Laser excitation of fluorescent-labeled polypeptides in polyacrylamide gels

ANALYTICALBIOCHEMISTRY

179,291-298

(1989)

Laser Excitation of Fluorescent-Labeled in Polyacrylamide Gels Vivienne

E. Duffy,*

*Department

Received

of

September

Peter

Biochemistry

A. Stockwell,* and TDepartment

Don M. Warrington,f’ of

Physics,

University

of

and Brian

C. Monk*-’

Otago, P.O. Box 56, Dunedin,

New Zealand

81988

A laser beam at 488 nm, converted into a fan of light by a surface-coated mirror oscillated in response to a triangular wave, was inserted into the base of a polyacrylamide gel. The laser light was trapped by internal reflection and gave uniform illumination throughout the entire gel slab. Photography with color film detected 50 fmol of fluorescein covalently coupled to ovalbumin, gave 80-fold greater sensitivity than transillumination in detection of fluorescein-labeled polypeptides, and was about 25-fold more sensitive than protein staining with silver. Laser illumination visualized end-labeled &galactosidase, afforded quality control of such preparations, and demonstrated that the end-labeled derivative contained about 25-fold less fluorescein than uniformly labeled @-galactosidase. The latter result was confirmed by dot-blot analysis using a polyclonal antibody specific for fluorescein. The application of end-labeling to the location of features of protein primary structure is discussed. 0 1989 Academic Press,

Polypeptides

Inc.

Provided that fluorescence quenching is not significant, the proportion of excited fluorescent molecules is directly related to the input energy at excitation frequencies (1). This relationship holds until all the molecules of the fluorophore are in the excited state or until physical problems such as heat generation or photobleaching impose additional constraints on the system. One efficient way to excite fluorescent molecules is to use the coherent light emitted by a laser, since most of the energy available from this source can be adsorbed by the fluorophore while heat generation is minimized. Provided that reflection and scattering are also mini’ To whom all correspondence lecular Biology Laboratory, 6900 Heidelberg, FRG.

should Postfach

0003.2697/&39 $3.00 Copyright 0 1989 by Academic Press, All rights of reproduction in any form

Inc. reserved.

be addressed at European 10.2209, Meyerhofstrasse

Mo1,

mized, the use of the laser beam has the advantage that the appropriate filters can efficiently screen out the incident wavelength and allow the recording of only the fluorescent emission frequencies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)2 is frequently used to separate proteins and polypeptides. In many instances it would be useful to detect with sensitivity fluorescent polypeptides in fractions separated by this method, for example, after the labeling of the exposed surfaces of cells or vesicles with impermeant fluorescent probes, in the probing of antigens or glycoproteins with fluorescent antibodies and lectins, or in the comparative analysis of relatedproteins by alignment of end-labeled fragments generated by partial digestion (l-4). Cotrufo et at. (1) used a slightly diffused laser beam, inserted at either the side or the base of polyacrylamide gels, to excite single tracks or regions of interest containing fluorescent probes. They reported the detection of 50 ng of a commercially available preparation of FITC-labeled Con A by laser illumination compared with visualization of 1 pg by transillumination. We have modified their technique by inserting the laser beam as a fan of light at the base of the polyacrylamide gel. The fan of light allows the uniform illumination of the entire gel. The incident light is trapped by internal reflection and a simple filter affords transmission of the fluorescent light but allows only minimal passage of the exciting light to a camera which employs standard color film. The system detects 50 fmol of FITC covalently coupled to ovalbumin and has been used in the analysis of Ftc-end-labeled Ptc-blocked desamino acid 1-/3-galactosidase.

’ Abbreviations used: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; FITC, fluorescein 5’-isothiocyanate; Ftc-, fluoresceinthiocarbamyl-; TFA, trifluoracetic acid; BSA, bovine serum albumin; PBS, phosphate-buffered saline; Con A, concanavalin A; PITC, phenylisothiocyanate.

