ANALYTICAL

65, I37-

BIOCHEMISTRY

Isoelectric Gel

Focusing

Combined Detecting

i 52 (1975)

in Thin Layer

Polyacrylamide

with a Zymogram Method Enzyme Microheterogeneity: Sample

for

Application.

CYRIL J. SMYTH'ANDTORKEL

WADSTROM

DepartmerIt of Bacteriology, Statens BakteriologisXo Laboratorium. S-l 05 2 1 StocXholm. Suvden Received

July

30. 1974:

accepted

November

22.

1974

In the study of the microheterogeneity of the phospholipase C of Clostridium perfringens using combined zymogram gel isoelectric focusing, different methods of sample application were tested. Of fifteen filter papers tried Whatman 3MM, Whatman No. I and Schleicher & Schull 2043B were least adsorptive and had sufficient absorptive capacity. Their absorptive capacity was about I PI/~ mm’. However, with the sensitive egg yolk zymogram procedure used even these filter papers were shown to adsorb 1 pg protein/ 12 mm’), significant levels in relation to the sensitivity of the detection technique. Application of small samples directly onto gel surface increased the sensitivity of the system 200-400 times over loading on filter paper squares. It was observed that enzymic components were selectively adsorbed to filter paper when applied below their pl values on gels with preformed pH gradients, thus reducing the apparent microheterogeneity observed. Problems associated with other application procedures were noted.

Since the initial description of thin layer polyacrylamide gel isoelectric focusing (l), the technique has been subjected to methodological improvements (2- 10). The use of cooling plates for thin layer techniques (3,6) and of a constant wattage high voltage technique (4,g) have made it possible to perform separations within one hour thus reducing the probability of enzyme inactivation entailed in the use of prolonged focusing times (12). Zymogram procedures originally developed for the detection of enzymic activity after electrophoresis in agarose, starch gel, cellulose acetate, paper and polyacrylamide gel have been adapted to gel electrofocusing. These have involved enzyme staining in situ by soaking the gel in histochemical reagents, the use of agar or agarose overlayers containing appropriate substrates and contact techniques between a substrate gel or paper impregnated with substrate (9,lO). Furthermore, a simple approach circumventing pH and inhibition problems associated ’ Present address: New York University 550 First Avenue, New York. NY 10016.

Medical

137 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

Center.

Department

of Microbiology.

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AND

WADSTROM

with the presence of carrier ampholytes (11,12) in the gel, for the detection of enzymic activities by zymogram procedures has recently been described (13). Various methods for sample application have been used with thin layer electrofocusing: filter papers of various grades (14-16); slots, basins or wells in or at the edge of gels (17-20); basins on the surface of the gel (21); polymerisation of the sample in situ (22); cellulose acetate membrane (23); dried down polyacrylamide pieces containing Ampholine (3); cover slips (24); application of small volumes of liquid over rectangular areas of the gel surface, or as streaks or directly as small drops (1,3,25). Most workers appear to have made their choice of method empirically or this was determined by the design of the particular apparatus used. No attempt to evaluate different application techniques systematically with respect to thin layer polyacrylamide isoelectric focusing has been reported. During zymogram gel electrofocusing studies on the phospholipase C of Clostridium per-ringens (EC 3.1.4.3.) observations of general significance were noted indicating that the method and site of application of enzymes to be subsequently detected by a zymogram procedure after thin layer polyacrylamide gel isoelectric focusing can affect the amount of activity detected and, more important, the degree of heterogeneity observed. MATERIALS AND METHODS Chemicals. Phospholipase C (EC 3.1.4.3.) from Clostridium perfiingens, labeled PLC 2CA, 2.56 mg/ml, 86 U/mg, was obtained from Worthington Biochemical Corporation, Freehold, NJ. Carrier ampholytes, Ampholine, were purchased from LKB-Produkter, StockholmBromma, Sweden; acrylamide and N,N’-methylene bisacrylamide, from B.D.H. Chemicals Ltd., Poole, England; riboflavin, sorbitol, CaCl, and ZnCl,, from E. Merck AG, Darmstadt, Germany; agarose, from I’Industrie Biologique Francaise, Gennevilliers, Seine, France; Coomassie Brilliant Blue R-250, from Svenska ICI Aktiebolag, Goteborg, Sweden; standard phosphate buffer, from P-H Tamm, Altuna, Sweden; human hemoglobin, from Nutritional Biochemicals Corp., Cleveland, OH; ovalbumin from Miles-Seravac Ltd., Maidenhead, Berks., England. All other reagents used were of analytical grade. Equipment. Glass cooling plates for apparatus constructed in this laboratory (8) were made by AB Wicklunds Glasinstrument, Stockholm, Sweden. A LKB Multiphor 2117 prototype was made available to us by H. Davies and C. Karlsson of LKB-Produkter. Applicator materials. The following brands of filter and chromatography paper were employed: Whatman No. 1, No. 3, 3MM, 542

