Preparative labeling of proteins with [35S]methionine

ANALYTICAL

204,85-89

BIOCHEMISTRY

(19%)

Preparative Labeling of Proteins with [35S]Methionine Leon W. Browder, Department

Received

Jillian

of Biological

November

Wilkes,

Sciences,

and David

University

of Calgary,

I. Rodenhiserl Calgary,

Alberta,

Canada

MATERIALS

Inc.

During the course of a study of in vitro protein synthesis using lysates of Xenopus laevis eggs, we observed intense labeling of distinct polypeptides when using different ATP regenerating systems as energy sources. These ATP regenerating systems utilize either pyruvate kinase plus phosphoenolpyruvate or creatine kinase plus creatine phosphate. Our initial interpretation was that these reagents were inducing specific protein synthesis in the lysates. This interpretation became questionable when we observed that labeled proteins appeared when the enzymes alone (without their substrates) were added to the lysates, and it became untenable when the proteins were labeled in the presence of either cycloheximide or chloramphenicol, inhibitors of cytoplasmic and mitochondrial protein synthesis, respectively. We searched for an alternative explanation of our results and demonstrated that the enzymes themselves were being labeled with [35S]methionine before being added to the lysates. We have examined this labeling reaction in more detail and have demonstrated that it has potential as a technique for rapid, simple, and efficient preparative labeling of proteins.

1 Present Address: Molecular Research Institute, Children’s Ontario, Canada N6C 2V5. 0003.2697192

Copyright All rights

IN4

27, 1991

The most common technique for preparative labeling of proteins with radioisotopes for experimental pur“‘1 . This isotope has certain limitations, poses utilizes including the emission of y- and X-irradiation, the release of gaseous “‘IZ from solutions of Na12’I, and the potential for concentration of 12’1 in thyroid glands. We have discovered a means for labeling proteins rapidly and simply with [35S]methionine. The technique is applicable to a wide variety of proteins. Antibodies labeled by our technique remain functional. ~1 iwz Academic Press.

T2N

Biology Laboratory, Child Health Hospital of Western Ontario, London,

$5.00

Cl 1992 by Academic Press, Inc. of reproduction in any form reserved.

AND

METHODS

Labeling Protocol Proteins to be labeled were dissolved in distilled water, added to [35S]methionine containing 0.1% 2-mercaptoethanol (Amersham, Cat. No. SJ.204; sp act, >800 Ci/mmol) in 0.5- or 1.5-ml microfuge tubes, and lyophilized. Typical labeling protocols utilized 60-90 &i [35S]methionine, 2-3 Fg protein, and distilled water in a total volume before lyophilization of 18-20 ~1. After lyophilization, the proteins were redissolved in 10 ~1 of buffer. Initial experiments utilized 50 mM HepesNaOH, pH 7.4, containing 50 mM KC1 and 5 mM MgCl,. Subsequently, it was learned that the KC1 and MgCl, are not essential, and they were no longer included in the buffer. SDS-Polyacrylamide Fluorography

Gel Electrophoresis

and

Each sample containing labeled proteinswas added to an equal volume of a solution containing 3.2% SDS,’ 20% mercaptoethanol, 16% glycerol, and bromphenol blue in distilled water (2~ SDS sample buffer) and was boiled for 3 min. Equal volumes of solutions of labeled proteins were loaded into wells of 10 or 12% polyacrylamide gels with 4.5% stacking gels and electrophoresed using the discontinuous buffer system of Laemmli (1). After electrophoresis, gels were fixed in 10% acetic acid, 45% methanol in distilled water and stained using either Coomassie brilliant blue R (0.05% in 50% ethanol, 10% acetic acid) or the silver staining procedure. For silver staining, fixed gels were soaked for 5 min at room temperature in a solution containing 0.1% K,Cr,O, in 0.2% nitric acid, rinsed four times with distilled water, soaked for 20 min in 0.1% AgNO, while being agitated, and rinsed first with distilled water, then with 3% Na,CO, containing 0.05% formalin for 30 s. Stain was developed

‘Abbreviations phate-buffered

used: saline.

