63, 249-2.54 (1975)
N. P. GROOME Bacteriology
Department, University Universit.v Street.
Received March 19. 1974: accepted July 30. 1974
Technical details and preliminary results are described for a new flat bed gel isoelectric focusing method to study charge heterogeneity in neuraminidases. The method combines simplicity and high resolving power with the capacity to analyse multiple samples in a single experiment. Isoelectric points are obtained which agree favourably with those gained by other methods.
Recent work suggests that viral neuraminidases can occur in multiple molecular forms differing in charge ( 1,2). Isoelectric focusing in density gradients is a useful tool for accurate isoelectric point (~1) determinations, and for quantitative work, but has some disadvantages. One problem is the interference which all nonionic gradient materials cause in the enzyme assay for neuraminidase. Sucrose, sorbitol, ethylene glycol, and ficoll all consume periodate, and produce a chromophore in the Aminoff assay (3). This fact makes it necessary to remove the gradient material from column fractions before enzyme assay. The possibility of using an alternative assay procedure, such as the detection of methoxyphenol, with methoxyphenyl neuraminic acid as a substrate (4). is complicated by the finding that ampholytes themselves produce a blue chromophore with the Folin-Ciocalteau reagent (5). A further limitation of column electrofocusing is its inability to compare multiple samples in a single experiment. For these reasons a procedure was developed to study neuraminidases by electrofocusing in polyacrylamide gels. Vesterberg (6,7) has summarised the advantages of flat bed gel isoelectric focusing over the use of gel rods. The following experiments were performed with an L.K.B. Multiphor apparatus (L.K.B. Instruments. Inc.). METHODS
The solution used for the polyacrylamide Acrylamide N,N’, methylene
3g bisacrylamide 249
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0 1975 by Academic Press. Inc. Printed of reproduction in any form reserved.
gel was as follows:
in the United
Sucrose (analytical grade) 40% w/v Ampholine pH 3- 10 (L.K.B. Instruments, Inc.) Distilled water to
2.8 ml 56.0 ml After thorough degassing 4.0 ml of 0.025% riboflavin solution was added, and the mixture pipetted into the space between two glass plates separated by a rubber gasket. The dimensions of the gel were approximately 125 X 260 X 2 mm. Polymerisation was achieved by exposure to a fluorescent lamp for about 30 min. After polymerisation one glass plate was carefully removed to leave the gel attached to the other. Any air bubbles which crept under the gel at this stage were worked to the edge and expelled. The gel plate was either used immediately, or could be stored for several weeks at f4”C covered with polythene to prevent evaporation. When ready for use, it was carefully lowered on to the cooling plate of the Multiphor taking care not to trap air bubbles. Samples absorbed in a 10 X 15 mm piece of Whatman 3MM Chromatography paper were applied after dialysis against distilled water. Since only small volumes (0.1 ml) could conveniently be added in this way, it was often necessary to concentrate samples beforehand. Electrode solutions were applied in paper strips as described by Vesterberg (7). The gel was cooled from beneath by a flow of tap water ( 10°C 4 literslmin). Focusing was conducted across the gel width with an initial application of 300 V. The voltage was then raised in a series of 20 V steps at 10 min intervals until maximum power was obtained. This was always within 2 hr. Thereafter the voltage was set at 1000 V and maintained at this level for a further 2.5 hr. At no time did the power exceed 30 Ws. An article describing detailed procedures for isoelectric focusing in gels has recently been published (7). Detection
Protein was detected by Coomassie brilliant blue as described by Vesterberg (6). Neuraminidase activity was detected using the chromogenic substrate MPN (2-(3 methoxyphenyl)-N-acetyl neuraminic acid) as described by Tuppy and Palese (8). Zones of gel requiring enzyme staining were isolated using a 5% agar solution to form a ridge around the area. This served several functions which were essential to ensure good results: (1) it prevented access of MPN to extremes of pH where it is hydrolysed; (2) it ensured complete coverage of areas to be stained with minimal wastage of expensive substrate. The latter point is particularly important, since unless the pH 3-10 ampholytes are supplemented by other ranges (6). gels at the completion
of focusing possess a wavy upper surface unsuitable for the simple application of staining solution. The staining solution contained 1 mgiml of MPN and 1 mg/ml of the diazonium salt of 4 amino-25 dimethoxy-4nitro azo-benzene (Black K Salt) (Koch-Light, Colnbrook, England) in 0.1 M phosphate buffer pH 6.0. The exact volume of staining solution required depended on whether the approximate location of the active bands was known, or whether a larger area needed to be covered. Also, as discussed below, dilute samples requiring up to 1 hr of incubation with substrate solution were more extravagant since it was necessary to change the solution every 20 min. Under optimal conditions as little as 0.25 ml could display a neuraminidase band. However, usually 1.0-2.0 ml was applied, and spread out as a thin film with a piece of glass rod bent into a right angle. The plate was covered and incubated at 37°C. In all cases, zones of enzyme activity were indicated by a deep red colouration which was stable for long periods. Washing the gel in 7% acetic acid enhanced the contrast by lowering the brown background caused by the diazonium salt. Particular care was necessary to prevent breakage of the gel once it was free of the glass plate. No attempt was made to measure the pH gradient across the whole gel width. However, the pI value corresponding to a given neuraminidase band was estimated by leaving space between adjacent samples on the slab. After focusing, agar sealing, and enzyme staining, a rectangular piece of gel of dimensions approximately 15 x 2 mm was excised from a region adjacent to the band, and placed into a stoppered tube containing 2 ml of degassed. deionised water. The tube was flushed with nitrogen, and the following day the pH measured at the same temperature as the run. The agar sealing method referred to above is particularly important if pH measurements are to be attempted. It prevents any alteration in pH, due to the enzyme staining solution, by physically separating the sample lanes from those used for pH measurement. Preparation of Neuraminidases Clostridium perfringens neuraminidase and horse ferritin were Sigma reagents. Influenza virus strain X7Fl was obtained by courtesy of Dr. G. C. Schild, World Influenza Centre, Mill Hill, England, and propagated in lo-day-old fertile hens eggs. The virus was concentrated by isopycnic banding in a B15 zonal rotor and the neuraminidase released using the protease nagarse (9). Residual virus particles were removed by centrifugation and enzyme purification achieved on a column of Sephadex G200. Full details of this procedure will be described in a forthcoming communication (5).