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from Whatman Biochemicals Ltd., Maidstone, Kent, England; Schleicher & Schull No. 2043B, as supplied by LKB-Produkter with their LKB 3276 paper electrophoresis equipment; Munktells No. 00, OB, 3, 5, 20R, and 1300, from Grycksbo Pappersbruk AB. Grycksbo, Sweden; LKB electrofocusing strip, supplied with the LKB Multiphor 2117, from LKB-Produkter; Beckman Blotters, No. 324326, from Beckman Instruments, Inc., Palo Alto, CA: Absorbent strips No. 5 1291, from Gelman Instrument Comp., Ann Arbor, MI; Millipore AP 100420R, from Millipore Filter Corporation, Bedford, MA. Cellulose acetate membranes were obtained from the following: Beckman electrophoresis membranes, from Beckman Instruments Inc.: Sepraphore III cellulose polyacetate electrophoresis strips, from Gelman Instrument Comp.; Millipore membrane filters HAWP 14250, (0.45 pm), from Millipore Filter Corporation. Glass fibre filter paper, Grade GF/B, was obtained from Whatman Biochemicals Ltd. Oxford Sampler micropipettes for lo- and 20-~1 volumes were purchased from Oxford Laboratories International Corporation, Dublin, Ireland and Wiretrol capillary pipettes (10 and 20 ~1) from Drummond Scientific Company, Broomall, PA. A water repellent plastic strip was kindly supplied by H. Davies and C. Karlsson of LKB-Produkter. Prepamtiotl of fiat bed polywrylamidc gels. The pH 5-7 Ampholine mixture used comprised pH 5-7, pH 3-6, and pH 6-8 Ampholine in the ratio, parts by volume, 3: 1: 1, made from 40% (w/v) solutions as supplied by the manufacturer. The following recipe was used to prepare four gels 100 X 195 X 1.5 mm (8) or two gels for the LKB Multiphor 21 17, (115 X 250 X 2.0 mm): 30% (w/v) acrylamide, 19.8 ml; 3% (w/v) methylene bisacrylamide, 10.4 ml: pH 5-7 Ampholine mixture, 6.25 ml: sorbitol, 12.5 g; riboflavin (5 mglml), 1.25 ml: distilled water to a final volume of 125 ml. Thus the final concentration of Ampholine was 2% (w/v), and acrylamide, T = 5.0% (w/v) and C = 5.0% (w/v) (26).” Gel isoelectric f&wing. Gel electrofocusing was performed at 60 W using a constant power regulator (26) to a final potential of 1800-2000 V. The separation time was 60 min.:{ In all experiments the electrode L It should be emphasized that the particular gel concentration used was found suitable for C. p@?r~gens phospholipase C, but is not necessarily minimally restrictive for enzymes of all molecular weights. Large pore gels can be obtained by choosing C values of 10, 1.5, or 20% (w/w). Such gels may be suitable for zymogram studies with high molecular weight enzymes. but present certain problems in handling (37). ” To gauge the time to optimal focusing for any particular protein. sperm whale myoglobin and the protein under test should be applied near both electrodes in single tracks of the gel. After fusion of identical sperm whale myoglobin bands. slices of the gel containing the test protein can be stained at intervals to determine when focusing has been achieved. From such experiments the minimal time to focusing for the test protein can be judged with reference to myoglobin which can be included with each run (7-9).