SDS,

sodium

dodecyl

sulfate;

PBS,

phos-

85

86

BROWDER,

WILKES,

in 3% Na,CO, containing 0.05% formalin for 5 min under reduced light conditions. The staining reaction was stopped by placing the gel in a solution containing 1.5 g citric acid/300 ml of the developing solution, and the gel was rinsed with distilled water. After staining, gels were soaked in Enlightning (New England Nuclear) and dried. Kodak XAR-5 film was exposed to the gel at -80°C until it was developed. Peptide Mapping

of Labeled Pyruvate

RODENHISER

A12345678

4s36-

Kinase

Labeledpyruvate kinase was fragmented with Staphylococcus aureus V8 protease using the procedure of Cleveland et al. (2). For the labeling step, 150 &i of [35S]methionine (SJ.204) was added to a sample of pyruvate kinase (3 pg in distilled water), and the mixture was lyophilized. After lyophilization, the labeled pyruvate kinase was rehydrated in 53 ~1 of a solution containing 0.125 M Tris/HCl, pH 7.4,0.1% SDS for 15 min at room temperature, followed by the addition of SDS in glycerol to yield final concentrations of 0.5% SDS, 10% glycerol, and 0.05 PgIwl protein. Samples were then boiled for 3 min. Proteolytic digestion of aliquots of labeled protein was carried out for 30 min at 37°C with Staphylococcus aureus V8 protease in Hepes buffer at the concentrations indicated in the legend for Fig. 5. After the addition of an equal volume of 2~ SDS sample buffer, each sample was boiled for 3 min. Samples were then loaded onto sample wells of a polyacrylamide gel and electrophoresed as described above. Immunoblotting

AND

B12345678

FIG. 1. Labeling of proteins by colyophilization with [35S]methionine. (A) Requirement for colyophilization (fluorograph of 12% polyacrylamide gel). Creatine kinase (2.25 pg; lanes l-4) or pyruvate kinase (2.0 pg; lanes 558) was colyophilized with 60 &i [?S]methionine (lanes 1 and 5), was lyophilized alone before being added to [?S]methionine (lanes 2 and 6), or was not lyophilized and was either added to lyophilized [3”S]methionine (lanes 3 and 7) or to unlyophilized [?S]methionine (lanes 4 and 8). Total volume of each sample before lyophilization was 18 yl. Mobility of molecular weight markers in the gel is shown on the left (in kDa). (B) Coomassie blue staining pattern of the fluorograph shown in A, which demonstrates that equal amounts of protein were present in each lane.

with Labeled IgG

Samples containing pyruvate kinase were loaded onto wells of 10% polyacrylamide gels and subjected to electrophoresis as described above. After electrophoresis, gels were soaked in transfer buffer (0.025 M Tris, 0.192 M glycine, 20% methanol) for 30 min, and the proteins were transferred onto nitrocellulose (Schleicher and Schuell BA83) at room temperature using 24 V for 45 min in a Genie Blotter (Idea Scientific, Corvallis, OR). The nitrocellulose was blocked in phosphate-buffered saline (PBS) containing 0.3% Tween for either 1 h at 37°C or overnight at 4°C. Blocked nitrocellulose was probed with rabbit anti-chicken pyruvate kinase (1:200 dilution) in PBS-0.1% Tween for the same duration as for blocking, followed by rinsing three times in buffer. The blot was next probed with labeled goat IgGs (1:lOO dilution; see below), rinsed three times in buffer, and followed by rinsing with distilled water. After rinsing, the blot was dried completely at room temperature and sandwiched with Kodak X-Omat AR film in a cassette without an intensifying screen at -80°C. Care was taken to prevent the nitrocellulose from sticking to the film. In order to avoid static electricity, the nitrocellu-

lose was peeled slowly from the film before the film was developed. Goat anti-rabbit immunoglobulins (Jackson Laboratories) were dialyzed against distilled water and lyophilized in 150-Fg aliquots. In order to label an aliquot of IgG with [35S]methionine, a tube of lyophilized IgG was rehydrated in 50 ~1 distilled water, 500 &i of [35S]methionine (SJ.204) was added, and the sample was lyophilized. After lyophilization, the sample was rehydrated in 200 ~1 of 50 mM Hepes-NaOH, pH 7.4, for 30 min, diluted to 1 ml with the buffer, and dialyzed overnight against PBS at 4°C. RESULTS

Labeling of proteins was accomplished by colyophilizing the proteins and [35S]methionine in distilled water, after which they were dissolved in buffer and subjected to SDS-gel electrophoresis and fluorography. Figure 1 compares a fluorograph of pyruvate kinase and creatine kinase with the Coomassie blue staining patterns for these proteins. The fluorograph demonstrates

[%]METHIONINE

Coomassie

Blue

Silver

Stain

LABELING

Fiuorograph

FIG. 2. Comparison of sensitivity of protein staining tive labeling. Matched samples of labeled pyruvate 0.134, and 0.0268 pg, respectively) were electrophoresed stained with Coomassie blue (lanes l-3) or silver stain subjected to fluorography (lanes 7-g).