291

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AL.

A(i)

12k

,lm=-

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NOTES I-21 2N5496 a3,5,7 BC557.EC177 09,!7 ED140 (111.15 80139 013 PN3643 Cl19 MJ2955 0212N3055

FIG. 1. Configuration of the laser-based polyacrylamide gel illumination system. (A) Low-frequency power oscillator. (i) Circuit diagram for triangular wave generator. (ii) Circuit diagram for modified conventional power amplifier. (B) Setup for laser illumination: 1, Prefocusing lens (1.5-m focal length); 2, low-frequency power oscillator; 3, modified speaker with oscillating mirror; 4, leveling table with holder for the polyacrylamide gels; 5, camera stand; 6, incident laser beam; 7, fan of reflected laser light feeding into slit of gel holder. (C) Detail of speaker with oscillating surface-coated mirror: 1, Speaker cone; 2, microscope slide mounted on a sprung axle which causes the free end of the slide to rest against a reinforced portion of the speaker cone; 3, surface-coated mirror mounted symmetrically above the axle. (D) Detail of holder for polyacrylamide gel: 1, leveling table; 2, slotted support for gel cassette glass; 3, polyacrylamide gel on cassette glass; 4, slit.

MATERIALS

AND

METHODS

Materials Acrylamide, N,N’-methylene bisacrylamide, sodium dodecyl sulfate, high-molecular-weight protein markers, Escherichia coli P-galactosidase (grade IX), fluorescein isothiocyanate (FITC, isomer l), and Tween 20 were obtained from Sigma Chemical Co. (St. Louis, MO); gelatine from Difco (Detroit, MI); Bio-Gel PGDG from Bio-Rad Laboratories (Richmond, CA); nitrocellulose (Hybond C), carrier-free Na1251, donkey biotinylated

anti-sheep IgG, and streptavidin-horseradish peroxidase preformed complex from Amersham (Buckinghamshire, England); and phenylisothiocyanate (PITC) from Applied Biosystems (Foster City, CA). All other chemicals and reagents were of the best available quality. Low-Frequency

Power

Oscillator

Coupling of a triangular-wave generator to a slightly modified conventional power amplifier (Fig. 1A) produced a triangle wave, linear above 15 Hz with a peak-

LASER-BASED

DETECTION

OF

FIG.

FLUORESCENT

POLYPEPTIDES

l-Continued

to-peak voltage of 38 V (peak current of about 2.4 A into 8 ohms). The 566-function generator gave a triangle wave from near dc conditions to 1 MHz, the frequency of which was controlled by a capacitor connected at pin 7, a resistor at pin 6, and a control voltage at pin 5. The triangle wave output at pin 4 had a negative dc offset with respect to the power supply ground and required a coupling capacitor with the polarity shown. The audio power amplifier was a modified commercial kit (Dick Smith Electronics, Sydney, Australia, Kit K-3440). The circuit gave ample power handling and high linearity with output. It included two modifications to reduce lowfrequency rolloff which would otherwise be unacceptable at the operating conditions of about 20 Hz. Cl was therefore increased from 4.7 to 100 PF to prevent a rise in

input impedance at low frequencies while C3 was increased from 100 to 1000 PF to lower the frequency at which feedback increased (to lower the gain to unity under dc conditions). Larger scale circuit diagrams of the low-frequency power oscillator will be made available on application to the authors. The loudspeaker controlled by the oscillator was a low-frequency woofer of 200-mm diameter with a long-throw voice coil and a free air resonant frequency around 25-30 Hz. This allowed cone excursions in excess of 10 mm peak-to-peak before the onset of obvious nonlinear movement. The usual cone displacement during laser scanning was about 2 mm and should certainly be within the linear range for this speaker. Any low-frequency speaker with a long-throw voice coil should suffice for the purpose.

