Sequence homology analysis of proteins by chemical cleavages: Using a mono and two dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Im. J. Biochem. Vol. 18, No. 12, pp. 1073-1082, Printed in Great Britain. All rights reserved

1986 Copyrtght

0020-7 I I X/86 $3.00 + 0.00 f“ 1986 Pergaman Journals Lid

MINIREVIEW

SEQUENCE HOMOLOGY ANALYSIS OF PROTEINS BY CHEMICAL CLEAVAGES: USING A MONO AND TWO DIMENSIONAL SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS KIA-KI HAN, CLAUDERICHARD, GUI-Yr ZHANG and ANDRE DELACOURTE Laboratoire des Neurosciences, Uniti: INSERM N” 16, Place de Vcrdun, 59045 Lille Ctdex. France [Tel. 20-53-45-291

(Receiaed 14 April 1986)

Abstract-l. The examination of possible sequence homology in proteins using SDS-PAGE systems after chemical cleavage is described. 2. After SDS-PAGE, the establishment of amino acid compositions. the techniques of staining gel and five different methods of chemical cleavages (cyanogen bromide, BNPS-skatole, hydroxylamine, formic acid and nitrothi~yano benzoic acid) have been used for peptide mapping studies. 3. Potential aunlications of this technique are discussed from both the biochemical and immunochemical

point of view. __

INTRODUCTION Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is one of the most effective, flexible and sensitive methods for fractionating and characterizing complex mixtures of biopolymers. It has almost unrivalled ability to resolve mixtures of proteins and polypeptides and has become one of the most important and widely-used microanalytical methods in biochemistry. This powerful analytical tool is used frequently for physico-chemical studies, molecular weight determinations, examination of protein patterns in normal and pathological conditions and investigations in biochemical genetics in short, for almost every aspect of biochemistry. Protein and polypeptide mapping is frequently used for identification, preliminary characte~zation and assessments of interrelationships. Cleveland et ul. (1977) have applied SDS-PAGE to one dimensional peptide mapping of partial protease digests of purified proteins or protein bands cut from gels for the purposes of identifying the precursor-product relationships. The success of this approach has led to wide-spread application. However this technique requires proteases that can generate specific banding patterns that depend strongly upon amino acid sequence and these proteases will generate peptide fragments too small to separate well on SDS-gels. Furthermore, the method of Cleveland et al. (1977) has encountered several problems: first, for a Coomassie Biue staining pattern, the sensitivity is about 5-1Opg of purified protein. It is difficult to obtain such quantity of protein because usually it is a small spot cut from a complex 2 dimensional gel pattern. It could be overcome by autoradiography (Fairbanks et

al., 1965) or by fluorography (Banner and Laskey, 1974). Obtaining labelled proteins is not always feasible and even if it is feasible, the process is expensive and time consuming. Therefore the silver-staining techniques for detecting proteins on SDS-PAGE (Oakley et al., 1980; Wray et al., 1981) have been developed. These techniques are capable of resolving less than 0.5 ng/protein mm’ of gel (Ochs et al., 1981) but because of this sensitivity, the procedure of Cleveland et al. (1977) is often difiicult to interpret: the enzyme preparations invariably contain a multitude of contaminating proteins that can be detected by silver staining and that may obscure the peptide map (Lischwe and Ochs, 1982). Since the pioneering work of Gross and Witkop (1961) on the chemicai cleavage of methionyl-X bonds in proteins by CNBr, the chemical reagents that can cleave peptide bonds adjacent to particular residues offer an alternative means of fragmenting proteins for such fingerprint analysis. Usually, the methionine content in proteins is low, so that its adjacent peptide bonds offer singular targets for cleavage that can yield a discrete small set of peptide fragments and which are ideally suited to peptide mapping. Recently, Han et al. (1983) have extensively reviewed the principles, applications and mechanisms of reactions of different chemical cleavage procedures. The more recent works of Mahboub et ai. (1986), have used chemical fragmentation and the fingerprint method analyse the sequence homology analysis of the triplet neurofilament proteins of beef. In this paper, we shall deal with the techniques of mono and/or two-dimensional gel electrophoresis, their application to molecular weight determination,

