Amino-terminal Sequence Analysis of Proteins Purified on a

THE Jorrnr;a~ OF UIOLOG~C~L CHIMISTXY Vol. 247, No. 10, Issue of May 25, pp. 3212-3251, 1072

Printed in V.S.A.

Amino-terminal Sequence Analysis of Proteins Purified on a

Nanomole Scale by Gel Electrophoresis”

(Received for publication, February 11, 1972)

ALAN AI. \VEIKER,$ TERRY PLATT,$ AND T<LAUS WEBER

Front the Biological Laboratories, Hawad r/niversity, Cambridge, Massachusetts 02138

SUMMARY

A simple, rapid, manual technique is described for determining the amino-terminal amino acid sequence of proteins on a nanomole scale. In this modification of the 5-dimethylaminonaphthalene-1-sulfonyl-Edman degradation, inorganic carriers permit convenient manipulation of small amounts of protein, and use of the detergent sodium dodecyl sulfate throughout the procedure maintains protein solubility. Nanomole quantities of pure protein for such sequence analysis are readily isolated from multicomponent systems by analytical scale polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Proteins are recovered quantitatively from the gel by elution. The method is therefore suitable for characterization of the proteins derived from multichain enzymes and viruses.

The decision to determine a protein’s amino-terminal sequence depends on availability of the purified species in sufficient quantity. Techniques such as the recently automated Edman degradation (1) have been extensively developed for proteins which can be purified in milligram amounts. Since many proteins can be purified only in microgram quantities, due to limitations in starting material for the purification or lack of convenient milligram scale preparative techniques, amino-terminal sequence analysis often requires a nanomole scale procedure.

The dnnsyll technique, introduced by Gray (a), is sufficiently sensitive for end group determination at the 0.1 nmole level (3). A combination of this technique with stepwise degradation by the Edman procedure has been successfully used for peptide sequencing by Gray and Smith (4) and by Bruton and Hartley (5). However, microtechniques have not been developed for purifying the amino-terminal peptide derived from nnnomole amounts of protein by chemical or enzymatic cleavage.

The da&-Edman combination has rarely been applied to intact prot,eins. Although tlansylation has been used to identify the a.mino-terminal residue of proteins directly, the traditional

* This work was supported by Grents GM-17602 and GlLI-09541 of the National Institrytes of Health.

t PIJtttional Science Foundation Predoctoral Fellow. ;The abbreviations used are: dansyl, 5-dimethylnminonaphthn-

lene-1-sulfonyl; SDS, sodium dodecyl sulfate; PTC: phenylthio- carhamyl.

manual Edman degradation generally has not worked well. One of the main difficulties is that, in contrast to peptides, many proteins become insoluble after one or two cycles of degradation. Since insoluble material does not react completely in subsequent cycles, unambiguous sequence determination is impossible.

We have developed a reliable manual dansyl-Edman procedure which can be used to determine an amino-terminal sequence of 5 to 10 residues for any protein with an unblocked amino terminus which can be purified on a nanomole scale. We have overcome protein solubility problems by replacing the aqueous organic solvents of the traditional Edman procedure with an aqueous inorganic buffer containing the detergent sodium dodecyl sulfate. SDS, which is known to denature and to solubilize proteins in aqueous solution (6-S), has also proved useful in the dansylation rea.ction.

Electrophoresis on analytical polyacrylamide gels in buffers containing SDS or urea is a widely used tool to characterize the individual polypeptide chains of multichain enzymes, viruses, and membrane preparations by charge or molecular weight. The high resolving power of these gels and their simple experimental design make them very attractive as a preparative technique for the study of multicomponent systems on a nanomole scale. We have successfully applied our SDS-dansyl-Edman procedure to nanomole quantities of protein separated 011 and quantitatively eluted from SDS polyacrylamide gels.

Proteins of both known and unknown amino-terminal sequence have been subjected to the SDS-dansyl-Edman degradation. For proteins of known sequence, our results agree with those found by conventional techniques. For proteins of unknown sequence, such as minor capsid proteins from the coliphages Qfi and R17, our amino-terminal sequence data are compatible with known RNA sequences from the phage genome.

MATERIALS AND METHODS

Chemicals-Reagents for polyacrylamide gel electrophoresis have been described previously (9, 10). Dowex 501-X8 was obtained from Bio-Rad.

l’henylisothioeyanate (sequenation grade) was an Eastman product. Sodium dodecyl sulfate, trifluoroacetic acid (both sequanal grade), and dansyl chloride (lo%, w/v, in acetone) were purchased from Pierce Chemical Company (Rockford, Illinois). Guanidine hydrochloride was obtained from Schware/ Mann. A Beckman amino acid calibration mixture (Type 1) was used. Cheng-Chin polyamide layer sheets were obtained from Gallard-Schlesinger (Carle Place, New York). Parafilm

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is an American Can Company product (Marathon Products Division). All chemicals used were reagent grade if not other- wise specified.

Proteins-Aspartate transcarbamylase from Escherichia coli was purified according to the method of Gerhart and Holoubek (11). &Galactosidase from e. coli was purchased from Boeh- ringer as a crystalline suspension. Egg white lysozyme (two times crystallized) was a Schwarz/Mann product. Lac repressor from E. coli was purified by the procedure of Gilbert and Mueller-Hill (12). Histone Hfl was obtained from Worthington. Subunit II of Q/3 replicase (gift of Dr. T. Blumenthal) was re- purified by gel electrophoresis. Tryptophan synthetase A protein was a gift from Dr. C. Yanofsky. Bovine serum albumin (Pentex recrystallized) was obtained from Miles Laboratories. Q/3 and R17 bacteriophage were purified as described previously (13).

Equipment-A heated vacuum desiccator (Fisher Scientific) is maintained at 60” and connected to an oil pump through a Dry Ice-acetone cold trap and a trap containing potassium hy- droxide pellets to keep water and acids out of the pump oil. A good water aspirator can be substituted for the oil pump, if drying times are increased by about 50%.

-4 desk top clinica, centrifuge (International) is used throughout the procedure, since centrifugation at higher speed is unnecessary. The centrifuge must have swinging buckets, so that precipitates will be packed on the bottom of the tube and super- natants can be cleanly withdrawn with a Pasteur pipette. The reactions are done in either disposable culture tubes (10 mm x 75 mm) or in 15.ml conical centrifuge tubes. The disposable culture tubes (6 mm x 50 mm) which are used for dansylation can be centrifuged inside a larger tube by using a paper towel insert.

Preparative Gel Electrophoresis in Sodium Dodecyl Sulfute

The 10% polyacrylamide SDS gels were prepared according to the method of Weber and Osborn (10). For 5% gels, both acrylamide and methylene bisacrylamide were halved. After polymerization, the gels were aged at room temperature for at least 4 days. They were then removed from the casting tubes by rimming and pre-eluted with stirring at room temperature into three changes of running buffer (0.1 11 sodium phosphate, 0.1% SDS, pH 7.2) at 12.hour intervals.2 Gels were sucked back into the casting tubes by drawing on the opposite end of the tube with a 50.ml plastic syringe connected by a short length of vacuum tubing. The pre-eluted gels should fit snugly, or wall effects will cause protein bands to smear.

Sample Preparation-Samples are heated to 95” for 2 min in loading buffer (0.01 M sodium phosphate, 1% SDS, 1% 2-mercaptoethanol, 10% glycerol, 0.001 y0 bromphenol blue, p1-I 7.2) supplemented with sufficient SDS to maintain a 2-fold weight excess over protein, then covered with I’arafilm and completely reduced for 4 hours at 37”. When there is reason to suspect t.hat the sample is contaminated with proteases, special precautions may be taken to avoid proteolysis during sample preparation as discussed by Pringle (14).