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Laser Illumination The experimental configuration for laser illumination of polyacrylamide gels is shown in Figs. lB-D. The argon ion laser (Spectra-Physics Model 166) was set to the optimal wavelength (488 nm) for excitation of fluorescein. The prefocused laser light incident on the loudspeaker-driven vibrating (about 20 Hz) surface-coated mirror (Figs. 1B and 1C) was converted into an oscillating beam functionally equivalent to a fan of light of uniform intensity. The acrylamide gel, retained on one side of the glass cassette used for electrophoresis, was placed on the stage illustrated in Fig. 1D. The stage was a purpose-built leveling table which included a slotted guide to support the edges of the gel plate and a horizontal 0.5 mm by 10 cm slit aligned to allow the fan of laser light to impinge, near its focal point, on the gel between about 0.15 mm above the supporting glass cassette plate and 0.15 mm below the upper surface of the O.&mm-thick polyacrylamide gel. Gels illuminated by the laser or by uv transillumination (Chromato-Vue Transilluminator, Ultra-Violet products, San Gabriel, CA) were photographed using ASA color film (Kodak, Auckland, New Zealand) in a standard 35mm single-lens reflex camera fitted with a Wratten No. 12 filter (Kodak). Fluorescent

Labeling of P-Galactosidase

FITC-labeling of the N-terminus of P-galactosidase was by a manual end-labeling procedure developed from related procedures described by Weiner et al. (5), Gill and Agabian (2), Jay (3), and Jue and Doolittle (4). Aliquots (50 pg) of P-galactosidase in plastic Eppendorf tubes were either uniformly labeled or end-labeled after dissolution in 50 ~1 of 2% SDS, 0.5 M sodium bicarbonate, pH 9. For uniform labeling 12.5 /*l of FITC (10 mg/ ml in dimethyl formamide) was added and for end-labeling 12.5 ~1 of 100% PITC was added, and the reaction mixtures were sealed under nitrogen. During the 30-min incubation at 50°C the samples were vortexed every 510 min to ensure saturation with the isothiocyanates. Equivalent amounts of FITC or PITC were added and the incubation was repeated. Nonbound isothiocyanate was removed by centrifugation through two Bio-Gel PGDG minicolumns (6) and the final eluate taken to dryness using a Speed Vat concentrator. The thiocarbamate derivative of the N-terminal amino acid of each sample was removed by treatment with 15 ~1 of anhydrous trifluoracetic acid (TFA) for 5 min at 50°C under nitrogen. After addition of 1 mg of solid SDS to facilitate subsequent dissolution, the TFA was evaporated using the Speed Vat concentrator. The dry protein residue was rehydrated in 100 ~1 of 0.5 M sodium bicarbonate, pH 9, and the free amino termini of the des-amino acid 1 derivatives were reacted with 15 ~1 of 10 mg/ml FITC in dimethylformamide for 45 min at 37°C. The excess

--El'

