245. Native Enzyme Mobility Shift Assay. (NEMSA): a new method for monitoring carboxyl group modification of carboxymethylcellulase from. Aspergillus niger.
Biotechnology Techniques, Vol 11, No 4, April 1997, pp. 245–247
1
1
Native Enzyme Mobility Shift Assay (NEMSA): a new method for monitoring carboxyl group modiÞcation of carboxymethylcellulase from Aspergillus niger Mohammad Hamid Rashid, Abdul Aala Najmus Saqib, Muhammad Ibrahim Rajoka and Khawar Sohail Siddiqui* National Institute for Biotechnology and Genetic Engineering, P.O. Box # 577, Jhang Road, Faisalabad, Pakistan
1
A simple, sensitive, accurate and more informative assay for determining the number of modiÞed groups during the course of carboxyl group modiÞcation is described. Monomeric carboxymethylcellulase (CMCase) from Aspergillus niger was modiÞed by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide. The different time-course aliquots were subjected to non-denaturing PAGE and the gel stained for CMCase activity. The number of carboxyl groups modiÞed are directly read from the ladder of the enzyme bands developed at given time. This method showed that after 75 min of modiÞcation reaction there were Þve major species of modiÞed CMCases in which 6 to 10 carboxyls were modiÞed.
24 pts min base to base from Key words to line 1 of text
1
1
1
Introduction Endo-b-1,4-glucanase (EC 3.2.1.4) or carboxymethylcellulase (CMCase) is a component enzyme of most cellulase systems which converts cellulose into oligosaccharides (Siddiqui et al., (1996). Chemical modification of different side chains in an enzyme is an important and widely used tool for studying structure-function relationship (Lundblad, 1995). The extent of modification is usually followed by aminoacid analysis (Lundblad, 1995), radioactive uptake (Siddiqui et al., 1993) or spectrophotometric analysis (Clarke and Yaguchi, 1985). All these methods either require specialized equipment or exposure to radiation. More importantly these methods, though sensitive, but only give average and non-integral values of modified residues because heterogeneous populations of modified enzyme are always present. Moreover the enzyme of interest should be very highly purified. A method for determining total number of modified residues of cysteines and lysines as well as heterogeneity of protein molecules has been described (Hollecker, 1989), but the conditions used are harsh. The enzyme is modified and electrophoretically run in the presence of 8 M urea, therefore the enzyme activity is lost. Moreover, because the gel has to be stained for protein rather than for activity, therefore, comparatively larger amounts of highly purified enzyme are needed. © 1997 Chapman & Hall
In this paper we report a sensitive new method [Native enzyme mobility shift assay (NEMSA)] for determining the number of modified carboxyl groups as well as heterogeneity of CMCase from Aspergillus niger. This method could be applied to any enzyme for whom a zymographic method is available, therefore, simultaneous determination of both enzyme activity and extent of modification is possible. Materials and methods Monomeric carboxymethylcellulase from Aspergillus niger NIAB280 was purified to homogeneity using FPLC (unpublished results) although it is not a precondition for the success of this technique. Native enzyme mobility shift assay (NEMSA) Carboxyl groups of CMCase from A. niger were activated by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide as a nucleophile (Hoare and Koshland, 1967; Siddiqui et al., 1993). Glycinamide (1 M) was added to two ml CMCase (11 U/ml) solution. The pH was adjusted to 5.5 with 12 M NaOH. The reaction was initiated by adding 0.02 g (50 mM) EDC. After appropriate time intervals, 150 ml of the reaction mixture were withdrawn and added to 150 ml of 0.5 M sodium acetate Biotechnology Techniques · Vol 11 · No 4 · 1997
245
M.H. Rashid et al. 1
1
buffer, pH 5.5 containing 1 M hydroxylamine to stop the reaction. The reaction was carried out for 75 min and then the samples were dialyzed against 10 mM Tris/HCl, pH 6.8, buffer to remove the excess reagents. All the samples were mixed with 43 native sample buffer in 3:1 (v/v) ratio and were subjected to 7.5% non-denaturing PAGE (Goldenberg, 1989) at a constant voltage of 100 volts. This gel was then used to develop overlay agarose gel for CMCase activity (Coughlan, 1988). The polyacrylamide gel was transferred to Coomassie blue G250 fast stain for total protein staining (Merril, 1990). Results and discussion The technique of Native Enzyme Mobility Shift Assay (NEMSA) has been successively applied to monitor the carboxyl group modification of CMCase from A. niger at different times (Fig. 1). The modification of a single