Determining dfa&& Loading-On SDS gels, an overloaded protein band “trails” severely, although the leading edge of the protein band remains sharp with a slightly increased mobility. A single gel which is overloaded with respect to one or more pro-

2 Use of highly purified reagents throughout may eliminate the need for pre-elution.

teins may nonetheless give narrow symmetrical bands for other proteins present in lower concentration. This situation often arises when intact virions are subjected to preparative electrophoresis. Minor viral components can be clearly resolved despite smearing of the major capsid proteins, as can be see11 in Fig. 2. Maximal loading depends on how well the protein of interest is separated from adjacent protein bands. In the sirn- plest case, a protein which cannot be resolved from adjacent bands on 6-cm gels may be cleanly separated on gels of 12- or l&cm length. This is possible because electrophoresis is much faster than diffusion of the protein bands. If proteins with simi1a.r mobilities are also present in much greater quantity than the protein of interest, trailing will be more of a problem. In this case, even longer gels may not give adequate separation and loading must be reduced. In the worst possible case, a. protein which cannot be purified in a single step must be subjected to a second preparative run. After preparative electrophoresis and elution of the protein bands, a small aliquot of each protein is always checked for purity by analytical polyacrylamide gel electrophoresis in SDS.

Removing Nucleic Acid-Intact viral DNA’s and RNA’s are excluded from 5% and 10% polyacrylamide gels. At current densities exceeding 6 ma per cm2 of gel cross-sect’ion, such highl) charged polyanions may cause the top of the gel to collapse. For this reason, when intact virions are disrupted for preparat,ive gel electrophoresis by heating with SDS and 2-mercaptoethanol, the gels are run at a current density not exceeding 6 ma per cm” until the tracking dye has moved at least 1.5 cm into t,he gel. The nucleic acid is then flushed from the top of the gel and removed with a Pasteur pipette, taking care not to touch the gel surface. After this, the current is raised to its normal level of 25 ma per cm2.

Localizing Protein Bands in Preparative Gels--M:e have used the following three techniques.

a. A lightly loaded analytical gel is run in parallel with each batch of preparative gels. Since protein mobilit.ies vary slightly, depending on the precise conditions of polymerization (for a review see Reference 15), the analytical gel must be poured at the same time and in the same diameter tube as the preparative gels. After electrophoresis, the middle of the tracking dye band, as judged by eye, is marked by inserting across the gel diameter a 26 gauge hypodermic needle which has been dipped in India ink (16). We have found that this procedure permits more accurate determination of mobilities than slicing through t.he dye band with a razor blade. The analytical gel is stained in Coomassie brilliant blue (10) for 2 to 12 hours and destained by diffusion at 37” on a roller in a culture tube which contains destaining solution and sufficient Dowex 501-X8 to absorb unbound stain irreversibly (17). A 4-ml gel requires approximately 2 g of Dowes. Gels destained in this fashion are clear and free of the artifacts sometimes produced by electrophoretic destaining. Since both mobilities and band width increase slightly with heavy loading, analytical gels should be used only as a guide to determine an ap- proximate mobility range for each protein band.

b. After electrophoresis, a single longitudinal guidestrip is cut from the edge of the preparative gel with a modified version of the gel slicer described by Fairbanks, Levinthal, and Reeder (18). The bulk of the gel is kept at 4” while the guidestrip is stained for 15 min and destained as in a. Because guidestrips cut from 10% polyacrylamide gels may stretch as much as 5% in length, this method is less accurate than the first, but prefer-


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3244 Amino-tern&al Sequencing of Proteins on 1Vanomole Scale Vol. 247, No. 10

Chromotography /

FIG. 1. Schemst,ic representat,ion of the SI)S-dansyl-Edman degradation.

able for preparative gels with diameter of 1.0 cm or larger. The method should not be used for 5 y0 polyacrylamide gels, since 5 y0 polyacrylamide guidestrips do not stretch uniformly.

c. When adjacent protein bands are too poorly resolved for mobilities to be a reliable guide, the entire preparative gel should be frozen and sliced transversely into l-mm sections. These sections are then eluted individually into suflicient 5 mM NaIIC03 containing 0.05% SDS to cover the slice, and an aliquot from each eluate is rerun on small analytical gels which are stained as usual to localize the protein bands. Eluates containing a single protein species are pooled, and those containing a mixture are rerun on preparative gels.

Eluting Protein Bands-Gel sections no thicker than 2 mm are eluted with agitation at 37” into 10 volumes (up to 5 ml) of 5 m&f NaHC03 containing 0.05$& SDS. After 12 hours, acrylamide fragments are spun out in a clinical centrifuge and the supernatant is lyophilized. The lyophilized residue can then be dissolved directly in coupling buffer as described below.

Stepwise Sodium Dodecyl Sulfate-Dansyl- Edman Procedure

Each cycle of SDS-dansyl-Edman degradation removes the NHzmterminal amino acid and exposes a new amino-terminal residue, which is identified by dansylating an aliquot of the protein. A complete cycle consists of four steps, as shown in Fig. 1: coupling, precipitation, cyclization and cleavage, and finally removal of an aliquot of protein for dansylation before the cycle is repeated. Allow enough starting material so that 0.25 to 0.5 nmole of protein can be withdrawn after each cycle for the de- sired number of cycles.

The residue from trifluoroacetic acid treatment is taken up immediately in 0.2 ml of coupling buffer and at least 20 ~1 of 10% SDS solution. The sample foams as bicarbonate neutralizes any remaining trifluoroacetic acid. Heating to 50” may be required to bring all the SDS into solution, if too large a crystal was added after cyclization and cleavage. Clumps of partially insolubilized protein are divided as finely as possible by tritura- tion with the tip of a Pasteur pipette. The pH is checked with pH paper which has been standardized in a buffer of known pH containing SDS. Indicator dyes have higher pK values when dissolved in SDS micelles. An aliquot (0.25 to 0.5 nmole in 10 to 100 ~1, including a small portion of any insolubilized material) is withdrawn for dansylation into a disposable culture tube (6 mm X 50 mm). The degradation may be continued by adding 10 ~1 of phenylisothiocyanate to the remaining solution, and proceeding as described under “Coupling.”

Before attempting to sequence an unknown, practice is strongly recommended with several cycles of SDS-dansyl-Edman degradation on 5 nmoles of a protein of known sequence such as lysozyme. To avoid difficulty during chromatography, 1-nmole aliquots should be withdrawn at each step for dansylation.

Dansylation-The stock solution of dansyl chloride (lo%, w/v, in acetone) is diluted 1: 20 into acetone to give a few milli- liters of working solution at 5 mg per ml. Small aliquots of stock solution are withdrawn from the “Hypovial” using a hypodermic syringe. At 4” in the dark, the stock solution may be used for at least 1 year, but the working solution is replaced weekly.

Coupling-The sample is dissolved in 200 ~1 of coupling buffer One-half volume of the working dansyl chloride solution is (0.5 M NaIIC03 adjusted to pI1 9.8 with NaOH) and no less than added to the protein aliquot withdrawn for dansylation, making 20 ~1 of lo70 SDS solution (w/v). For efficient detergent ac- it 33 % in acetone. A precipitate may form, which will not inter- tion, SDS must be present in at least 1.4-fold weight excess over fere with dansylation. The tube is covered with Parafilm and protein (6-8). Then 10 ~1 of phenylisothiocyanate are added to incubated at 37”. Prewarming the tube is unnecessary and may the tube, which is immediately warmed to 50” in a water bath, even cause evaporation of the acetone, which is needed to solubil- flushed with nitrogen, and sealed with several layers of Parafilm. ize the dansyl chloride. The reaction is terminated after 20 Most of the phenylisothiocyanate will remain as a separate or- min by acid precipitation, following the suggestion of Gros and ganic phase at the bottom of the tube. The reaction proceeds Labouesse (19). At protein concentrations greater than 50

for 30 min at 50”, with occasional vortexing every 5 or 10 min to keep the coupling buffer saturated with phenylisothiocyanate.

Precipitation-Nine volumes of acetone are added while vortexing the reaction tube, and the flocculent white precipitate is spun down for 2 min in the clinical centrifuge. The yellowish supernatant is withdrawn with a Pasteur pipette and discarded; the precipitate is washed with 2 ml of 100% acetone, spun down, and the colorless supernatant again discarded. The precipitate is spread around the walls of the tube by vortexing, and the tube is set nearly horizontal in the heated vacuum desiccator where the damp slurry is dried under vacuum for 20 min. The slurry must remain on the walls of the tube when it is set in the desiccator. Rapid evaporation of acetone under reduced pressure will cause lumps on the bottom to explode and be blown out the mouth of the tube.