1074

KIA-KI

the amino acid composition of proteins eluted from gels, different techniques of staining, different protocol of chemical cleavages (cyanogen bromide for methionyl bonds; BNPS-skatole for tryptophanyl bonds, the hydroxylaminolysis for the asparaginylglycyl bonds; partial acid hydrolysis of aspartyiprolyl bonds and the cleavage of X-cysteinyl bonds by NTCB-acid of proteins. We will take the cleavage on the tryptophanyl bonds by BNPS-skatole on the sequence homology analysis of the triplet neurofilament “L”, “M” and “H” subunits of beef by peptide mappings on the SDS-PAGE systems as an original example. This is a critical review with extensive discussions based on our personal unpublished work. Monodimensionul SDS-PAGE generation qf protein sampIes

electrophoresis

and

Electrophoresis and staining with Coomassie Blue are usually performed in the standard fashion. We take an example reported by Nikodem and Fresco (1979) and the conditions currently applied by us (Richard et al., 1985). Proteins (2-20 pg) are dissolved in 25 1.11water and mixed with an equal volume of buffer containing 0.125 M Tris-HCI, pH 6.8, 20% glycerol, 2% SDS and 0.001% bromophenol blue, then 10 ~1 of fi-mercaptoethanol is added. This sample is boiled for 2 min cooled and transferred to a sample well of IO-15% SDS-PAGE slab depending upon the size of proteins being separated and electrophoresed in the standard manner using the discontinuous solvent system as reported by Laemmli (1970). The 5-l 5% acrylamide reticulation gradient as described by Thorpe er al. (1979) are frequently used by us (Richard et al., 1985). Bidimensional electrophoresis O’Farrell (1975) has described a technique for the separation of proteins by two-dimensional polyacrylamide gel electrophoresis (ZD-PAGE). The principles are that the proteins are separated according to isoelectric point by isoelectric focusing in the first dimension and according to molecular weight by SDS-PAGE in the second dimension. Since these two parameters are independent, the possible uniform distributions of protein spots across a two dimensional gel may be obtained. The more recent works of Duncan and Hershey (1984) have considerably improved the conditions reported by O’Farrell (1975) by Anderson and Anderson (1978) and by Bravo and Celis (1980). Duncan and Hershey (1984) have changed conditions and optimized isoelectric focusing conditions. These authors have varied five major IEF-parameters: volthours. concentration of acrylamide. NaOH, H, PO, and equilibration time in order to determine the effect of each 2D-IEF/SDS-PAGE gel patterns and to optimize IEF conditions. A new PAGE-qstem peptidef

,fix the separa~~an af smail

Tsugita ef al. (1982) have reported a new method for the separation of small peptides in a volatile buffer system after modification with a strongly acidic fluorescent amino groups reagent. This new tech-

HAS ri ai. nique has been developed in order to facilitate both separation and extraction of peptides and core with proteins ranging in MW from 200 up to 100,000 dalton. These authors have used a volatile buffer: trimethylamine-formic acid pH 11.7 and a reaction with a reagent which binds covalently to amino groups reagent (1~3.4-tr~sulfonylpyrene-8-isothi~)cyanate). Because of the addition of strongly negative charges to proteins and it allows migration according to MW. The chemically modified proteins arc fluorescent and require neither fixation nor staining for their detection. This is very important for dealing with small peptides. Another advantage is the increased solubility of the chemically modified proteins which makes their extraction easier and more efficient from the gel. The method avoids the use of salts, dyes or SDS.