It is well known that electrofocusing of large molecules to equilibrium in gels requires prolonged run times due to the sieving effect of the gel. The present work used two different purified neuraminidases. To ensure that the run conditions were adequate to focus these enzymes. a molecule larger than either of them (horse ferritin, Stokes radius 79 A) (9, IO), was focused and detected by protein staining in addition to the two neuraminidases which were detected by enzyme staining. The results are shown diagramatically in Fig. 1. Ferritin focused sharply into four major and two minor bands in the acidic region of the gel. An identical pattern was obtained when the sample application position was adjusted, suggesting that focusing was complete. Clostridium perfringens neuraminidase gave one major component of isoelectric point 5.1 and two very minor components. The viral enzyme gave at least four rather diffuse components with isoelectric points of 5.7, 6.5, 7.1, and 7.25. The possibility of additional minor components could not be excluded. DISCUSSION
The isoelectric point obtained above for the C. perfringens neuraminidase was in good agreement with that obtained (4.95) in density gradients ( 1). However, values for the four main components of the
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FIG. 1. Diagram of separations obtained using the flat bed gel isoelectric focusing procedure described in the text. Samples were applied on paper strips at the positions indicated. A - Ferritin - 100 pg: B -C/ostridium perfiingens neuraminidase - 100 pg; C - X7F1 neuraminidase - 10 pg. The specific activity of the Clostridium enzyme preparation used was 0.08 unitslmg protein. One unit of neuraminidase is the amount which releases 1 pmole of N-acetyl neuraminic acid from an excess of sialo-lactose in 1 min at 37°C in 0. I M acetate buffer pH 5.0.
viral enzyme were in complete disagreement with those obtained previously (2) for the same strain of influenza. This was not due to the gel electrofocusing procedure as in the authors experience X7FI enzyme, purified by their method (5). and focused in a density gradient consistently showed a major component of isoelectric point 7.2 and a partially resolved minor component of isoelectric point 5.7. In fact, the results obtained by the present authors are very similar to those given for the neuraminidase from A2/Aichi/68 (I). It is not possible on the basis of the available information to explain these differences. Further experiments, to discover the significance of these multiple molecular forms, are in progress. No attempt was made in the present study to obtain a quantitative estimate of the amount of enzyme present in a band. A method has previously been suggested to adjust the pH of the gel after focusing to provide optimal conditions for enzyme assay ( 11). However, it has been found by us that the diazonium salt rapidly precipitates out of s$rtion at 37°C. This is due to the conversion of the azo grouping R-NrN to R-OH with the release of nitrogen (12). and is characteristic of all diazonium salts. Within approximately 20 min the staining solution fails to produce a colour when added to pure methoxyphenol. This effect would selectively favour development of colour in high activity bands unless the staining solution was frequently replaced. In view of the difficulty in obtaining MPN most workers will wish to avoid such extravagance. Clearly, any worker requiring more than a simple visual estimate of the amount of enzyme present in a band will have to study very carefully the factors which affect colour production. The precipitation phenomenon referred to above, can also impair resolution by producing broadened bands. Best results are to be expected if the enzyme activity applied in the sample is enough to produce bands inside 20 min. For the guidance of other workers we suggest 100 pg of C. perfringens neuraminidase and 50 pg of X7FI neuraminidase. It is as well to practise the manipulations with the bacterial enzyme before using any other neuraminidase. A narrower pH gradient could be used if required, and would be expected to give enhanced resolution. ACKNOWLEDGMENTS The authors wish to thank Mr. P. Scargill of L.K.B. tiphor apparatus and the Research Resources branch Bethesda, Maryland for the gift of MPN.
Instruments for the loan of the Mulof the National Institute of Health.
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