AL. *-

FITC was removed by centrifugation through two minicolumns and the samples were stored at -20°C in the dark until required for analysis. Chemical studies, which used acid hydrolysis or cleavage with anhydrous TFA to give FITC and dansyl chloride derivatives for analysis by thin-layer chromatography (7-ll), showed that the products of the steps of the manual Edman degradation procedure were as expected. Thus, uniformly Ftc-labeled des-amino acid l+galactosidase and the comparable Ptc-blocked species recovered after a single round of Edman degradation had the diagnostic N-terminal amino acid (methionine) available for reaction with FITC or dansyl chloride. In the Ptc-blocked des-amino acid 1 sample, the fluorescein was subsequently covalently attached only via the N-terminal methionine. In the uniformly labeled des-amino acid 1 sample most of the fluorescein was associated with the c-amino group of lysine and, as expected, a small proportion of the label was recovered as the methionine derivative. High-molecular-weight protein markers labeled with FITC (Ftc-protein markers) and Ftc-ovalbumin were prepared as described by Monk (12). The content of fluorescein associated with ovalbumin was determined spectrophotometrically (13). Preparation and Application of a Sheep Anti-Fluorescein Serum A sheep polyclonal serum which specifically recognizes fluorescein was prepared by a standard immunization regime using multiple-site subcutaneously injected Ftc-bovine serum albumin (Ftc-BSA) as the immunogen. The serum obtained following the second of two boosts (at 3 and 6 weeks) was fractionated using ammonium sulfate to give a crude IgG fraction (20-35% saturated ammonium sulfate cut) which was stored in 50% glycerol in phosphate-buffered saline (PBS) at -20°C. Dot blot analysis showed that the IgG fraction failed to recognize the BSA carrier used for immunization, ovalbumin, /3-galactosidase, or Ptc-blocked des-amino acid l-P-galactosidase, but reacted with Ftc-BSA or Ftcovalbumin. Dot blot analyses were carried out using standard procedures with a l/1000 dilution of the sheep anti-fluorescein serum IgG fraction. After antigen application, blots were blocked using 1% gelatine, 0.05% Tween 20 in PBS; washed and incubated with the IgG fraction (l/1000 dilution), biotinylated donkey antisheep second antibody, and streptavidin-horseradish peroxidase preformed complex (at concentrations recommended by the manufacturer) in the same buffer; and developed using 4-chloro-1-naphthol and hydrogen peroxide as substrates (14). Analytical Techniques SDS-PAGE was carried out in the Tris-bicine system described by Johnstone and Thorpe (15). Protein was

LASER-BASED

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determined by the method of Lowry et al. (16). However, FITC and PITC interfere with such assays. Therefore trace levels of lz51-labeled @-galactosidase were included with samples undergoing derivativization, and the protein content of samples was calculated from the lz51 recovered. The P-galactosidase was iodinated using iodogen as catalyst (12) and the nonincorporated label removed by column chromatography over Bio-Gel PGDG.

FLUORESCENT

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POLYPEPTIDES

A

-

RESULTS

With a laser beam power of 400 mW, at least 30 mW/ cm of the fan of light passed through the slit and was uniformly distributed along the length of the slit. The light incident on the flat bottom of the 0.8-mm-thick SDS-PAGE slab gel was mostly trapped by internal reflection and uniformly illuminated the entire gel. The bulk of the light emerged from the end of the gel opposite to its point of entry. The uniform illumination of the gel is demonstrated by the uniform excitation across the width of the gel of a display of equivalent loadings of SDS-PAGE-separated Ftc-protein markers (Fig. 2A). The uniformity of the excitation is further illustrated in Fig. 2B. This shows the same gel as that in Fig. 2A except that the gel was rotated 180” in the horizontal plane, so that the comb of the sample wells was the site of insertion of the laser beam into the gel. Slightly more light was scattered in this case, but the ratio of fluorescence in each band was maintained in the two configurations, showing that the illumination was still uniform through the entire length of the gel. Transillumination of the same gel (Fig. 2C) illustrates the considerable increase in useful fluorescent signal afforded by laser illumination. When the laser system was used to detect a dilution series of the Ftc-protein markers (Fig. 3A), 0.67 ng of protein per band was readily detected (arrow). The background of fluorescence streaking down some of the lanes appears to be due to some aggregation of labeled markers and to protein heterogeneity. The problem can be reduced by centrifugation of the SDS-solubilized markers immediately prior to application to the gel. Some background can also occur if the laser light is not properly aligned and scratches in the cassette glass are illuminated. Transillumination of the same gel (Fig. 3B) detected about 55 ng of protein per band (arrow), before the background of transmitted uv light became excessive. Figure 3 shows at least an 80-fold difference in sensitivity between laser illumination and transillumination. Tracks l-4 in Fig. 4A show that with about 200 mW of incident light, the laser illumination successfully detected 0.4 ng of Ftc-ovalbumin in a uniformly illuminated gel, although the Ftc-ovalbumin migrates as a relatively diffuse band during SDS-PAGE. Transillu-