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solutions were 1 M H,PO, (anode) and 1 M NaOH (cathode) applied to the surface of the gels on IO-mm wide filter paper strips (Gelman Absorbent Strips Cat. No. 5 129 1). The cooling water was at 4°C. Applicators and sample application. Filter paper, and cellulose acetate and polyacrylamide squares were 8 X 8 mm. The latter were cut from gels cast with and without pH 5-7 Ampholine mixture (vide supru), from which 10% by weight of water was allowed to evaporate at room temperature. The plastic strip was cut into 15 x 15 mm pieces in which 8 x 8 mm wells were cut. Samples of phospholipase C of 10 or 20 ~1 (1 mg/ml and 0.5 mglml, respectively) were soaked into these applicators immediately after positioning on the surface of the gel. In addition equal volumes were placed directly onto the surface of the gel in the form of rectangular drops or streaks using Oxford sampler micropipettes and Wiretrol capillary pipettes. Three different sites on gels were tested with each application technique, as recommended by some authors (3,4.10). Applicators were removed after the first 20 min of each run. Measurement ofpH gradients. A flat membrane Ingold surface electrode Lot 403-30 (24) was obtained from W. Ingold AG, Zurich, Switzerland. pH measurements were made with a Radiometer type PHM 29b pH meter (Radiometer A/S, Copenhagen, Denmark) calibrated with standard sodium phosphate buffer. Measurements of pH on the gel surface were performed at 4°C with the gel plate in position on the cooling plate, at intervals of 5 mm on a line from anode to cathode, 3-4 cm in from the edge of the gel. Washing the gel for overlayering. This was performed as previously described ( 13) with certain modifications. The gel. still attached to its glass base, was soaked in 0.4 M Tris-HCl buffer, pH 7.4 made 1 mM with respect to CaC12 and ZnCl, (28). Soaking proceeded at 20°C for 8 min. After equilibration the gel was removed, excess buffer allowed to drain from the surface, fluid at the edges mopped off with absorbent paper and the gel surface “dried” off in a draught of cool air for l-2 min at room temperature. The equilibration technique in 0.4 M buffer adjusts the pH of the gel rapidly except at the anode (13). Even the 8 min equilibration period used in this study did not achieve this. On overlayering the polyacrylamide gel with lecithin- or egg yolk-containing agarose, a zone of opacity some 5-10 mm wide developed in these overlayers where the filter paper wick soaked in 1 M H,PO, had been located. If this zone was not excised by cutting off approximately 1 cm of gel plus overlayer from the anode edge of the gel, it developed and spread. Drying the surface of the gel prior to overlayering with substrate-containing agarose was instituted to avoid blurring effects resulting from

ISOELECTRIC

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rapid spread of the enzyme and/or its products in a thin film of moisture between the polyacrylamide and agarose gels. It was also essential to have good contact between the polyacrylamide and the agarose otherwise slipping of the overlayer on the polyacrylamide gel surface occurred during subsequent handling of the plate, leading to the production of blurred or an increased number of zones. Zymogram procedure. The gels on their glass supports were placed in Plexiglass molds 4-mm in depth made to fit the appropriate glass plates. After preliminary experiments final substrate concentrations of 1.5% (w/v) lecithin and 10% (v/v) egg yolk emulsion in the 1% (w/v) agarose overlayers were chosen. Decreasing the substrate concentrations below these values gave opacity zones of decreased contrast with respect to the substrate overlayers with diffuse indistinct edges. Lecithin was emulsified in Tris-buffered saline (TBS) (0.15 M sodium chloride in 0.02 M Tris-HCl buffer, pH 7.4, made 1 mM with respect to CaC12 and ZnC12), using a MSE ultrasonic disintegrator (Amplitude 8 pm for l-2 min, 20°C). The overlayer was prepared by mixing equal volumes of lecithin emulsion and 2% agarose dissolved in TBS and cooled to 56°C. An egg yolk was separated and washed carefully with 0.85% (w/v) NaCl to remove residual albumin. It was then mixed with 25 ml of TBS and centrifuged at 20,OOOg for 30 min at 4°C. The supernatant when diluted 1: 1 with 2% agarose in TBS gave the overlayer. Gels were incubated at 37°C in a humid chamber and observed for the development of opacity zones, which were recorded photographically or drawn carefully on graph paper. Kodak photomicrography colour film (PCF-36 2483) was preferred as it seemed to give higher contrast than several black and white films tested. Staining of gels. The gels were fixed, stained with Coomassie Brilliant Blue R-250 and destained as previously described (8). Preranning of gels to establish pH gradients. To establish the pH gradient and attain the normal end-of-run conditions in the gel prior to application of samples, gel electrofocusing was performed as described for 1 h. The electrode strips were removed and the pH gradient measured. Thereafter fresh electrode strips were applied at the anode and cathode and samples applied at predetermined pH values on the pH gradient. Electrofocusing at 2000 V recommenced for a further 1 h, the applicators being removed after the first 20 min. Preparation of reconstituted gels. These gels were prepared with the normal composition, but without Ampholine using 0.02% (w/v) ammonium persulphate as catalyst. The gels were then soaked in 1 liter of 10% (w/v) sorbitol overnight at 4”C, weighed on the glass plates and the weights of the gels reduced by 10% by allowing water to evaporate from them at 20°C. They were placed in 3% (w/v) of pH 5-7 Ampholine mix-