Cyclization and Cleavage-Tubes are allowed to cool to room temperature, and 0.2 ml of trifluoroacetic acid is added to each. If the resulting solution is not clear, the precipitate from the coupling step was not completely dry. The degradation is continued in any case, but drying times should be increased by 5 to 10 min in subsequent cycles. The tubes are warmed to 50”, flushed with nitrogen, sealed with Parafilm, and incubated for 5 min at 50”. The Parafilm is removed, a small crystal of dry SDS (about 1 mg) is added, and while still warm the tube is vortexed to dissolve the SDS. The trifluoroacetic acid is blown away in the hood under a stream of nitrogen, so that a residue forms on the walls of the tube. The tube is then set nearly horizontal in the heated vacuum desiccator and dried in oucuo for 20 min.


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pg per ml (for example, 5 pg/lOO pl), the dansylated protein can be precipitated directly by cautious addition of ice-cold 20% trichloroacetic acid. The trichloroacetic acid solution must be added dropwise and with constant vortexing, or evolving carbon dioxide will cause uncontrollable foaming in the detergent solution. Below 50 pg per ml, 5 pg of succinylated carrier protein3 are added before trichloroacetic acid precipitation to assure quantitative recovery.

In either case, the precipitate is spun down and the supernatant withdrawn with the finely drawn tip of a Pasteur pipette4 and discarded. The precipitate is washed once with 0.2 ml of 1 N HCl to remove dansylic acid. After centrifugation the supernatant is withdrawn, and 50 ~1 of 6 N HCl (reagent grade con-

centrated HCl diluted 1:1 with distilled water) are added. A neck is drawn on the tube using a fine osygen torch, and the tube is sealed under water aspirator vacuum (oil pump unnecessary). After hydrolysis for 4 to 6 hours at 105”, as recommended by Gros and Labouesse (19), the tubes are opened and the hydrol- ysates dried down under vacuum in the heated vacuum desiccator. NHS-terminal isoleucine, leucine, and valine occasionally require longer hydrolysis, as discussed under “Chromatography.”

Chromatography-Chromatography is performed essentially according to the method of Hartley (3) as modified by Neuhoff (cited in Reference 3). Polyamide plates coated on both sides are cut to 5 cm x 5 cm with a scissors from the full 15.cm square plate. The hydrolysis residue is dissolved in 1 to 3 ~1 of 50% aqueous pyridine (v/v). Using a X pipette mouthpiece and the finely drawn tip of a melting point capillary,4 the unknown is repeatedly spotted onto one side of the plate by touching the capillary tip to the polyamide layer. This does not damage the plate, which is tough and virtually unaffected by small scratches or compression. The application spot should not exceed 2 mm in diameter. The plate is dried for a few seconds between ap-

plications in the hot air stream of a hair drier. Only about 10% of the sample is applied, since the dansyl spots will trail severely if the plate is overloaded. With experience, overloading can

3 Succinylated carrier protein was prepared according to the method of Habeeb et al. (20). Protein was dissolved at 10 mg per ml in 8 i% guanidine hydrochloride buffered with 1 M Na&Oa, and a 36-fold molar excess of solid succinic anhydride over amino groups was added in three equal aliquots at l-hour intervals. The solution was dialyzed extensively into 0.05 M NaHCOa and stored in the cold. Succinylated carrier protein should be dansyl-negative in the following test. An aliquot of 0.05 M sodium bicarbonate solution containing about 5 &of succinylated carrier protein is added to 50 vl of coupling buffer made 1% in SDS. The succinylated carrier protein is dansylated, precipitated, hydrolyzed, and chromatographed as usual. No dansyl derivatives other than a faint ti-dansyl-lysine should be detectable even on overloaded polyamide plates. Any protein may be used as carrier (for example, bovine serum albumin or lysozyme). Protein purity is irrelevant since reactive amino groups are irreversibly succinylated, and succinylation assures solubility even in the absence of detergent or denaturing agents (20).

4 Cawillaru Microwiwetles-Kimax melting point capillaries and 1 ” L .

a microburlier without flame-spreader are-ised. The center of the capillary is rotated just above the flame, taking care to heat as short a length of tubing as possible. When the glass has soft- ened, the capillary is removed from the flame and immediately drawn out. If the capillary is drawn in the flame, it will melt in two. With the capillary resting on a clean towel, a single scratch is made at the midpoint with a diamond pencil. When the capillary is bent slowly into a “U” with the scratch on the convex side, the resulting break is even. Finely drawn Pasteur pipettes are made similarly.

by judged at the time of application by the amount of brown hydrolysis residue present on the white polyamide sheet..

In general, the smallest sample is applied which permits unambiguous identification of the unknown dansyl-amino acid. A larger sample must be applied when chromatography reveaIs no dansyl derivatives other than e-dansyl-lysine. A smaller sample may be advantageous by reducing background when multiple spots are observed. Incomplete hydrolysis of an ali-

phatic NHz-terminal dansyl derivative may result in two bright

spots without other significant background (2, 19). In this case the sample should be dried down, taken up in 6 N HCI, and rehydrolyzed for an additional 8 hours.

A standard mixture containing about 0.02 nmole of each dansyl-amino acid (see below for preparation of this mixture) is spotted on the back of the plate opposite the unknown. The plate is held in a 250-ml beaker under a hot air stream for at least 5 min. Incomplete evaporation of the pyridine will produce trailing in the first dimension, due to local neutralization of the 1.5% formic acid.

Ascending chromatography in four successive solvents is performed according to the method of Woods and Wang (21) as modified by Hartley (3). Solvents II, III, and IV are run in the same direction perpendicular to Solvent I.

Solvent I 1.5% formic acid in water Solvent II benzene-acetic acid, 9:l Solvent III ethyl acetate-acetic acid-methanol, 2O:l:l Solvent IV 0.05 M NasP04 in 25% aqueous ethanol

The four solvents can be made in advance and stored in tightly capped bottles at room temperature. Solvents should be kept covered at all times and never used for longer than 3 hours, since differential evaporation affects their chromatographic properties. Solvents should be discarded whenever they cease

to resolve the standard dansyl-amino acid mixture adequately. Altogether, chromatography requires less than an hour: 5 min

each for Solvents I, II, and III; 15 min in Solvent IV; at least 10 min of drying after Dimension I and 5 min each after Dimen- sions II and III. Dimension IV requires only brief drying before examination under ultraviolet light. Solvents II, III, and IV need a solvent-saturated atmosphere, and can be run in 150-ml beakers covered with a stretched sheet of Parafilm. The solvent should just cover the bottom of the beaker evenly. Plates are placed in beakers and withdrawn using a tweezers. The resulting abrasion marks are not harmful, however, since the solvent

front is never allowed to run to the edge. Plates are kept as vertical as possible during chromatography. The lower edges

of the plate must not touch the walls of the beaker or capillarity may distort the solvent front.

Chromatography is not difficult but the manipulations require practice. Before several trial cycles of SDS-dansyl-Edman are attempted on a protein of known sequence, reliable chromatographic identification of the dansyl derivatives must be possible. When the standard dansyl-amino acid mixture is applied to both sides of the plate, registry of the spots should be sufficiently ac- cura.te to distinguish dansyl-isoleucine from dansyl-leucine after Dimension II; dansyl-alanine from dansyl-amide, dansyl-thre- onine from dansyl-serine, dansyl-glutamic acid from dansyl

aspartic acid, cy-dansyl-histidine5 from e-dansyl-lgsine after

5 We have found that 0.dansyl-histidine cannot be resolved from e-dansyl-lysine in Dimension III unless (a) the polyamide thin layers are presoaked for at least 10 min in Solvent I and (b)


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3”46

From E. coli P-Galactosidnse. lac repressor.

Aspartate tlnllsca~bam~~lase catalytic suburiitc. Tryptophan synthetase .4 protein.,

Minor capsid proteins from ItNA coliphage KIT matura.tion protein”. Q3 maturation protein (IIn)c. Q@ read-through protein (IIb)r..