and preparakinase (0.67, and either (lanes 4-6) or

that protein labeling is dependent upon colyophilization of the protein with [35S]methionine, since minimal labeling is detected if the components are not colyophilized. Figure 2 is a comparison of the sensitivity of detection by protein staining and preparative labeling. Protein bands that are barely detectable by Coomassie blue are readily detectable on the fluorograph after short-term exposure. Silver staining and preparative labeling have apparently equivalent sensitivity. Since both of the proteins used in our initial experiments are kinase enzymes, we attempted to label nonkinases and nonenzyme proteins by this procedure. Figure 3 shows fluorographs of molecular weight standards (A) and goat immunoglobulins (B). All of these proteins are labeled, and the intensity of the bands corresponding to the molecular weight standards is roughly proportional to the intensity of their staining with Coomassie blue on the polyacrylamide gels (data not shown), suggesting that all proteins in the mix of molecular weight standards have roughly the same efficiency of labeling. The grade of [35S]methionine that we used in these experiments is relatively unstable, being subject to radialytic decomposition (3). We have examined the possibility that this instability is related to the ability to label proteins nonbiologically. Amersham markets a grade of [35S]methionine that is stabilized with 0.1% 2-mercaptoethanol and 15 mM 3,4-pyridinedicarboxylic acid (SJ. 1515). As shown in Fig. 4A (lane 2), labeling with this grade of methionine is ineffective, suggesting that radialytic decomposition of [35S]methionine is essential to achieve protein labeling. The necessity for radiolytic decomposition to label proteins by colyophilization was further tested by using either 3,4-pyridinedicarboxylic acid (15 mM) or the reducing agent dithiothreitol (1 mM) to stabilize unstable [35S]methionine (SJ. 204). As shown in Figure 4B, both of these reagents substantially reduce labeling. These

OF

87

PROTEINS

results support the hypothesis that the instability of [35S]methionine is related to its effectiveness in preparative labeling of proteins (see Caution). In our initial experiments, proteins to be labeled were dissolved in distilled water. Because stabilization of pH reduces the extent of radiolytic decomposition of methionine (3), we anticipated that the presence of buffer would reduce the effectiveness of preparative labeling. As shown in Fig. 4C, labeling is very inefficient in the presence of buffer. After proteins are colyophilized with label, they are routinely dissolved in buffer. Since buffering at moderately alkaline pH stabilizes proteins against radiolytic decomposition, we anticipated that label would be lost from proteins that are rehydrated in low pH solutions or in distilled water. As shown in Fig. 4D, the nature of the solution has a dramatic effect on the recovery of labeled pyruvate kinase. Lyophilized proteins dissolved in low pH buffer (pH 6.1; lane 1) or distilled water (lane 3) are much less radioactive than those dissolved in pH 7.4 buffer. In additional experiments, pyruvate kinase rehydrated in buffers in a pH range from 7.0 to 12.0 had equivalent radioactivity (data not shown). In order to determine whether the labeling of pyruvate kinase is restricted to terminal addition of label or is more generally distributed on the molecule, we subjected labeled pyruvate kinase to partial proteolysis with V8 protease. A fluorograph of the SDS-polyacrylamide gel is shown in Fig. 5A. The label is clearly present in multiple proteolytic fragments of pyruvate kinase, indicating that the label is generally distributed. The utility of this technique depends upon the retention of the biological properties of proteins after they have been labeled. Thus, we have examined the ability of immunoglobulins labeled by our technique to retain their ability to recognize their antigens in immunoblot

B

A

- Bo&i;;eswum

-

albumin -Heavy

Egg albumin (45 kDa)

chain (50 kDa)

“:&y .e ~a

- Glyceraldehyde-Sphosphate dehydrogenase (36 kDa) - Cadxmic anh drase (29 kDa) - Trypsinogen 724 kDa)

- LigM chain (25 kDa)

-Soybean lrypsin inhibitor (20.1 kDa) - Lactalbumin

(14.2 kDa)

FIG. 3. Preparative labeling (A) and goat IgGs (B).

of Sigma

molecular

weight

standards

88

BROWDER, 12

WILKES,

3

AND

RODENHISER

a consequence of radiolytic decomposition of the methionine, which is known to increase proportionately with an increase in P-radiation intensity (4,5). The progressive reduction in volume during lyophilization of an aqueous solution of [35S]methionine would progressively increase radiation intensity in the remaining material, producing radioactive decomposition products that would react with the protein molecules. Once formed, the labeled proteins themselves are apparently subject to radiolytic decomposition. Buffering reduces the extent of both the initial decomposition of methionine and the subsequent decomposition of labeled protein. This technique is a rapid, simple procedure for producingproteins labeled with 35Sthat should have a number of applications in analytical work. It is particularly noteworthy that the antibody that we labeled retained its specificity. Thus, this technique will facilitate production of functional 35S-labeled antibodies.

