in the detergent against the salt solution at the concentration that will be present at the conclusion of the gradient.

Two other problems often arise following the use of HPLC for purification. The first has to do with the volume of the sample used for assaying activity. Upon successful completion of the ion-exchange step, it is necessary to determine enzymatic activity. These determinations are performed on samples taken from a series collected during the course of the purification. With HPLC purification, however, the volume of each sample collected will probably be no more than a few hundred microliters, and often less. Further, the number of samples is usually small. This situation in HPLC is in contrast to that found in traditional chromatography, where the volume of each sample can be in the milliliter range and the total number of samples or fractions collected can be in the hundreds.

Since to locate the enzyme, it is necessary to assay each of the fractions for activity, the concentration of salts will not be the same in each fraction when gradients are used. And since salts often inhibit activity, false data may be obtained on the distribution of activity across the column if salts remain in the sample.

Finally, the pH at which the purification is carried out may not be suitable for the assay. To solve this type of problem, many investigators, particularly those more familiar with fractions containing volumes such as 10 to 20 mL, usually adjust the sample solution to the assay conditions, either by dilution of an aliquot of the fraction into the buffer used for the enzyme assay or by removing 1 to 2 mL from each and, by dialysis, changing the buffers.

Of course, with the small volumes involved, dialysis is often out of the question. Also, dilution usually consumes most of a given fraction. Therefore, it is necessary to be prepared to carry out the separation step more than once.

Most if not all the problems associated with salt, pH, and the presence of organics can be minimized if not eliminated following concentration by using one of the newly developed microconcentration systems.

5.9 SUMMARY AND CONCLUSIONS

Two considerations dominate the development of a strategy for the purification of enzymatic activities: the choice of the sample to be used as the source of the enzyme and the extent of the purification.

The samples that can be used as a source of starting material can be divided into three groups: multicellular (I), cellular (II), and subcellular (III).

For samples in group I, which includes tissues, organs, biological fluids, and cultured cells, one task is to separate the cellular from the extracellular compartment and another is to obtain a homogeneous population of single cells. These cells constitute the samples of group II. Group III contains samples obtained after cell lysis and includes organelles and purified proteins.

HPLC can be useful for the purification of proteins to homogeneity. It can also be useful as an analytical tool to monitor the purification of proteins using other more preparative procedures.

When HPLC is used to purify enzymes and the enzymes must be located by an analysis of the fractions collected during the separation, the solvents used in the purification may cause problems to develop.

GENERAL REFERENCES

Cell separation techniques

Owen CS (1982) Magnetic cell sorting. In Cell Separation: Methods and Applications, Vol. 2, TG Pretlow and TP Pretlow, Eds. Academic Press, Orlando, FL.

Pretlow TG, Pretlow TP (1981) Sedimentation of cells: An overview and discussion of artifacts. In Cell Separation: Methods and Applications, Vol. 1 TG Pretlow and TP Pretlow, Eds. Academic Press, New York, 1981.

Quintner MI, Kollar EJ, Rossomando, EF (1982) Exp. Cell Biol 50:222.

Regnier FE (1984) HPLC of membrane proteins. In Receptor Purification Procedures, Vol. 2, JC Venter and LC Harrison, Eds. Liss, New York.

Waymouth C (1981) Methods for obtaining cells in suspension from animal tissues. In Cell Separation: Methods and Applications, Vol. 1, TG Pretlow and TP Pretlow, Eds. Academic Press, New York.

Protein and enzyme purification by HPLC

Hearn MTW (1994) Reversed-phased high performance liquid chromatography. In Methods in Enzymology, Vol. 104, WB Jakoby, Ed. Academic Press, Orlando, FL. Regnier FE (1984) High performance ion-exchange chromatography. In Methods in

Enzymology, Vol. 104, WB Jakoby, Ed. Academic Press, Orlando, FL. Schmuck MN, Gooding KM, Gooding DL (1984) / Liquid Chromatogr 7:2863. Unger K (1984) High performance size-exclusion chromatography. In Methods in Enzymology, Vol. 104, WB Jakoby, Ed. Academic Press, Orlando, FL.

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