El

c

FIG. 2. Laser illumination and transillumination of an SDS-polyacrylamide gel displaying separated Ftc-protein markers. (A and B) Laser illumination with the direction of illumination indicated. (C) Transillumination. A total of 1 pg of the Ftc-protein marker mixture was applied to each lane of the gel and separated.

mination of this gel barely detected the top loading (50 ng) of Ftc-ovalbumin. Silver staining of the same material detected 10 ng of Ftc-ovalbumin (Fig. 4B). Each ovalbumin molecule contained 5.7 mol of fluorescein (determined spectrophotometrically), indicating the

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123456709 4

B

FIG. 3. Sensitivity of laser illumination. (A) Laser illumination, photography for 5 s. (B) Transillumination, photography for 2 s. The indicated levels of each Ftc-protein marker band (assuming an equivalent contribution of each protein to the mixture) were separated by SDSPAGE. Lanes 1,5, and 9,166 ng; Lane 2,55 ng; Lane 3,18 ng; Lane 4, 6 ng; Lane 6,2 ng; Lane 7,0.67 ng; Lane 8,0.22 ng.

presence of 50 fmol of fluorescein in 0.4 ng of Ftc-ovalbumin. The laser technique had a further practical advantage over transillumination which gave additional sensitivity: Transillumination allowed photography of fluorescent gels for up to about 2 s before the transmitted light gave an excessive background whereas photography of the laser-illuminated gel allowed exposures of at least 25 s. Even with the extended photography, prints of the latter image yielded an advantageous black background to the yellow-green fluorescence.

FIG. 4. Laser detection of Ftc-ovalbumin. (A) Laser illumination, photography for 25 s. (B) Silver staining. The indicated loadings of Ftc-nrotein markers or Ftc-ovalbumin were separated by SDS-PAGE. Lane 1,0.4 ng Ftc-ovalhumin; Lane 2,2 ng Fm-ovalbumin; Lane 3,10 ng Ftc-ovalbumin; Lane 4, 50 ng Ftc-ovalbumin; Lane 5, 300 ng Ftcprotein marker mixture.

FIG. 5. Relative incorporation of FITC in uniformly and end-labeled P-galactosidase. (A) Laser illumination and silver staining. Lanes l-5 were illuminated with the laser and lanes 6-10 were the same gel after silver staining. Lanes 1 and 6, Ftc-protein marker mixture, 300 ng; Lanes 2 and 7, uniformly labeled Ftc-des amino acid l-@galactosidase, 28 ng; Lanes 3 and 8, uniformly Ftc-labeled des-amino acid l-pgalactosidase, 140 ng; Lanes 4 and 9, uniformly Ftc-labeled des-amino acid 1-B-galactosidase, 800 ng; Lanes 5 and 10, Ftc-end-labeled Ptcblocked des-amino acid 1-@-galactosidase, 800 ng. (B) Dot-blot comparison of FITC incorporation into fi-galactosidase and its derivatives. Rows 1 and 2, uniformly Ftc-labeled des-amino acid l-fl-galactosidase; Rows 3 and 4, Ftc-end-labeled Ptc-blocked des-amino acid l-bgalactosidase; Row 5, untreated @-galactosidase. The indicated samples were dotted onto nitrocellulose in fivefold dilution series beginning at 100 ng. The blot was probed with the sheep anti-fluorescein serum (l/1000 dilution) and developed as described under Methods.