Gels are stained with Coomassie Blue 0.05% in propanol-acetic acid-H20 (5:2:13 by vol) and then destained with 10% acetic acid and may be stored in this solution until required. Siher sfainings ,fi)r proteins in PAGE Several ultrasensitive silver staining techniques for detecting polypeptides on one and/or ZD-PAGE have been reported in the literature (Switzer er uf.. 1979: Oakley et al., 1980: Merril et al., 1980; Sammons et al.. 1981; Wray et al., 1981; Merril et ul., 1981). Ochs et al. ( 1981) have compared the six of the most recent silver staining methods for detecting polypeptides on PAGE. They have found the method of Sammons et al. (I 98 1) to be least expensive and most reproducible. For staining gels of I .5 mm in thickness, it is also the most sensitive method. The other methods are preferable for staining gels of 0.8 mm or less in thickness.

Recently, Bahrman and Thiellement (1985) have reported a new method for staining proteins after high resolution ZD-PAGE. The PAGE-matrix is stained blue by photoreduction of nitro-bluetetrazolium into formazan while the spots of polypeptides remain unreacted. The sensitivity of this method is comparable to that obtained by silver staining. The proteins spots in ZD-PAGE are therefore stained “negatively” and suitable for their localization. Amino acid composition ($ proteins eluted.firom poly acr$amide gels Brown and Howard (1980) have established the amino acid composition of proteins eluted from gels with a small quantities (2&5Opg) of protein. These authors have shown that a measurable background contamination resulted from the elution of slices of blank polyacrylamide gels. They have corrected the composition for background contribution based on the volume of gel eluted and then used resulting composition with accuracy. Usually, proteins were eluted from gels by either electrodialysis by the procedure of Stephens (1975) or by diffusion into 0.02% SDS (I ml:1 cm3 gel slice) for 24 h with one change of eluate. The pooled eluates were dialyzed

Sequence

homology

analysis

for 2 days against water with frequent changes of the dialysate. Sreekrishna et al. (1980) have modified the amino acid analysis with ninhydrin of Coomassie Blue stained proteins from polyacrylamide gels since the method reported by Houston (1971) in which the stained ge1 slices are directly hydrolyzed by HCI impairs and prevents the determination of basic amino acids. The reason for this is that a large amount of ammonia derived from the hydrolysis of polyacrylamide is present in the hydrolysate. Sreekrishna PI at. (1980) have extracted the stained protein from the gel prior to acid hydrolysis. These authors have submitted the Coomassie Blue protein SDS complex present in the gel to elution by homogenization of the gel in 2 ml of 0.05 M NH,HCO, containing 0.05% SDS followed by incubation at 37°C for 10 hr on a shaking water bath. The gel suspension was centrifuged and the supernatant was corrected. The pellet was vortexed again with another 1 ml of 0.05 M NH,HCOl containing 0.05% SDS and recentrifuged. The pooled supernatant was dialyzed extensively at 4:‘C against 0.005% SDS. The dialysate was lyophilized repeatedly to remove NH4HC0,. The dry sample was ready for acid hydrolysis. The recovery of the protein extracted from the gel was greater than 90% of the starting material. Manabe ef al. (1982) have described a method of extraction of proteins from fixed, stained, two-dimensional polyacrylamide gels (2D-PAGE) and subsequent amino acid microanalysis. After 2D-PAGE, the stained spots were punched out and the proteins in each piece of gel extracted with 0.1 M NaOH 2% thiodiglycol. The extracted proteins were then hydrolyzed and analyzed by the fluorimetry after reaction with O-phthaldialdehyde. This method has reduced the amount of background contaminants and the amino acid analysis of I pg or less of extracted proteins has become possible.

of proteins by chemical cleavages

1075

1076

KIA-KI HAN ef a/.

The more recent works of Richard et al. (1985) and Mahboub ef al. (1986) are a great interest for the chemical cleavage on Trp-X bonds followed by peptide mappings. Mahboub et al. (1986) have reported the comparative studies of neurofilament triplet proteins after chemical cleavage at both preparative and analytical scales. Because of its wide applicability, we describe the protocol experimental as reported by Richard et al. (1985). Experimental (1985)

protocol according

to Richard et al.