Major capsid proteins from RNA coliphnge 1~17 coat proteinc. &p coat proteinc

Other proteins Qp replicase subunits IIC. Hen egg white lysozyme

-

I

1

,

Iz?lWlt?.,

3

3 1.2

1 3

5 5

0.7 0.35

_-

390 135,000 38,000

100 33,000 36 29,000

40 100

70 70

45 5

38,000 hlet-Arg-Ala-Phe-Ser-Ala-Leu-Asxe 41) 000 Pro-Lys-Leu-Prof 36,000 Ala-Lys-Leu-Glx-Thr-Val-Thr-Leue

14,000 Ala-Ser-Asx-Phe-Thr-Glx-Phe-Val-Leu-Val* 14,000 Ala-Lys-Leu-Glx-Thr-Val-Thr-Leui

64,000 Ser-Lys-Thr-Alai 14,000 Lys-Val-Phe-Glyk

Mel wt Amino-terminal sequence

Thr-Met-Ile-Thr-Asx-Ser-Leu-Alan Met-Lys-Pro-Val-Thr-Leu-Tyr-Asx-Val-Ala-Glx-Tyr-

Ala-Gly-Val-Serb Ala-Asx-Pro-Leu-Tyr-Glx-Lys-Ile-Ile Met-Glx-Arg-Tyr”

-

Vol. 247, No. 10

D Amino-terminal sequence agrees with that, determined with conventional techniques by I. Zabin and A. V. Fowler (manuscript in preparation).

* Sequence agrees with that determined with conventional techniques by Platt et al.6 c Protein purified by gel elect.rophoresis. Sequence analysis and gel purification are described under “Materials and Methods.” Cl Sequence agrees with t,hat det.ermined with convent,ional techniques by Guest et aE. (23). e Smino-terminal sequence corresponds to RNA sequence of Steitz (24) and Thompson.? f Sequence corresponds to H.NA sequence of Billeter et al. (25). g Sequence agrees with that determined with conventional techniques by Moore et al. (26). h Sequence agrees with t)hat determined with conventional techniques by Weber (27). i Sequence agrees with that determined with conventional techniques by Konigsberg et al. (28). j Sequence corresponds to KNA sequence of Hindley and Staples (29). k Sequence agrees with that determined with conventional techniques by Canfield (30).

Dimension III; and dansyl-arginine from edansyl-lysine after Dimension IV. The effect of overloading on chromatography can be observed by applying increasing amounts of the standard dansyl-amino acid mixture to Oue side of the plate until resolution deteriorates.

Chromatograms are interpreted under ultraviolet light as in Hartley (3) and in Fig. 3. Plates are inspected with a minera- logical (not germicidal) lamp. Protective goggles must be worn. Dansylic acid is bright blue, dansyl-amide greenish blue, and the dansyl-amino acids yellowish green except for O-dansyl-tyrosine derivatives which are bright yellow. Dansylation of a protein with NHz-terminal lysine normally yields (Y, e-didansyl- lysine. In contrast, when an internal lysine becomes NHz-terminal in the course of SDS-dnnsyl-Edman degradation, it yields the fluorescent derivative c-phenylthiocarbamyl-ar-dansyl-lysine and a degradation product. The predominant derivative remains near the origin in the first three dimensions, while the other moves with leucine in the first two dimensions and with methionine in the third.

Dan&Amino Acid Standard Mixture-This is prepared according to Gray (a), but the dansylntion reaction is terminated by adding excess formic acid. In this way the bicarbonate

thoroughly dried in a hot, air st,re:tm before applying the sample. This problem does not, arise (3, 5) when polyamide thin layers are reused after regeneration according to t,he method of Wang and wu (22).

0 T. Platt, J. Files, aud K. Weber, manuscript in preparation. T T. Thompson, personal communication.

buffer is not applied to the polyamide sheet, where it may cause smearing in the first dimension by neutralizing the 1.5% formic acid solvent.

A loo-p1 aliquot of an amino acid standard mix (Beckman Instruments amino acid calibration mixture, 2.5 mM in each amino acid in 0.01 N HCI) is dried down in the heated vacuum desiccator, redissolved in 0.5 ml of 0.1 M NaHC03, and dansylated by adding 0.25 ml of fresh working dansyl chloride solution at 5 mg per ml in acetone. The tube is covered with Parafilm and incubated at 37” for 30 min. Reaction is terminated by adding 25 ~1 of 88% formic acid. The dansylated standard mixture is used directly for spotting (0.1 ~1 contains 0.025 nmole of each amino acid) and stored in the cold. This dansyl standard mixture contains didansyl-histidine, which is converted to oc-dansyl- histidine by acid hydrolysis. To obtain oc-dansyl-histidine, an aliquot of the standard mix may be dried down and hydrolyzed, or alternatively a stock solution of histidine can be dansylated with a 2-fold excess of amino acid over dansyl chloride as described by Gray (2).

RESULTS

Sequence Results--Amino-terminal sequence results obtained with the SDS-dansyl-Edman microtechnique are presented in Table I. Proteins ranging in molecular weight from 14,000 (Q/3 coat protein) to 135,000 (fi-galactosidase) have been stud- ied. Those purified by gel electrophoresis in the presence of SDS are indicated in the table with a superscript c. No more than 0.25 nmole of protein per step was needed for amino-termi-


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nal sequence analysis, although more was used in several in- stances when sufficient starting material was available.

As an illustration of the SDS-dansyl-Edman procedure as np- plied to proteins purified on a nanomole scale by gel electrophoresis, we describe here a model experiment xhirh yielded the amino-terminal sequences of the two minor capsid proteins of bacteriophage Q/% The major coat protein species accounts for 95% of the capsid protein, and the two minor protein species IIb and IIa for 4% and less than 1 %, respectively (31). In the model experiment, 6 mg of purified Qfi virions mere disrupted for 2 min at 95” with a 2-fold weight excess of SDS and 1 To 2- mercaptoethanol. After reduction for 4 hours at 37”, equal aliquots of the denatured virus were layered onto sis “pre-eluted” 10% polyacrylamide SDS gels (0.8 cm x 12 cm). About 5% of the sample was run in parallel on a fifth gel for use as a guide during elution. After electrophoresis at 3 ma each for 1 hour, viral RNA which had been excluded from the gel was removed with a Pasteur pipette. The current could then be raised to 8 ma each, and the gels run for 16 hours. The preparative gels were frozen at - 20” while the guide gel was stained and destained as usual. IJsing mobilities determined from the guide gel, the IIa and IIb bands were cut out separately from each preparative gel and eluted into 4 ml of 0.05% SDS buffered with 5 mM NHI- HCOI for 12 hours at 37” with agitation. Elution under these conditions permits quautitative recovery of the minor phage proteins after gel electrophoresis (see Table II). To determine purity, 5% of each eluate was rerun on analytical SDS polyacrylamide gels. Fig. 2 demonstrates that after elution, each minor phage protein is completely free of the other and of the major coat protein species. The eluates were then lyophilized, redissolved in coupling buffer, and subjected to three cycles of SDS-dansyl-Edman degradation. Using 40 pg of IIa protein we obtained the amino-terminal sequence Pro-Lys-Leu-Pro, and using 100 pg of IIb protein the sequence Ala-Lys-Leu-Glx- Thr-Val-Thr-Leu (Table I). The biological implications of these sequence data are considered in the “Discussion.”

Preparative Gel Electrophoresis-Proteins for SDS-dansyl- Edman sequence analysis can be quantitatively recovered from ‘Lpre-eluted” gels. In the model experiment described in Table II, greater than 99% recovery was obtained for Qfl coat protein (molecular weight 14,000) run on 10% polyacrylamide SDS gels and for @-galactosidase (molecular weight 135,000) run on 5% gels. These gels combine experimental convenience, estraor- dinary sensitivity (less than 0.5 pg of protein is easily visible after staining), and high resolution, although they separate polypeptide chains almost entirely on the basis of molecular weight (10, 33, 34).