A further indication of the sensitivity of the laserbased system is given in Fig. 5A in which the levels of fluorescence of uniformly and end-labeled P-galactosidase are compared. The end-labeled 116K molecule contains about 25-fold less fluorescein than the equivalent amount of the uniformly labeled species. The ratio of fluorescence labeling is consistent with the numbers of amino groups available in each preparation for labeling. A quantitatively identical estimate was made by immune dot-blot analysis (Fig. 5B). The fluorescein associated with each of the FITC-labeled molecules was detected using a fluorescein-specific polyclonal antibody raised in sheep. The Ftc-end-labeled des-amino acid 1 derivative of /3-galactosidase contains about 25-fold less immunoreactive material than the uniformly labeled sample. In control experiments the antiserum reacted faintly with unlabeled /3-galactosidase (Fig. 5B) and failed to react with the Ptc-blocked des-amino acid 1 derivative (data not shown). Preimmune serum gave no reaction with /3galactosidase or its derivatives (data not shown). The analysis was confirmed by Western blots (data not shown) which showed that about 90% of the fluorescent hapten in the two fluorescein-labeled preparations was recovered with the 116K P-galactosidase band and that the immunoreactive fluorescein was recorded in the same 25/l ratio expected from the dot blots and laser illumination.

LASER-BASED

DETECTION

OF

DISCUSSION

Instead of a relatively narrow beam of laser light penetrating limited selected regions of the acrylamide gel (l), the fan of laser light generated by an oscillating mirror uniformly illuminates the entire gel in both dimensions and with sufficient coherent light to detect levels of fluorescein-labeled molecules at least 80-fold lower than those possible with conventional transillumination. Fifty femtomoles of fluorescein attached to ovalbumin was detected with ease in laser-illuminated gels, giving a 25-fold increase in sensitivity over silver staining of the relatively diffuse glycosylated ovalbumin band. The laser-based system offers higher signal-to-noise ratios than transillumination because of several factors. The laser has a single wavelength (488 nm) close to the excitation maximum of the fluorophore while the transilluminator emits a broad spectrum of predominantly uv light. The laser therefore efficiently excites the fluorescein, and scattered incident light is easily blocked with the appropriate choice of filter. In addition, the bulk of the laser light is trapped by internal reflection as it is guided through the gel at 90” to the detector camera, significantly reducing the levels of incident light that must be blocked out by the filter. Neither photobleaching nor excessive heat generation occurs with the power level (about 300 mW) incident on the gel. In contrast to the limited exposure time possible with transillumination, the recording of the laser-induced fluorescence with commercially available color film gives a yellow-green fluorescent image on a background which blackens with the length of exposure. This suggests that even greater sensitivity could be obtained with increased exposure times, with higher speed films, and with optimization of the laser intensity used to illuminate the gel. One disadvantage of the laser-based system is the use of a relatively powerful argon ion laser. In the present study only about 10% of the available power of the laser was used. For many purposes a much lower powered laser could replace of the argon ion system. For example, a small (5 mW) laser combined with the use of films with higher ASA ratings could give sensitivities similar to those reported here. The laser-based system was used to detect an end-labeled molecule and provided further evidence of the specific end-labeling of the Ptc-blocked des-amino acid 1 derivative of /3-galactosidase. In addition to independent chemical data which showed that the fl-galactosidase preparation subjected to one round of manual Edman degradation contains a single N-terminal amino group available for reaction with FITC, laser illumination of the SDS-PAGE-separated polypeptides allowed the direct visual demonstration of the appropriate relative levels of fluorescein incorporated into denatured P-galactosidase (24 available FITC labeling sites) and the