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ture for 24 h at 4°C. The gels usually peeled off their glass plate supports at this stage, but could be made to readhere. Finally, gels were reweighed and water perevaporated until the original weights were regained (29). RESULTS

Assessment of application techniques. With the preparation of phospholipase C used in these studies three characteristic opacity bands appeared in the overlayers with pIs of 5.6, 5.3, and 5.2 (average values from 20 determinations). Of these, that with the lowest pI was weakest and usually developed after the appearance of the other two. Fifteen different filter papers were tried as applicators. It can be seen from Fig. la-c that opacity zones failed to develop when samples were applied on some of the filter papers towards the anode edge of the gel. The nature of the filter paper applicator and its position on the gel affected the rate of development of opacity bands in other cases. Some filter papers had inadequate absorptive capacity, e.g., Munktells 00 and 20R. Of those tested Whatman 3MM and Schleicher & Schiill 2043B possessed adequate absorptive capacity for these experiments and gave the best results with samples applied near the anode. In order to test the general applicability of the findings with filter papers similar experiments were carried out on broad pH 3.5-10 gradients (8) using ovalbumin and hemoglobin loaded in lo-pug amounts. With the LKB electrofocusing strip no stained bands were detected with either protein whether applied close to the anode or cathode. With Millipore AP 100420R, Beckman Blotters and Gelman absorbent strips no protein bands were detected after staining when samples were applied anodically. Whatman No. 1, on the other hand, showed no obvious adsorption over control fluid spots when samples were applied near the cathode, but there was a marked reduction in the intensity and number of stained bands when the filter papers were applied near the anode. The other applicators and direct application of samples onto the gel surface were compared with Whatman 3MM filter paper pieces (Fig. 2). With the two different micropipettes employed, zymogram bands detected in tracks of the gel where the samples had been applied towards the anode, developed at the same rate and to the same intensity as bands developing in relation to other sites of application. This contrasts clearly with Whatman 3MM as an applicator. In virtually all experiments when material was applied directly onto the gel surface there was a tendency for stained protein bands or zymogram bands to appear crescent-shaped where application was near the anode, whereas samples applied at the cathode nearly always gave straight bands. The polyacrylamide pieces with or without Ampholine gave results

ISOELECTRIC

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143

FOCUSING/ZYMOGRAM

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FIG. 1. Development of phospholipase C zymograms applicators. Dotted squares mark the original positions of removal after the first 20 min of each electrofocusing run. agarose with TBS. Time of incubation was 4 h at 37°C. applicator was 10 pg (860 mu). Opacity zones denoted dotted area; distinct zone. open area; intense zone. shaded.