When we attempted to apply our SDS-dansyl-Edman technique to microgram quantities of protein eluted from such gels, the first results were disappointing. Gel electrophoresis and subsequent elution of the protein reduced the sensitivity of the sequencing technique by 5-fold, so that 2.5 nmoles of protein instead of 0.25 nmole were required per step. Since we had expected quantitative recovery of protein, we attributed the decrease in sensitivity to either (a) chemical modification of the NHQ-terminus of the protein during electrophoresis, or (b) direct interference with the SDS-dansyl-Edman degradation by material rhlted together with the protein from the polyacrylamide. Pre-electrophoresis with runnin, 0’ buffer or with methyl green (34) to eliminate persulfate artifacts (for review see Reference 15) did not increase sensitivity, nor did pre-running the gels

TABLE II

Recovery of proteins by elution from polyacrylande gels after electrophoresis in presence of SDS

Bacteriophage Qp coat protein and p-galactosidase were labeled with [3Hlformaldehyde by the procedure of Rice and Means (32), using as solvent 8 M guanidine hydrochloride buffered with 0.1 M

sodium borate at pH 9.0. Two parallel gels were run for each protein, one to follow the course of elution and the other as a control. After electrophoresis, a gel section containing the protein was eluted as described under “Materials and Methods” into four 5 ml changes of buffer. To follow the course of elution, an aliquot of eluate was wit,hdrawn after each buffer change and counted in Aquasol. After the fourth change of buffer, the cl ted gel slices and the uneluted control sections were burned in a Packard sample oxidizer, and the tritiated water counted in a modified Bray’s cocktail. The extent of elution was determined by calculating the radioactivity remaining in the eluted gel slices as a percentage of the counts in the uneluted control. The net radioactivity eluted into SDS was then normalized to the extent of elution.

Counts per min in uneluted control gel. . 79,400 10,400 Counts per min in eluted gel.. 600 100 Efficiency of elution after 10 hours 95yc 95% Efficiency of elution after 16 hours 99% 99%

Q,9 coat protein

FIG. 2. Purification of minor capsid proteins from bacteriophage Qp by polyacrylamide gel electrophoresis in the presence of SDS. This model experiment is described under “Sequence Re- suits.” A, complete Qfi virion; Li, purified IIa maturation protein: C, purified IIb read-through protein.


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with a low molecular weight protein (9). Because the interfering material seemed to be electrophoretically neutral, we resorted to diffusion as suggested by Weber and Kuter (9). In a model experiment, 100 pg of lac repressor were recovered from such a pre-eluted gel (see “Preparative Gel Electrophoresis” under “XIaterials and Methods”), and four steps of SDS-dansyl-Edman degradation were successfully completed by withdrawing 10-pg (0.25 nmole) aliquots at each step for dansylation. We do not know whether the interfering substance is residual acrylamide monomer, polyacrylamide, or polyacrylic anid, a contaminant in one of t,he reagents, or a by-product of the polymerizat,ion reaction (for a review see Referenre 15) ; however, it appears to be removed by diffusion.

Stepwise Sodium Dodecyl Sulfate-Dansyl-Edman Deyradation- A complete cycle of SDS-dansyl-Edman degradation consists of four steps as shown in Fig. 1.

Chpliny Reaction-In this step the protein is coupled with the water-insoluble Edman reagent, phenylisothiocyanate, to form a l~l~enylthiocarbamyl derivative. The manual Edman degradation has usually been restricted to peptides, since many proteins are insoluble in aqueous organic solvents such as 50% pyridine which have been used to dissolve the Edman reagent. Other proteins become insoluble after one or two cycles of degradation.

The detergent SDS is known to solubilize and denature proteins iu aqueous solution (6-8). Preliminary attempts to avoid solubility problems by adding SDS directly to the aqueous organic solvents of the traditional manual Edman procedure were unsuccessful. Model experiments revealed that the detergent action of SDS was greatly reduced in 507, pyridine. However, above its critical micellar concentration (7) SDS has been shown to bring not only proteins but also water-insoluble organic dyes into aqueous solut,ion (for a review see Reference 6). We therefore used SDS in an aqueous inorganic coupling buffer to solubilize both the prot,ein and the water-insoluble Edman reagent. When excess pllell2’lisothiocyarlate is present and forms a two- phase system, SDS micelles presumably transport molecules of phenylisothiocpanate from the denser organic phase to SDS- protein compleses in the aqueous phase.

Precipitation-It is necessary to remove excess phenylisothio- cyailate a.ild nonvolatile by-products of the coupling reaction (4) before cyclization of the PTC-protein in anhydrous acid (1). Tncomplebe removal can lead to elevated backgrounds during identification of the dansylated NHr?-terminal amino acid. SDS in the aqueous coupling buffer precludes repeated extrac- tions with an immiscible organic solvent to remove phenyliso- thiocyannte and its by-products, since (a) the detergent action of amphipliilic SDS prevents formation of a sharp interface, and (5) 1”L’C-I)rot.ein might partition into the organic phase in the preseme of high roucentrations of detergent. We therefore chose l)recGl)itatioil with uine parts acetone since SDS, phenyl- isothiocynnate, and the by-products of the coupling reaction are all soluble in aqueous acetone.

When a solution of protein iu aqueous SDS is buffered with an organic compound, the protein precipitates from 90% acetone as a gummy residue while the buffer remains soluble. In model experiments with lysozyme and R17 coat protein, the I’TC-protein was precipitated with nine parts acetone from aqueous SDS buffered with dimethylallylamine-trifluoroacetic acid (35). The precipitates were thoroughly washed with acetone and dried under vacuum at 60”. In neither case would the dry residue

dissolve completely in anhydrous trifluoroacetic acid, even after prolonged vortexing at 50”. Preliminary performic acid oxida- tion of the proteins (36) did not affect these results.

Use of a strong 0.5 M sodium bicarbonate-carbonate buffer in the coupling reaction solves this problem, since the inorganic salt NaHC03 gives a flocculent precipitate in aqueous acetone. When PTC-protein and NaHC03 co-precipitate from 90% acetone, the flocculent inorganic precipitate acts as a carrier to keep the insoluble PTC-residue finely dispersed. This co-precipitate, when thoroughly washed with acetone and dried briefly in vucuo at 60”, dissolves immediately in anhydrous trifluoroacetic acid. Moreover, the acetone precipitation extracts sodium trifluoroacetate formed in the previous cycle of degradation by neutralization of NaHC03 and Na2C03. Since sodium trifluoroacetate is readily soluble to at least 1 M in trifluoroacetic

acid and to at least 4 ivr in 90 y0 acetone, there is no accumulation of salt during the degradation.*

Co-precipitation from sodium bicarbonate-carbonate buffer has a second, unexpected advantage. In model experiments with R17 coat protein and histone Hfl, we found that 90% acetone quantitatively precipitates the PTC-derivatives of these two proteins from coupling buffer at final concentrations as low as 10 pg of protein per ml.9 Histidine Hfl contains 27% lysine on a molar basis (371, in contrast to 50/O in R17 coat protein (27). Since the e-amino group of lysine reacts with phenylisothiocyanate to form a PTC-derivative, thereby converting a positively charged polar residue to an uncharged hydrophobic derivative, the PTC-histone would be expected to have greater solubility than most PTC-proteins in 90% acetone. Quantita- tive recovery of both histone Hfl and R17 coat protein therefore suggests that the precipitation procedure is perfectly general.

Cyclization and Cleavage-Incubation with trifluoroacetic acid catalyzes cyclization and cleavage of the PTC-protein to produce the 2-anilino-5-thiazolinone derivative of the NHt-terminal amino acid and to expose a new NH2 terminus. Edman (for re-

* The presence of sodium carbonate in the coupling buffer slightly complicates this picture. Model experiments show that solutions of N&03 at concentrations of 0.1 to 1.0 M separate into two phases when made 90% in acetone. The viscous lower phase, with less than 5% of the total volume, contains greater than 957, of the original sodium carbonate, as judged by constant weight after extensive drying under vacuum at 60”. When 0.5 M coupling buffer at pH 9.8 is made 90y0 in acetone, the two salts seem to be- have independently; a flocculent precipitate characteristic of sodium bicarbonate can be spun out, while a second phase characteristic of sodium carbonate coats the test tube wall with small oily beads. The sodium carbonate does not interfere with the :tbilit,y of sodium bicarbonate to act as an effective protein carrier.