BNPS-Skatole cleavage on the tryptophanyl bonds. The gels were equilibrated with 70% acetic acid (v/v) containing 0.1% of phenol during IO min. Then the gels were cut off into small pieces and ground in a potter. For each band, about 10ml of 70% acetic acid containing 0.1% phenol (w/v) were used. BNPS-Skatole cleavages, carried out as described by Hunziker el al. (1980) were performed in 70% acetic acid and 0.1% phenol for 48 hr at room temperature in the dark by using IO mg (IO-fold excess) of the reagent per gel. After the addition of P-mercaptoethanol (20~1 per gel) a further incubation for 5 hr at 37°C was performed. The solution containing the peptide fragments was then dialyzed against the electrophoresis buffer (Tris-Gly-SDS pH 8.3) (4 x 100 ml containing 20% glycerol) for 48 hr. Finally, the solution was dialyzed against the electrophoresis buffer containing 20% glycerol and 2% p-mercaptoethanol. The solution was finally concentrated by putting the bag in close contact with Sephadex G-200 for 72-96 hr at 4-C on a paper sheet. Hydroxylaminolysis proteins in PAGE

of usparaginyl-Rl)~c:,~l bonds qf

Hydroxylaminolysis of Asn-Gly bonds has been in the study of proteins since 1969 as first reported by Bornstein (1969). The reaction mechanism of this cleavage is such that cleavage occurs specifically at a limited number of sites (Asn-Gly) and has made it suitable for the generation of polypeptide fragments on a preparative scale to be used in sequence studies and/or immunological studies. Its application to peptide mapping using SDSPAGE systems was firstly reported by Lam and Kasper (1980) and more recently by Saris rr al. ( 1983) and by Mahboub et al. (1986). Saris et al. (1983) have reported a modification of the hydroxylamine cleavage technique in which proteins were cleaved while immobilized in the matrix of polyacrytamide gel. The reaction under these conditions retains its high specificity for Asn-Gly bonds and has the advantage that the gel matrix, acting as a carrier contributes to a high recovery of the cleavage products. These authors (Saris et al., 1983) have also deveIoped a ZD-version of the cleavage method which allows rapid recogni~tion of interrelationships between proteins in a complex mixture. The versatility of the procedure is demonstrated in a number of applications by these authors. Owing to its wide applicability, we describe the experimental protocol reported by Saris PZ ul. (1983).