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with different filter papers as the filter paper squares before Overlayer was 1.5% lecithin in Amount of enzyme applied per as follows: faint visible zone,

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AND WADSTRtiM

- 5.50 - 5.15

PH

- 6.15 - 6.45 - 6.70 - 7.10 - 11.6

3.50 4.35 4.65 5.15 5.55 5.75 6.15 6.45

pH

6.70 7.10 11.2

FIG. 2. Development of phospholipase C zymograms with micropipettes and other materials as sample applicators. Dotted squares and rectangles indicate original application sites of removable applicators or the surface area of samples applied as free liquid onto the gel surface. The surface basins formed with the plastic strip are appropriately indicated. Overlayer was 1.5% lecithin in agarose with TBS. Time of incubation 4 h at 37°C. Amount of enzyme applied per applicator was IO pg (860 mu). Opacity zones denoted as in Fig. 1.

equivalent to those obtained with the micropipettes. As with dry filter paper squares, the polyacrylamide squares, once placed on the gel surface, could not be removed or repositioned without tearing the gel surface, unless they were soaked with sample or distilled water. Even when dried down slightly the polyacrylamide applicators did not readily absorb samples and were more difficult to manipulate than other applicators. Furthermore, when they were placed on the gel in the region where the phospholipase C finally focused, no opacity zones developed (Fig. 2). This was shown to be due to setting up of the pH gradient in such applicators. The polyacrylamide pieces unlike the filter papers have poor porosity so that the protein has to migrate off the applicator gel onto the separating gel surface. The Sepraphore and Beckman cellulose acetate gave identical results

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to those obtained with micropipettes. However, no opacity zones developed with samples applied on Millipore filter membranes. The cellulose acetate applicators also had a poorer absorptive capacity in comparison with Whatman 3MM filter paper. Millipore filter membranes should be used with caution as those normally supplied contain significant amounts of Triton X-100 as a wetting agent, about 2% (w/w). It is, however, possible to obtain membranes devoid of this agent if requested. Its presence in our case probably inactivated the enzyme and could explain the nondetection of opacity zones. The plastic strip was easy to handle, cut, apply and remove from the gel surface. However, tests with human hemoglobin showed that material applied in these surface basins leaked between the applicator and the gel surface resulting in broadened protein zones on focusing and subsequent staining. This explains the broader zymogram bands shown in Fig. 2. Wells, basins or slots in the gel were not considered suitable means of applying samples as with the high voltage technique it is essential to have intact gels of even thickness. Breaks in the gel result in local overheating at these points of increased resistance resulting in evaporation of water from the gel and burning or cracking of the gel. Application of samples OH preformed gradients. From the foregoing experiments there seemed to be a correlation between different applicators, different application positions, the initial pH of the gel and the pIs of the phospholipase C components. The initial pH of standard gel (5.5-5.7) was only marginally above the pIs of the three phospholipase C components studied. Gels were prerun to establish the pH gradients and samples applied on the preformed gradient every 5 mm from the cathode to the anode. The development of phospholipase C zymogram bands in relation to the initial sites of sample application on Whatman 3MM filter papers is shown in Fig. 3. When samples were applied from near the cathode to the region where the phospholipase C components finally focused, the pattern of bands was consistent with those observed in Fig. la-c. A sequential decrease in the activity and the disappearance of components was then noted as the filter papers were placed anodic to the pIs of these enzymic forms. After incubation for 24 h, zones of opacity developed in relation to all sites of application albeit very weak in the case of those nearest the anode. However, by this time a complete loss of resolution of zones that developed in tracks at earlier incubation times was observed. Identical findings were obtained with Whatman glass fibre and No. I filter papers. To exclude the possibility that the riboflavin catalyst and/or unpolymerized monomer in the gel contributed to the disappearance of zymogram bands with the use of filter paper squares. the experiment in Fig. 3

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SMYTH

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6.5 6.7 7.2 11.6

FIG. 3. Zymogram development following application of phospholipase C with Whatman 3MM filter paper squares onto a gel with a preformed pH gradient. (a) Opacity zones indicating phospholipase C activity photographed after incubation for 5.5 h at 37°C. Ten micrograms of enzyme (860 mu) was applied to each applicator. Overlayer was 1.5% lecithin in agarose with TBS. (b) Diagram showing relationship between opacity zone development, the pH gradient and the sites of application of the filter paper squares on the preformed pH gradient. The pH gradient profile did not change during the experimental run. The zymogram arrowed in (a) is represented by the extreme right pattern in the diagram. Opacity zones denoted as in Fig. 1, except that hachured zones were intermediate in intensity between those indicated by shaded or open areas.