9 RI7 coat protein and histone Hfl were dissolved at 1.0 mg per ml in coupling buffer containing 1% SDS. A 20-4 aliquot of each protein solution was diluted into 0.2 ml of the same buffer, coupled with phenylisothiocyanate, precipitated with acetone, and dried as described under “Materials and Methods.” The dry acetone precipitates were taken up in 4 ml of 6 N HCl with a crystal of phenol, sealed under oil pump vacuum, and hydrolyzed for 24 hours at 105”. Since the presence of inorganic salts during acid hydrolysis can lead t.o reduced recovery of certain amino acids, the following controls were included. Tubes containing coupling buffer and SDS but not protein were carried in parallel through the coupling, precipitation, and drying steps. The protein-free precipitates of NaHC03 and NazCOa were taken up in 6 N HCl with phenol, and 20 ~1 of the appropriate protein solution were added before hydrolysis. The efficiency of acetone precipitation, as judged by recovery of alanine and aspartic acid, was normalized to these controls.


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view see Reference 1) emphasized the use of anhydrous acids for the cyclization and cleavage reaction, in order to avoid unwanted hydrolysis of internal pepMe bounds. Since lleutralizat.ion of the sodium bicarbonate-carbonate carrier by trifluoroacetic acid results in an 0.5 to 1.0 M solution of water in trifluoroacet,ic acid, depending 011 the precise 111-T of the coupling buffer, it was necessary to show- that 1 to 2 (;; moisture in the trifluoroacet,ic acid does not interfere significallt,ly with the SDS-dansyl-Edman degradation.

The data of Piskiewicz et nl. (38) 011 the extraordinary lability of aspartyl-proline bonds (aid usE)aragi~~~l-prolirle bonds after dea.midation) suggests that a protein rich in such bonds would be especially sensitive to moist trifluoroacetic acid. The coat prot,ein of coliphsge Q/3 contains 2 aspartyl-proline and 3 as- par:lgillvl-pl,olirle bonds in a polypeptide chain of 131 amino acids (28). In a model esperiment, 1” incuba.tion of Q/3 coat protein for 15 min at 50” in trifhmroacetic acid containing 2.5% water revealed no significant hydrolysis of internal peptide bonds.

.is in the traditional Edman degradation, after rernoval of the trifluoroacetic acid either by evaporation or lyophiliza.tion, the protein residue will not redissolve completely in coupling buffer even in the presence of SDS. We solved this problem, however, by adding a small crystal of dry SDS to the trifluoroacetic acid solution just before evaporation. The resulting residue dissolves easily when heated to 50” in coupling buffer. Pre- sumably, a detergent-protein complex has already formed in the lyophilized material and need only be rehydrated.

The residue from trifluoroacetic acid treatment should always be redissolved as quickly as possible in coupling buffer. Reduc- ing the cumulative exposure to strong acid in this way will (a) reduce chrornatographic backgrounds due to nonspecific acid hydrolysis of the polypeptide chain, and (b) minimize tl-le danger of blocking the SDS-dallspl-Edma.tl degradation by acid-cata- lyzed cyclization of :L newly exposed NHz-terminal glutamine to pyrrolidone carbosglic acid (41). Mernal glutamine residues which have become NHQ-terminal in the course of degradation do not cyclize to an appreciable extent under our conditions. In a model experiment using 0.3 nmole of protein per step, we obtained an amino-terminal sequence Met-Glx-Arg-Tyr (Table I) for tryptophan synthetase A protein in agreement with the pub- lished sequence Met-Gln-Arg-Tyr of Guest et al. (23).

Dansylation-The new NHz-terminal amino acid available aft,er cyclization and cleavage in trifluoroacetic acid is identified

~0 A 5-rl aliquot of Qp bacteriophage at 17 mg per ml in buffer (0.1 x Tris-IICI-10 mu MgC12-0.1 M NaCl, pH 7.2) was added t,o 0.2 ml of anhydrous t,rifluoroncetic acid, flushed with nitrogen, and incubated at 50”. After 15 min a small crystal (about 1 mg) of dry SDS was added to the solution and the sample was dried down, taken up in coupling buffer, dansylated, hydrolyzed, and chromatographed as usual. In the control experiment, 5 pl of the &a solution were extensively dried in the heated vacuum desiccator before addition of the trifluoroacetic acid. In both the model experiment containing 2.5% water and in the anhydrous control, incubat.ion for 15 rnin at 50” revealed less than 1% NHB-terminal proline relative t.o NH*-t.erminal alanine as judged by visual dilution analysis. A 100.fold dilution of the sample into 50% pyridine yields a dansyl-alanine spot of intensity comparable to the dansvl-proline contaminant in the undiluted sample. The NH,- t,ermInal proline can be att.ribut.ed entirely to the Qp maturation prot.ein (see “Sequence ltesults”) which constit.utes less than 1% of the t,otal capsid protein. This confirms earlier work by Ymyth, Stark, and Konigsberg (cited ill lieference 39) in which no uew end groups could be detected by the cyanate technique (40) after in- cuhation for 24 hours at, 25b in moist trifllloroacetic acid.

by dansylation. The thiazolinone derivative of the previous amino-terminal residue, resulting from trifiuoroacetic acid treatment, may be ignored when the Edman procedure is used in con- junction with dansylation (4). Presumably, thiazolinone derivatives of the amino acids do not react with dansyl chloride, or if they do, yield products which move with the solvent front during the second chromatographic dimension.

Excessive dansylic acid (the hydrolysis product of dansyl chloride) interferes with thin layer chromatography. The dansylated protein is therefore freed of dansylic acid by precipitation with trichloroacet.ic acid according to the method of Gros and Labouesse (19)) which simult,aneously extracts SDS and inorganic salts. At concentrations exceeding 0.05 mg per ml (10 pg/200 pl), the dansylated protein can be precipitated directly with 20% trichloroacetic acid. At lower concentrations, 0.05 mg per ml of succinylated carrier prot.ein should be added before trichloroacetic acid precipitation to assure quantitative recovery of the dilute dansylated protein. Succinylated carrier protein is dansyl-negative and introduces no spurious chromatographic background, since all reactke amino groups have been irreversibly succinylated.

Limitations of Sodium Dodecyl Sulfate-Dansyl-Edman Proce- dure-Edman (42) notes that during manual degradation the repetitive yield is only 90 to 95%, which he at,tributes primarily to oxidative desulfurization of the phenylthiocarbamyl group by small amounts of oxygen that are difficult to exclude in a manual technique. After desulfurization, the resulting pheuyl- carbamyl protein cannot cyclize and cleave in the trifluoroacetic acid treatment, and is effect,ively blocked from further degradation. Fortunately, as Hartley (3) has emphasized, peptide chains which have been blocked in this way do not react with dansyl chloride, so that a clean NH*-terminal identification can be made at each step, albeit in decreased yield. Oxidative desulfurization may be a major limitation on the sensitivity of the manual SDS-dansyl-Edman procedure, since we have occasioa- ally been unable to proceed beyond four cycles of degradation despite adequate starting mat.erial.

In the course of sequence analysis on E. coli Zac repressor, Platt et al. (43) have found that the dansyl-Edman technique of Bruton and Hartley (5) for pept,ides will proceed through an internal tryptophan residue, although no dansgl derivative of tryptophan could be identified. We have not sequenced an internal tryptophan using our SDS-dansyl-Edman technique for proteins, but the two procedures are sufficiently similar so that no difficulties are anticipated.

Dan&proline background may rise prohibitively after several cycles of degradation with proteins rich in aspartyl- and asparag- inyl-proline bonds, if the trifluoroacetic acid step is prolonged (see “Cyclization and Cleavage” under “Results”).