instrumental

Experimental protocol Saris et al. ( 1983) Protein bands in the gels were located first by classical methodology and excised. The gel pieces were swollen in 5% methanol. paper backing was peeled off and excess SDS in excess was removed by washing at 4°C in four changes of 3 ml 5% methanol. Pieces excised from low percentage gels (less than 10%) were swollen in 40% methanol, 5% acetic acid, before washing in 5% methanol. Gel pieces were brought to near-dryness in a vacuum dessicator and submerged in reaction in a closed vial and incubated for 3 hr at 45’C’. The reaction medium was made by dissolving 1.1 g hydroxylamine HCI (final concentration 2 M), 4.6 g guanidine HCI (final concentration 6 M) and 15 mg Tris base (final concentration 15 mM) in 4.5 M LiOH to give a pH of 9.3 and final volume of 8 ml. To prevent formation of insoluble carbonates when making 4.5 M LiOH, it is important to use freshly boiled water. After the reaction, gel pieces were blotted on tissue paper and washed with 4 changes of 3 ml 5% methanol. Here again. the low percentage gel pieces were washed in 40% methanol. 5% acetic acid prior to the 5% methanol washing in order to prevent the loss of small fragments. The gel pieces are again brought to near-dryness and then submerged in electrophoresis sample buffer (30 jll of buffer per 3 pl of gel material) and kept at 37 C for 2 hr and 95’C for 5 min. At this point, protein was released from the gel matrix either by electroelution or by direct analysis on a second SDS-PAGE. Partial acid hydro~~.s~.~ of a.~pariyf--pr~)l~lho&5 Aspartyl~~prolyl bonds have been found to be hydrolyzed during exposure to low pH (2 2.5) under conditions where other aspartyl-X bonds are stable. Jauregui-Adell and Marti (1975) and Landon ( 1977) have studied the acid lability of Asp-Pro peptide bonds and established procedure for fragmentation of proteins for sequence studies. The average frequency of AspPro bonds is one bond per 400 amino acid residues. Owing to the rarity of AspPro bonds, their selective hydrolysis usually yields relative large peptides that are suitable for analysis by SDS-PAGE. Sonderegger ei al. (1982) and more recently Rittenshouse and Marcus (1984) and Mahboub et a!. (1986) have demonstrated that cleavage occurs predominately at aspartyl-prolyl bonds during the partial acid hydrolysis. Ritthenhouse and Marcus (1984) have reported that the case with which small amounts of protein, either in solution or in gel pieces. can be cleaved by heating and directly submitted to electrophoresis makes the method ideal for peptide mapping and for rapid detection of Asp--Pro peptide bonds. Experimental protocol of acidic cleacuge oj Asp--Pro bonds according to Sonderegger et al. (1982) Gel pieces containing the protein band were equilibrated with 5 ml 75% formic acid for 4 hr at 20 C. Subsequently, the acid solution was aspirated and the gel piece covered with 2 ml liquid paraffin in order to prevent evaporation of formic acid. The gel piece was incubated at 37 ‘C for 24 hr after which the paraffin

was then replaced by Tris buffer in order to remove the formic acid. After soaking in 10 ml of 250mM Tris-HCI, 0.5% SDS, pH 9.0 for 2 x 1 hr at 2O”C, the gel piece was kept in 125 mm Tris-HC1, 0.5% SDS, PH 6.2,20 glycerol for another 2 x 1 hr. An improved method in which the gel pieces were lyoph~liz~d 3 times and finaiiy reswollen in 125mM Tris-Hei, 0.5% SDS, pH 6.8, 20% glycerol for 15 min was also reported by these authors. Cleavage of X-cysteiny/ cyano-benzoic acid

bonds by 2-nitro .5-thio -

Degani and Patehornik (1974) reported that Xcysteiny1 residues were converted to S-cyanocyst~~ny~ residue by reaction with nitro-thiocyanobenzoic acid at pH 8.0. The S-cyanocysteinyl or fi-thiocyanoalanine was incubated at pH 9.0 for cleavage of the peptide bond. It was firstly applied to SDS-PAGE systems recently by Mahboub PZ nL in our laboratory (1986) on peptide mapping after chemical cleavage of neurofilament bovine triplet proteins. Experimental protocol according to Mahboub et al. performed in our laboratory (1986). The protein containing lanes were excised and each gel piece was first cut into small. cubes and incubated in 1Oml of 1OOmM Tris-HC1 buffer pH 6.8 (ccontaining OS M KCI, 3 mM EDTA, 6 M urea) for 4 hr. Subsequently, the solution was sucked off and fresh buffer was added. A IO fold molar excess of NTCB (10 mg per gel piece) was added and the pH was rapidly adjusted to 8.2 with NaOH. After incubation at 37°C for 40 hr under nitrogen with stirring, the reaction was stopped by lowering the pH to 4.0 with acetic acid. The mixture was centrifuged and the supernatant was dialyzed against the eiectrophoresis buffer (pH 8.3) for 24-48 hr and finally concentrated against Sephadex G-200 as described, above. Sequence homology analysis by chemical cleavages can be &unsiderably improved by immuno~h~i~~ techniques. This is one of the reasons we described the experimental protocol routinely performed in our laboratory. immunological techniques Hemon et al. (1986)