FIG. 4. Instability of [%]methionine is related to the effectiveness of preparative labeling. (A) Pyruvate kinase was colyophilized with either unstable [%]methionine (SJ. 204; lane 1) or stabilized [%]methionine (SJ. 1515; lane 2). (B) Pyruvate kinase was colyophilized with either unstable [35S]methionine (SJ. 204; lane 1) or unstable [35S]methionine stabilized with either 15 mM 3,4-pyridinedicarboxylic acid (lane 2) or 1 mM dithiothreitol (lane 3). (C) Pyruvate kinase was dissolved in either distilled water (lane 1) or 10 ~1 of 50 mM Hepes, pH 7.4 (lane 2), before lyophilization. After lyophilization, all samples (except for C, lane 2) were rehydrated in 10 ~1 of 50 mM Hepes, pH 7.4, an equal volume of 2X electrophoresis sample buffer was added, and the samples were boiled for 3 min and loaded on polyacrylamide gels. The sample in C, lane 2, was rehydrated in distilled water, which (because the sample was lyophilized in buffer) resulted in a buffered solution that was identical to that of the other samples. (D) The effects of the composition of the rehydration solution. Pyruvate kinase (2.4 pg) was colyophilized with 60 @i [%]methionine and rehydrated in 10 ~1 of 50 mM Hepes, pH 6.1 (lane l), 10 ~1 of 50 mM Hepes, pH 7.4 (lane 2), or 10 ~1 distilled water (lane 3). Samples were incubated in the above solutions for 30 min at room temperature, after which they were diluted in an equal volume of 2X SDS sample buffer, boiled for 3 min, and loaded onto the electrophoresis gel.

assays. As shown in Fig. 5B, 35S-labeled goat anti-rabbit immunoglobulins indeed retain their recognition of rabbit immunoglobulins when used as a secondary antibody on an immunoblot. DISCUSSION

The correlation between instability of methionine and the effectiveness of labeling suggests that labeling is

FIG. 5. (A) Partial proteolysis of rabbit pyruvate kinase with Staphylococcus aureus V8 protease. Final concentrations of protease were 0 (lane l), 0.0006 (lane 2), 0.019 (lane 3), 0.038 (lane 4), and 0.06 pg/pl (lane 5). Molecular weight markers are shown on the left. (B) Autoradiograph of immunoblot of rabbit pyruvate kinase using rabbit antipyruvate kinase and labeled goat anti-rabbit IgG (lanes l-3). Lanes 4-6 lacked rabbit anti-pyruvate kinase.

[35S]METHIONINE

LABELING

Caution. The radiolytic decomposition of unstabilized 35S-labeled methionine produces volatile radioactive products (3,6,7). Thus, we strongly recommend that safety precautions to minimize exposure to volatile emissions be used when handling the methionine. In particular, we suggest the use of an activated charcoal trap to adsorb the volatiles when lyophilizing the [35S]methionine and protein.

OF

89

PROTEINS

REFERENCES 1. Laemmli,

U. K. (1970)

3. Amersham Tech ucts of %Labeled Heights, IL.

This work was supported by a research grant to L.W.B. Natural Sciences and Engineering Research Council and fellowship to D.I.R. from the Alberta Heritage Foundation cal Research.

from the a research for Medi-

(London)

Tip No. 106 (1988) Amino Acids,

4. Evans, E. A. (1982) view 16 (R 821037),

ACKNOWLEDGMENTS

Nature

227,

2. Cleveland, D. W., Fischer, S. G., Kirschner, U. K. (1977) J. Biol. Chem. 252, 1102-1106.

5. Jocelyn, Academic

6. Meisenhelder, 120. 7. Simonnet,

Volatile Decomposition ProdAmersham Corp., Arlington

Self-Decomposition Amersham Corp.,

P. C. (1972) Biochemistry Press, London. J., and

Hunter,

F., and Simonnet,

680-685. M. W., and Laemmli,

of Radiochemicals, Arlington Heights,

of the SH Group, T. (1988) G. (1990)

Nature

ReIL.

pp. 100-101, (London)

Radioprotection

335, 25,

273.