Small polyamide sheets are ver) sensitive to overloading. Since chromatography on polyamide thin layers can detect 0.01 nmole of dansyl-amino acid (31, SDS-dansyl-Edman sequencing on this scale should in principle present no problem. However, even 1 pg of total hydrolysis residue on an application spot of l-mm diameter will cause dansyl-arginine, dansyl-histidine, and e-dansyl-lysine to smear.” The reason for this is that the first

11 In a model experiment, 5 pg (0.35 nmole) of hen egg white lysozyme were subjected to four cycles of SDS-dansyl-Edman degradation. Using 0.2.ml volumes of coupling buffer throughout, I-pg aliquots (0.07 nmole) of protein were withdrawn for dansylation at each step in volumes of 40 to 100 ~1. The first t.hree


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Solvent I - Solvent I - Solvent I -

FIG. 3. Chromatography of dansylated amino acids on polyamide thiu layers. M&O,, methionine sulfone; cys a, cysteic acid. The derivative a-dallsyl-c-phenylthiocarbamy-lysine runs a-ith lencine in the first two dimensions, and with methionine in the third. ;t , after I>imensions I and II; B, after Dimension III; C, after 11imensioil IV.

dimension clirolnatogl~~L~,hic solvent must extract these dansyl derivatives from the total hydrolysis residue applied to the origin. Since smearing is proportional to the product of the mobility of a species and the time required to extract it, species which move with the solvent front will trail most severely. Conse- quently, loading must be increased about 5-fold for NHZ-terminal arginine and histidine, despite the fact that overloaded polyamide plates resolve all other dansyl-amino acids well. Thus 0.25 nmole of protein per step becomes the realistic maximum sensitivity for the SDS-dansyl-Edman degradation.

Chromatography presents other difficulties which may reduce the sensitivity of the tecahnique. The dansyl derivatives of as- parbic and glutamic acid are occasionally obscured by an O- dansyl-tyrosine smear resulting from dansylation at p1-T 9.8 instead of pH 8.1 as recommended by Gros and Labouesse (19). I-se of a roupling buffer at pH 9.0 instead of 9.8 would probably reduce the 0-dans~l-t~rosille smear without interfering with the degradatioii.

Dans~l-metl~iolliile may appear as both the unoxidized derivative or as the sulfoside, since the thioether is susceptible to air osidation (44). This can cause problems when workiug at the limits of the technique’s sensitivity, by reducing the intensity of both spots (see Fig. 3).

When an internal lysine becomes NHz-terminal in the course of degradation, it reacts with danayl chloride to form an ol-dansyl- e-I’TC-lgsine derivative. Uecause this disubstituted thiourea is unstable to acid hydrolysis (39), two spots may appear during clIromatograpli~. Nonetheless, the chromatogram can be interpreted easily, since one spot moves with leucine in Dimension II and with methionine in Dimension III, while t,he other spot remains very near the origin in Dimensions I, II, and III (Fig. 3).

DISCUSSION

11-e have presented :I simple, manual SDS-dansyl-Edman procedure using nariomole quantities of protein. Generally, an amino-terminal sequence of 5 to 10 residues can be obtained for any protein with an unblocked aminoterminus which is available on a nanomole scale. Nanomole quantities of protein for se- queiire analysis are easily purified from complex ~~iulticoriipoiieirt systems such as multichain enzymes and viruses by electrophoresis on polyacr;r-lmnide gels in the presence of SDS. I-llder favor- able circumstances, proteius from membrane preparations also can be purified by gel electrophoresis (16, 45). The individual proteins are recovered quantitatively from the gel by elution

cycles of degradntioll produced clearly interpretable result,s, but the fourth cycle failed to identify arginine in the sequence Lys- Va7 Phe-Gly-drg (30).

with SDS solution, and subjected directly to :Lmirlo-terlllirl;ll sequence analysis without removing the detergent,.

Recent work in our laboratory employing the SIX-dansyl- Edman microtechnique for amino-terminal sequence analysis suggests the generality of this procedure and the range of its potential application. Three examples are considered below.

a. RXA sequencing of several single-stranded RXA caolil)hages is well under way (see for example References 24, 25, and 29) and similar work can be anticipated with other systerrs and with purified messenger RNA species. Amiiio-terminal sequence data for the corresponding proteins can be used in toll-

junction with ribonucleotide sequence data to identify which of many potential initiation sites for protein synthesis actually function in viva. The SDS-dansyl-Edman microtechnique must be used when the protein of interest is available only in nanomole quantities. For instance, in the sequence of Billeter et al. (25) there are three XUG and two GUG codons within the first 175 nucleotides from the 5’.end of bacteriophage Q8 RNA, any one of which could potentially function as an initiation site (for review see Reference 46). The amino-terminal sequence of t,he Q/3 maturation protein obtained in the model experiment described under “Sequence Results” and presented in Table 1, enabled us to identify one of these codons (the AT’G rodon begin- ning at position 62) as the true initiation site and incidentallg to place the maturation protein cistron at the 5’-end of the RNA genome (31).

RNA sequences coding for the initiation of protein biosyllthe- sis can also be identified as ribosome binding sites, which are oligonucleotide tracts of messenger RNA protected from mild RN&e digestion by the initiation complex for protein synthesis In this complex the ribosome is specifically bound to an mR?;A molecule in the presence of initiatiou factors, flMet-tRi\jA, and GTl’ (46). Currently, 6 ribosome binding sites have been sequenced, all from the family of single-stranded RKA coliphages such as QB (29) and Rl7 (24). III general, ribosome binding sites can be assigned unambiguously to specific phage cistrons only by using the genetic code to correlate the RNA sequence with the corresponding amino-terminal sequence of the cistron’s protein product. For instance, the amino-terminal sequence of the phage-specific subunit II of &a replicase, which is presented in Table I, enabled Hindley and Staples (29) to assign their ribosome binding sequence to the &a replicase cistron. In a similar case, the amino-terminal sequence of the R17 maturation protein (Table I) not only confirmed the identity of the maturation cistron initiation site (24) but also estended the RN4 sequence at the initiation site.7

b. Amino-terminal sequence analysis can demonstrate relationships between Yiral 1)roteins. For illstance, sequence data obtained in the same model experiment described under “Se- quence Results” and presented in Table I reveal t,hat the majol coat protein (molecular weight 14,000) of bacteriophage Q/3 and the minor capsid component 111) (molecular weight 36,000) have identical amino-terminal sequenres for at least 8 residues.” This extraordinary fact led us to postulate that the IIb protein is a read-through product resulting from polypeptide chain elonga- tion past an inefficient IYGA stop signal at the natural t,ermina- tion site of the QS coat protein cistron. Growth of wild type QS on a high level ITGA suppressor confirmed this hypothesis by

1% Moore et al. (26) reached a similar conclusion illdependently, using conveutional sequencing techniques with 100 times ts mllrh starting material.


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increa.sing the molar frt&on of 111) protein in purified phage from 2 to 7a/, (31).

Precursor-product relationships between distinct capsid proteins have already been established by ot,her techniques for bacteriophage ‘1‘4 (47) and fol, poliovirus (48). In the future we can esl)ert that characterization of viral proteins by amino-term- iual sequence will yield similar results.

c. SDS-dilnsyl-Edriial~ sequenc+e analysis can also demonstrate relationships between mutant’ and wild type proteins. Work in progress on f<. coli lnc repressor h.1 L s shown that protein biosyn- t.hesis can reinitiate at an int,ernal methionine codon after poly- I)el)tide chain termination at an early amber block (43). The reit&iated fragment of lnc repressor was first isolated from a. crude cellular extract by precipitation wit’h :tntibody prepared against wild type repressor (4Q),13 and then separated from the antibod) polypel)tide chains by electrophoresis on 10% polyncr)-lamide SDS gels. Because the cross-reacting repressor fragment coti- st,itut,es only O.OZ”h of a crude cellular e&act, antibody precipitation yielded very small amounts of ma.terial which could be sequenced only by the Sl)S-dans~l-Ednlal1~ microtechnique. The results are presented in Table 1. When this sequence was compared with t’hat of wild type lac repressor which had been purified and partially sequenced by conventional means,6 it was found that residues 43 through 46 of t,he wild type sequence cor- respond to the amino-terminal sequence of t,he reinitiated fragment. This indicates that, an AlTG triplet which codes for the internal residue methionine 42 ill wild type lnc repressor can function as an initia.tion site for protein s@hesis when a.ctivated by an early amber mutation in the gene. Stewart et al. (50) have drawn similar conclusions in their work with a eukaryotic yeast, using conventional t,echniques for protein purifica.tion, peptide mapl)irlg, and peptide seyuencing.

