Covalent Labeling of a Protein An Experiment in Protein Technology Alexander J. Anderson Department of Medical Laboratory Science, Queensland University of Technology, GPO Box 2434, Brisbane 4000, Australia
An important area of modern protein chemistry is the covalent attachment of labels of various t m e s t o oroteins. Labeled proteins find application in m a n ~ ~ e c h n i ~such ues as radioimmunoassay, enzyme immunoassay, fluorescent antibody staining, and nucleic acid probes. T h e purpose of protein labeling is t o take a protein t h a t already possesses some specific property, for example, t h e ability to hind a particular ligand, and provide i t with a n additional readily measurable property by attaching a lahel. T h e labels may include radioisotopes, fluorescent dyes, haptens, specific ligands such as biotin derivatives, and enzymes. Many examples of this technology are too expensive or potentially hazardous for use in undergraduate laboratory classes. T h e original work of Rinderknechtl on the labeling of serum albumin with fluorescein isothiocvanate has been developed a s a simple experiment for undeigraduate teaching of t h e topic of protein labeling. T h e presence of the label is easily demonstrated b y its fluorescence, while bovine serum albumin is available a t reasonable prices, is readily separated from the reaction mixture by gel filtration, and its functional integrity can he measured b y its ability t o bind bromocresol green (BCG)2.
Experlrnental Detalls
investigation of the Labeled Product When the above reaction is complete, load 0.1 mL of the reaction mixtureonto the column,andelute withsaline. When the firstof the two colored zones approaches the bottom of the column, collect fractions of 5 drops (approximately 0.25 mL) until the zones are eluted. Load samples of the original protein, the reaction mixture, and both zones from the column onto the electrophoresis plate, and perform the electrophoretic separation. As soon as this is complete, view the plate under UV light while it is still wet, and note the position of any fluoreseent hands. Stain the plate with 0.2 g/L Ponceau Red in 50 g/L trichloroacetic acid for 5 min, then destain with successive rinses of 5% v/v acetic acid until the backaround ~~-~ loses all color. Note the position of any stained bands, and compare the positions of the protein bands with those of the fluorescent hands. Add 4 mL of BCG reagent to a 0.05-mL aliquot of the original protein and to a 0.1-mL aliquot of the first colored fraction eluted from the column. Serum albumin that retains its biological activity will produce a green color. Results and Dlscusslon When a protein is labeled, i t is important t o purify t h e product from unreacted protein and excess labeling reagent and to demonstrate t h a t the label has been incorporated and t h a t t h e specific biological activity of the labeled protein remains intact. Alterations t o t h e physical properties of pro-
Reagents The protein solution is preparrd hy dirsol\,ingbovinr serum albumin (Sigma Chemical Company, St. I.ouis, hlOl in phvriulogienl salinp(I1 151 mol L N\'nClltuatinalc~,ncmtratic,nof?Omgml..'l'he lah~lingrenpnr is prepared just hefore use by dianhing 20 mg of fluorescein iiuthioryanste (Sipma) in 1 mL of 50 g/L sudium hirarhmate. 'l'he BC'C reagent rur albumin consists of 6 X 10- md/L bn,mocrpadgrcrn in (1 u5 mol/L citrate buffrrpH :I.& contnining 3.0 mL of Brij 35 detergent per liter.
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Labeling Reaction To 0.2 mL of protein solution in a microfuge tube add 0.05 mL of the labeling reagent solution, and mix well. After incubation at 37 'C for 30min. stop the reaction by adding 5 mg of solid glycine, mix well, and incubate a further 5 min. While the above reaction is Droeeedinr. oack a 40- X 5-mm calmnn of Senhadex G-25 f~harkacia)inuohhvsioloeieal ~. . . .. saline. and prepare the elec~roph&is apparatus for use. 7 . h ~Helena < ~ e l e n a Lsburarcrler, Hmumont. 'l'X, Zip-Zone prucrdure fur swum electrophoresis on cellulose acetate plates is used in these laboratories; however, any reliable method for serum electrophoresis may he used. The advantage of the Helena system is that the position of fluoreseent bands can he marked on the Mylar hacking of the plate with a waterproof marking pen prior to staining for protein. 7~~~~~
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origin
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' Rinderknecht, H. Nature 1962, 193, 167-168.
Kachmar. J. F.: Grant, G. H. In Fundamentals of Clinical Chemis W;Teitz, N. W., Ed.; Saunders: Philadelphia, 1976: Chapter 7. Kabakoff, D. S. "Chemical aspects of enzyme lmrnunoassay". In Maggio. E. T.. Ed.; Emyme Immunoassay; CRC: Boca Raton. Fl., 1980; pp 71-104.
Diagram of the electrophoresis plate viewed under UV light (A) and after staining wim Ponceau Red (8). Lane 1: unreacted protein: lane 2: reaction mixture; lane 3: first colored zone eluted from the Sephadex column: lane 4: Second zone from the Sephadex column. Volume 66
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teins may result from labeling reactions, and these changes may affect the subsequent applications of the product or its purification from the reaction mixture3. Figure 1 is a diagram of the results obtained from the electrophoresis. When viewed under UV light the original protein is not fluorescent, hut the reaction mixture contains two fluorescent bands. Separation of these two hands on Seohadex G-25 demonstrates that the hand with lower electr&horetic mobility has a higher molecular weight than the faster movine hand. When the electro~horesis late is stained for protein, the original protein appears as a single shar~ hand while onlv the slower movine of the two fluorescent.hands is stained, appearing as a diffuse hand with a hirher mobilitv than the oririnal protein. This identifies the faker movingfluorescent band as excess reagent from the reaction and the slower moving fluorescent hand as labeled albumin and shows that no unreacted albumin remains a t the termination of the reaction. The faster migration rate and wider sample zone of the labeled protein shows that the physical properties of the protein have been modified. The faster migration rate shows that the ionic charge of the protein has been altered in the reaction, as would be expected since the coupling of fluores-
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cein isothiocyanate to the protein results in the loss of lysine amino groups and the acquisition of carboxyl groups, thus increasing the overall negative charge a t the pH of the electrophoretic separation. The increased width of the labeled protein zone compared to that of the original protein indicates that the number of label molecules attached to each protein molecule is variable, resulting.in a product with a &nge of ionic charges. The labeled protein retains its ability to hind BCG. The experiment thus demonstrates that a Gotein may he covalently modified in order to introduce the additional property of fluorescence while retainine its own s~ecifichindine function. I f t i m e permits, the experiment >an he extenzed to establish a quantitative relationship between fluorescence and dye binding capacity. I t is possible to quantitate the amount of dye incorporated through its absorbance a t 493 nm or by fluorescence with excitation a t 490 nm and emission measured a t 550 nm, and BCG binding capacity from the change in ahsorhance at 637 nm. Acknowledgment The author thanks T. Walsh for helpful discussion of the manuscript.