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147

was repeated using reconstituted gels. Again the opacity bands disappeared sequentially in order of pI and decreased in intensity as the site of application became more anodic. The same experiment was performed and the gel fixed and stained for protein bands after the pH gradient had been measured. A visual correlation between the loss of zymogram bands and the protein bands was apparent. Since in all experiments filter paper squares had been removed routinely at 20 min, it might be argued that zymogram bands would have developed had the filter papers been left in position longer when applied near the anode. To test this hypothesis filter papers were applied 10 mm from the anode wick on a normal gel and one with a preformed pH gradient. Applicators were removed at 5-min intervals. No opacity bands developed in overlayers except in tracks of the gels where control spots of enzyme had been applied directly on the gel surfaces. However, filter papers removed at zero time and after 5 and 10 min of electrofocusing had detectable enzyme activity bound to them. This was detected by placing the removed papers on lecithin agarose plates. Filter papers removed at later time intervals had no detectable bound activity by this method. When identical experiments were performed using 10 or 20 ~1 of phospholipase C spread over rectanglular areas of the gel surface or using Sepraphore cellulose acetate squares as applicators, bands of phospholipase C activity developed with equal rapidity and corresponding intensity in relation to all application positions. The stability of phospholipase C in Ampholine oj- varying pH. The stability of the phospholipase C to pH was tested by diluting the preparation to 500 &g/ml with 2% (w~v) Amphohne in distilled water of pH ranges 3-5, 6-8, and 9-l I. A control sample was diluted with distilled water. These mixtures were incubated at 4°C for 1 h and then 20-yl samples applied as rectangular drops over the gel surface at the same three application sites used in Fig. 1. On development of the zymogram, three bands corresponding in intensity to those of the distilled water control developed within each track of the gel irrespective of Ampholine pH or the positions of application. Thus no apparent inactivation was observed by prior exposure to acid or alkaline Ampholine. Comparative sensiti\lity of overlayers and detection limits oj. enzymic activity izith diflerent upplicatiort techniques. Amounts of phospholipase C from 10.2 pg to 0.15 ,ug (20 ~1) were applied in parallel runs to standard pH 5-7 gels, using Whatman No 1 and Whatman 3iMM filter paper squares and drops of enzyme spread over rectangular areas of the gel surface. All samples were applied on a line 10 mm from the cathode reservoir strip. It can be seen from Fig. 4 that the egg yolk overlayer was some eight fold more sensitive than the lecithin overlayer. With

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detection limits of phospholipase C with different substrate overFIG. 4. Comparative layers. Amounts of phospholipase C applied as samples spread over rectangular surface areas of the gel surfaces were, reading from right to left: 10, 5. 1.5, 1.35, 0.61, 0.3 1. 0. I5 r.~g (860-11.8 mu). Zymograms photographed after incubation for 2 h at 37°C. (a) Overlayer was 10% egg yolk in agarose with TBS. (b) Overlayer was 1.5% lecithin in agarose with TBS.

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prolonging of incubation up to 8 h as little as l-3 mU of enzyme could be detected with the egg yolk overlayer. With Whatman No 1 applicators the limits of detectable enzyme were 860 mU and 430 mU with the lecithin and egg yolk overlayers respectively. With Whatman 3MM applicators, the detection limits were 430 mU and 2 1.5 mU respectively. Increasing the incubation time did not allow the development of opacity bands at lower protein loading in both cases. It can be calculated from these detection limit data that even under the optimum conditions in this system Whatman 3MM and Whatman No 1 absorbed eO.5 ,ug/13 mm’ and Q 1 pg/ 12 mm”, respectively.