I%anomole qua,ntities of pro6ein eluted from SIX polyacrylamide gels can be hydrolyzed for amino acid analysis (51). It may also be possible to subject these ljroteins to carbosT-=termi- real anslysis, since both cnrboxypeptidase ;Z and 13 are active in SDS (52). The free amino acids released by carboxypeptidase digestion can be identified by dansylation.14 Moreover, these same proteins can also be charact,erized by enzymatic (9, 15) and immunological activity (53),13 since Weber and Kuter (9) have recently shown that many proteins can be renatured from SDS with at least partial recovery of activity after removing the detergent.

.-lch-nozubd37?2ents~~e thzlllk Dr. H. G. Khorana for use of his Packard sample oxidizer, and Miss C. IIering for cheerful, expert technical assistance.

1. 2. 3. 4. 5.

6.

7.

8.

I<UYAN, P., .\PZD B~~;ao, G. (lB(i7) h’ur. J. Hiochem. 1, 80 GRAY, W. R.. (1967) Methods Enry,uol. 11, 139 HAR.I’LJI:Y, B. S. (1970) Biocherrc. J. 119, x05-822 Gn:\Y, W. I<., .IxJ) SMITH, J. F. (1970) Anal. BiOchem. 33, 36 I~RUTON, C. J., AND H.\Rus~, 1~. S. (1970) J. ilfol. Biol. 62, X5-

178 PITT-RIVERS, 11.) ANI) IMPJOMH.~U), F. S. A. (1968) Biochem. J.

109, 8%25-830 I&YNOLDS, J. A., .\NI) T.\NFORI), C. (1970) 1’1.0~. n-at. A cad. SC?.

u. s. A. 66, 1002 FISH, W. W., ~CICYNOLDS, J. A., .AND TINFOHD, C. (1970) J. Biol.

Chewy 246, 51G(i-5168

13 J. Rosenbusch and Ii. Weber, manuscript in preparation.

9.

10.

11.

12.

13.

14. 15

16

WE~ER, EC., .\ND KUTJ:R, D. J. (1971) J. Bid. Chem. 246, 4504- 4509

WERER, K., AND &IIORI\., hl. (1969) J. Biol. Chew/. 244, 44O(i- 4412

GERHART, J. C., ,ZND HOLOUISJX, I~. (1967) J. Riol. Ch.enr. 242, 2886-2892

GILBICRT, W., AA-L) MUELLER-HILL, B. (1966) Proc. A’at. A cod. Sri. U. S. A. 66, 1891

OSBORN, M., WEINEH, A. M., AND WI.:RI<R, K. (1970) Eur. J. Biochem. 17, 63

PRINGLE, J. R. (1970) Biochem. Biophys. Res. Commun. 39, 46 CHRUUCH, A., AND KODBAJ~D, D. (1971) Science 172, MO-

451 FAIRBANKS, G., STJXK, T. L., AND WALLACH, I). F. IT. (1971)

Biochemistry 10, 2606 17. WRBER, K., PRINGLE, J. Jt., AND Ostroax, M. (1972) Methods

Eneymol. 11, in press 18.

19. 20.

21.

FAIRB.~NKS, G., JR., LISVINTH~L, C., AND REEDELI, R. 15. (1965) Hiochenc. Biophys. Res. Commun. 20, 393-399

GROS, C., .ANJ) L,+ISO~ESSJ;, B. (1969) Eur. J. Bzochewl. 7, 463 HAHEEB, A. F. 6. A., CASSIUY, H. G., ANJ) SINGI.GI~, S. J. (1958)

Biochim. Biophys. Acta 29, 587-593 WOODS, K. R., ASD WANG, K-T. (1967) Biochim. Bioph?;s. A c/a

133, 369-370 22. WANG, K-T., AND Wu, P-H. (1968) J. Chromatogr. 37, 354 23. GUEST. J. H.. DRAPE~U. G. 11.. CARLTON. B. C.. ANI) Y.\xoF~

24. 25.

SI<Y k. (1967) J. Biol. Chem.‘242, 5442-k446 ’ STJWTZ, J. A. (1969) Xalure 224, 957-964 BILLXTXR, M. A., DAHLBERG, J. IL, GOODMAX, H. M., HIND-

LEY, J., ANJ) WEISSMANN, E. (1969) Cold Spring Harbor Symp. Qvant. So!. 34, 635

26. MOORIC, C. H., F~RRON, F., BOHSXRT, D., ANJI WEISSM.\NN, C. (1971) Nalure 234, 203

27. 28.

WEIIER, K. (1967) Biochemistry 6, 3144 KONIGSBERG, W., XUT.~, T., KATZ~, J., AND WJ~ISEIX, K. (1970)

n‘alure 227, 271 29. HINDLIST, J., AND STAPLES, 1). H. (1971) ATature New Hiol. 234,

211 30. 31.

C.~NFIELD, 11. E. (1963) J. Biol. Chem. 238, 2691-2697 WI:ISI~;R, A. RI., AND WEUI~:R, K. (1971) Nature IVew Biol. 234,

206 32. RICE, IL. H., AND MEANS, G. E. (1971) J. Biol. Chem. 246,831-

832 33.

34. 35.

36. 37.

SHAPIRO, A. L., VIR~I~LA, E., AND MAIZEL, J. V., JJZ. (1967) Biochem. Biophys. Res. Commun. 28, 815

STR.AISS, E. G., AND KAESBERG, P. (1970) Virology 42, 437 NI~LL, H. D., KP:UTM,~NN, H. T., COPP, D. H., AND PUYJLS,

J. T., JR. (1969) Proc. Nat. Acad. Sci. U. S. A. 64, 771 EDM.IN, P. (1960) Ann. N. Y. Acad. Sci. 88, 602 BOXKER, J., .~XD T’so, I’. (Editors) (1964) The nucZeohis/ones,

Holden-Day, San Francisco 38.

39. 40.

PISKIEWICZ, D., LANDON, M., AND SMITH, E. L. (1970) Biochem. Biophys. Res Commun. 40, 1173

KONIGSRERG, W. (1967) Methods Enzymol. 11, 461 %.\J~Ic, G. K., AND &VYTH, D. G. (1963) J. Biol. Chem. 238, 214-

226 41. 42.

43.

B~oiw&x, R. (1967) Methods Enzymol. 11, 398 EDMAN, P. (1970) in S. B. NJCJEDLEMAN (Editor), P&ein se-

quence determination, Springer-Verlag, New York PLATT, T., WEB~R, K., G.INEM, D., .?ND MILLER. J. II. (1972)

44. 45. 46. 47. 48.

Proc’. Nut. Acad.‘Sci: U. S. A. 65, 897 HIRS. C. H. W. (1967) Methods Enzuwlol. 11. 197 B~~ET&H~;R, M. 6. (ld71) ,Vature Ne”w Bial. i31, 229 LENGYEL, I’., AND &LL, I>. (1970) Bacterial. Rev. 33, 264 Ir.4~<x~~r, U. K. (1970) Nature 227, 680 BALTIMORE, II., Hu.\NG, A., MANLY, K. F., REI~OSH, I>., .\ND

STAMPFJCR, M. (1971) The strategy of the viral genome, Ciba Foundntiotl

49.

50.

PLATT, T., MILLER, J. H., AND WEBER, K. (1970) Nature 228, 1154

51. 52.

~TE'IVART, J. W., SHERMAN, F., SHIPMAN, N. A., AND J.uxsors, M. (1971) J. Biol. Chem. 246, 7429

KYTE, J. (1971) J. Biol. Chem. 246, 4157-4165 ~UIDOTTI, G. (1960) Biochim. Biophys. Acta 42, 177 'ROBERTS, D. B. (1969) J. Mol. Biol. 45, 221-229


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