1
1
carboxyl by EDC in the presence of glycinamide neutralizes one negative charge. The CMCase molecules having one negative charge neutralized will migrate less compared with native band. Each lane of the gel shows a spectrum of CMCase (Fig. 1) molecules with 0, 1, 2 . . . Z number of modified carboxyl groups. As shown in Fig. 1 (different lanes), CMCase modified for any particular length of time shows a ladder of many bands indicating heterogeneity of the modified species. This information is not available when aminoacid, radioactive or spectrophotometric analysis is used to monitor chemical modification (Clarke and Yaguchi, 1985; Lundblad, 1995; Siddiqui et al., 1993). Moreover the use of above mentioned methods requires that the total concentration and molecular weight of the purified enzyme be known, whereas the only prerequisite for NEMSA is that an activity staining method of that enzyme be available. This also means that the enzyme need not be purified to homogeneity level for accurate results. Another important advantage of NEMSA is that one can correlate the enzyme activity and number of groups modified in one simple experiment with minute amounts of enzyme. No bands could be seen in the gel stained for protein by Coomassie Blue G250 (result not given) because of minute amount of protein. On the other hand as little as 1 ng of CMCase could be visualized by activity staining (Coughlan, 1988). In case of CMCase this method showed that after 75 min of modification there were five major species of modified enzyme in which 6 to 10 carboxyls were modified (Fig. 1, lane 7). Conclusions The simple technique of NEMSA was successfully applied to monitor the carboxyl group modification of CMCase from A. niger under native conditions but in practice it could be applied to any enzyme and modification method simply using a gel apparatus.
1
1
Figure 1 Native Enzyme Mobility Shift Assay (NEMSA) of CMCase modiÞcation. The enzyme was modiÞed by EDC in the presence of glycinamide. The aliquots taken at different time intervals were subjected to 7.5% non-denaturing PAGE. Electrophoresis was terminated when the tracking dye front reached the edge of the gel. The gel was then incubated for 30 min in 50 mM sodium acetate, pH 5.5 buffer with two changes. The gel containing CMCase was than overlaid on top of an 0.8% (w/v) agarose gel made in 50 mM sodium acetate buffer, pH 5.5 containing 0.1% (w/v) carboxymethylcellulose. The gel sandwich was wrapped in cling Þlm and incubated at 45¡C. After two hours the agarose gel was transferred to 0.1 (w/v) Congo Red solution. After 30 min the gel was transferred to 1 M NaCl solution for the visualization of spectrum of CMCase bands in each lane. The lanes from right to left shows time of modiÞcation reaction: 1, 5, 10, 15, 30, 50 and 75 min. The numbers from bottom to top of the lanes show the extent of modiÞcation.
246
Biotechnology Techniques · Vol 11 · No 4 · 1997
Acknowledgements The work described is part of the M.Phil (Biotechnology) research of Mr. A.A. Najmus Saqib. This work was financed in part by a grant made by the United States Agency for International Development under PSTC proposal 6-163, USAID grant no. 9365542-G00-89-42-00 and PAEC. We wish to acknowledge Dr. K.A. Malik, Director NIBGE, for providing research facilities. The technical assistance of G.A. Waseer is appreciated. References Clarke, A.J. and Yaguchi, M. (1985). Eur. J. Biochem. 149, 233–238.
Native Enzyme Mobility Shift Assay (NEMSA) 1
1
Coughlan, M.P. (1988). In: Methods Enzymol. 160, 135–144. Goldenberg, D.P. (1989). Analysis of protein conformation by gel electrophoresis. In: Protein structure, a practical approach, T.E. Creighton, ed. pp 225–250, Oxford: IRL Press. Hoare, D.G. and Koshland, D.E. (1967). J. Biol. Chem. 242, 2447–2453. Hollecker, M. (1989). Counting integral number of residues by chemical modification. In: Protein Structure, A Practical
Approach, T.E. Creighton, ed. pp 145–153, Oxford: IRL Press. Lundblad, R.L. (1995). Techniques in Protein Modification, Florida: CRC Press. Merril, C.R. (1990). In: Methods Enzymol. 182, 477–488. Siddiqui, K.S., Loviny-Anderton, T., Rangarajan, M. and Hartley, B.S. (1993). Biochem. J. 296, 685–691. Siddiqui, K.S., Azhar, M.J., Rashid, M.H. and Rajoka, M.I. (1996). World J. Microbiol. Biotechnol. 12, 213–216.
Received as Revised 25 February 1997
1
1
1
1
Biotechnology Techniques · Vol 11 · No 4 · 1997
247