DISCUSSION

Cellulose contains aliphatic alcohol groups which are easily oxidised to carboxyl groups and hence, in any of its forms, it behaves as a weakly acidic cation exchanger (30). The retention of solutes on cellulose involves adsorption and ion exchange processes (3 I ), adsorption being particularly marked at low pH (32). The adsorptive capacity of cellulose can be greatly affected by the method of preparation and purification which must ideally aim at removing impurities under conditions which do not bring about the above changes in cellulose structure (30). Cellulose present in most of the usual grades of filter paper has been reported to give rise to marked adsorption effects especially with basic proteins in the acid pH range (32). By contrast with application of samples for subsequent protein staining, the quantities of protein applied for zymogram procedures are often small, especially with purified enzymes, in order to balance enzymic activity with the resolution of components and the nature and sensitivity of the detection technique. Filter paper has been used with thin layer gel electrofocusing because of its absorptive properties and simplicity of application. At high protein loading adsorptive effects may be less obvious although the removal of minor protein components could still be important (33). Adsorption of sperm whale myoglobin and ovalbumin to filter paper has been noted at lower protein concentrations when these were placed anodically (4). Indeed in the case of sperm whale myoglobin this produced a completely different protein pattern. From these studies with phospholipase C the conclusion can be drawn that at low protein loading all the types of filter papers examined bound this enzyme. The problem was aggravated in this case by the use of a narrow pH gradient Ampholine mixture to resolve these components as this gave the gel an initial pH rendering the filter paper more adsorptive for the enzyme. Filter papers applied near the anode thus bound the en-

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zyme more tightly as the pH dropped. whereas, in most cases, those applied near the cathode released enzyme as the pH rose. The filter papers tested in this study differed considerably in both their absorptive and adsorptive capacities for 20 ~1 of material and 10 ,ug of protein, respectively. Several brands of filter paper rated highly in nonadsorptive capacity but could not absorb this small volume on the size of square used. As a general rule it would seem desirable to apply samples as small volumes to the gel surface. However. the use of Whatman 3MM, Schleicher & Schiill 2043B or Whatman No 1 employed by most authors can be recommended provided adequate preliminary studies are carried out with the system under study to discount the possibility of adsorption phenomena. About 3 mm’ of these filter papers should be allowed for each microlitre of sample. When large volumes are to be applied either because concentration of a large number of samples would be tedious or precipitation of proteins occurs, these can be applied as streaks from anode to cathode or in square glass basins applied firmly to the gel surface with this high voltage technique (3.2 1). The LKB electrofocusing strip and Gelman absorbent strip had high adsorptive properties. However, these effects could be markedly reduced by soaking squares in 2% (w/v) pH 6-8 Ampholine and drying them, thus allowing use of their excellent absorptive capacity (Smyth & Wadst~om, unpublished data). Different application sites on a gel between the anode and the cathode using filter papers as applicators have been shown to produce marked anomalies with narrow pH gradients. Robinson (16) observed that proteins did not focus with the same PI. Although not observed with phospholipase C, studies with other enzymes in this laboratory have revealed similar findings, e.g., subtilisin focused at pH 7.0 when applied towards the anode, but at pH 9.0 when applied near the cathode, as detected by a zymogram procedure (13, T. Wadstriim, unpublished data). It has also been reported that when proteins with pIs below 4.5 were placed near the anode denaturation and artefacts resulted, and that cathodic reduction of proteins with alkaline pIs could lead to inactivation or the production of artefacts also (34). The application of pH sensitive proteins at or near their pls on gels with preformed pH gradients has been recommended (35). Frater (36) concluded from his studies with wool protein that many of rhe bands formed on a gel in which Ampholine had been polymerised were due to artefacts caused by sample positioning and that this problem was alleviated by prerunning gels or using reconstituted gels. With the phospholipase C used in this study the pH of application did not affect the pattern of protein or zymogram bands except when material adsorbed to filter papers. One must agree, however, that using prerun gels is a useful aid in initial investigations with any particular enzyme being analysed by zymogram procedures.

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ACKNOWLEDGMENTS The authors would like to thank C. Karlsson and H. Davies of LKB-Produkter for their advice and LKB-Produkter for the use of a prototype production model of their LKB Multiphor for these experiments. The skillful technical assistance of Marianne Kjellgren and Peter Allestam is gratefully acknowledged. We are also grateful to J. So’derholm, 0. Vesterberg and R. Eriksson for stimulating criticisms and ideas. This study was supported by a grant from the Swedish Medical Research Council ( 16X-2562).

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