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Ptc-blocked des-amino acid 1 derivative (1 available FITC labeling site). The ratio of labeling seen with laser illumination was verified by a separate analysis using a fluorescein-specific polyclonal antibody raised in sheep. Both approaches indicate that the bulk of the fluorescein-labeled protein obtained after the single round of Edman degradation and saturation labeling with FITC is recovered as intact 116K /?-galactosidase. Only a small proportion of the Edman-degraded material appeared to be internally nicked and gave fluorescent products of lower molecular weight. Laser illumination therefore allowed ready quality control of end-labeled preparations. Other studies, which used iodinated P-galactosidase to monitor the recovery of protein during the end-labeling procedure outlined under Methods, showed average recoveries of 50-60% of the input protein as the final endlabeled product. The remaining 40-50% of the material was lost principally during the minicolumn centrifugation steps required to remove unreacted PITC and FITC. The use of laser technology and the fluorescein-specific antibodies make it possible to detect readily the products of the partial chemical and proteolytic degradation of end-labeled P-galactosidase. This approach could be applied to the detection of other polypeptides which have a free amino terminus, are amenable to a single round of Edman degradation, and could therefore be labeled specifically at the N-terminus. The partial degradation of such end-labeled molecules can give valuable information about the location of cutting sites within the protein primary structure, help to order fragments generated by more complete degradation, and allow the alignment of sequences in related polypeptides (2,3,4). When this approach is combined with a suitable affinity chromatographic technique, it may also be possible rapidly to locate glycosylation sites and epitopes between cutting sites of definable primary structure distances from the labeled N-terminus. A similar technique has recently been described by Jay (17). Both laser-based polyacrylamide gel illumination and the fluorescein-specific antibody technique avoid a requirement for radioisotopes (3), should significantly reduce the amounts of protein needed for experiments dependent on end-labeling regimes, and make such approaches more readily applicable. The apparatus described here, provided that an appropriate laser is available, is considerably less expensive than laser-based systems for DNA sequence analysis which are under commercial development. Alternatively, Western blots may be probed with the antifluorescein antibody to size end-labeled polypeptides. ACKNOWLEDGMENTS Mr. N. O’Brien of the Department of Physics is thanked for his assistance in constructing the oscillating mirror and gel holder. This work was supported in part by grants from the Medical Research

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Council of New Zealand and the Cunningham sity of Otago. B.C.M. was a Senior Research Research Council of New Zealand.

Bequest Fellow

ET

of the Univerof the Medical

8. Gray, W. R. (1972a) in Methods in Enzymology (Hirs, C. H. W., Ed.), Vol. 11, pp. 121-138, Academic Press, Orlando, FL. 9. Gray, W. R. (1972b) in Methods in Enzymology (Hirs, C. H. W., Ed.), Vol. 11, pp. 333-344. Academic Press, Orlando, FL. 10. Woods, K. R., and Wang, K. T. (1967) Biochem. Biophys. Acta

133,369-370.

REFERENCES 1. Cotrufo, R., Monsurro, M. R., Delfino, Anal. Biochem. 134,313-319. 2. Gill,

P. K.,

and Agahian,

3. Jay, D. G. (1984)

J. Biol.

4. Jue, R. A., and Doolittle, 5. Weiner, A. M., Platt, 3242-3251.

N. (1982) Chem.

G., and

Geraci,

11. Croft, L. R. (1972) Protein Sequence Analysis, 2nd New York. 12. Monk, B. C. (1987) J. Immunol. Methods 96,19-28.

G. (1983)

150.925-933.

J. Bacterial.

13. Brodrick, J. W., Glaser, C. B., Largman, ceffo, M., Fasset, M., and Maeda, H.

259,15572-15578.

R. F. (1985)

T., and Weber,

Biochemistry K. (1972)

6. Tuszynski, G. P., Knight, L., Pipemo, (1980) Anal. B&hem. 106,118-122. 7. Kawauchi, B&hem.

AL.

H., Tuzimura, K., Maeda, (Tokyo) g&783-789.

Chem.

G. L., and Walsh, H., and Ishida,

C., Geokas, M. C., Gra(1980) Biochemistry 19,

4865-4870.

24,162-170.

J. Biol.

ed., Wiley,

247, P. N.

N. (1969)

J.

14. Hawkes, R., Niday, E., and Gordon, J. (1982) Anal. Biockem. 119, 142-147. 15. Johnstone, A., and Thorpe, R. (1982) Immunochemistry in Practice, p. 159-161, Blackwell, Oxford. 16. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 17. Jay, D. G. (1986) Biochemistry 25,554-556.