according

to Mahbaub,

Preparative gel ~~e~trophores~s was performed using double thickness 3 mm slab gets and Ioading proteins (2-5 mg of proteins) across the full width of the gel. After electrophoresis, the gels stained and then destained as reported above. Preparation of antisera: pieces of acrylamide correm sponding to purified proteins were sliced out, and the po~ypept~d~s were e~~trophoretical~y &ted from the get and dialyzed against phosphate buKer saline (PBS}. 500 ji I of these proteins or chemically cleaved resulting polypeptides were emulsified in 500,~l of incomplete Freund’s adjuvant and injected intradermally into rabbit with intradermal injections. The first bleeding was performed 2 months after the beginning of~mmun~~at~on and then 8 days after each boost. ~rnrn~n~~~~~~~~~: after SDS-PAGE with a 5-15% acrylamide reticulation gradient, gels were washed in a 0.15 M Tris-glycine, pH 6.8 containing 20% of methanol for about 20min to remove SDS. Then

they were put in cIose contact with nitrocelfulose sheets (120 x 140 mm), 0.45 M pore size (Schleicher and Schull, Federal Republic af Germany) and the proteins were transferred with a Transblot apparatus (manufacturers. The nitrocellulose sheets were saturated during 2 hr at 20°C in 5% BSA (bovine serum albumin) in PBS buffer. After saturatjon, the nitrocellulose sheets were washed once more with PBS buffer and incubated overnight at 4°C with antibodies at a dilution of l/200 in PBS. Then the blots were washed 3 times in PBS and incubated with anti-rabbit antibodies labelled with peroxydase (Fasteur~ which were previously diluted to i/SO in PBS, The nitrocellulose were finally deveioped with diaminobenzidine 0.5 mg/ml in 0.1 M Tris buffer pH 7.6, 0.001 hydrogen peroxide. DlSCUSSION In general, the methods for fra~entation of proteins by chemical cleavage require elution of the protein from gel and thus relatively large amounts of material. Therefore, peptide mapping by enzymatic cleavage of proteins without prior elution from the gel has been described by Cleveland et el. (1977). However, some problems inherent to the method of Cleveland et al. (1977) limit its application. First, the Coomassie Blue staining gel technique is not very sensitive (5-1Opg of purified protein). Fairbanks et al. (1965) used autoradiography and Bonner and Laskey (1974) uses fluorography. The process is expensive and time consuming. Therefore the silverstaining techniques were proposed by Oakley ef al, (1980) and the sensitivity is capable of resolving less than 0.5 ng protein/mm’ of gel. However enzymatic digestion & situ is not easily interpretable by silverstaining, because the enzyme used contains a multitude of contaminating proteins that obscure the peptide mapping. Furthermore, the enzymatic digestion occurs in the stacking gel by ~~e~tro~horesing the substrate and enzyme into the stacking gel and then turning off the current for approximate’ly 30min. The stacking gel is porous and the small peptide fragments are able to diffuse during the incubation period and this diffusion may be detected by autoradiography (Lischwe and Ckhs, 1982). Takeda and Cone (1984) have improved the method of Cleveland er al. (1977) by using 2D analysis applicable to amounts, less than O.Sgg of proteins. These authors have used radioiodinated proteins which were digested enzymatically in the presence of 0.1% SDS and an excess of non-labelled bovine serum albumin {BSA) (0.2 mg/mlf reiative to labeled substrate in order to attain re~r~u~ibilit~ by maintaining a consistant substrate concentration among different samples. Kowit and Maloney (1982) have reported that the routine heating of protein samples at IOOVZ prior to SDS-PAGE can cause peptide bond cleavage. Therefore the least heating time should be performed 12% of protein wiH be cleaved by heating for 5 min at IOO’C in gel eiectrophoresis sample buffer (Ritthenhouse and Marcus, 198411. Therefore, the cleavage of proteins at methianyl bonds by CNBr is a valuable tool used in peptide mapping as firstly reported by Nikodem and Fresco

1078

KIA-KI HAN et cd.

(1979). Cleavage can be readily accomplished in SDS-PAGE using high concentrations of CNBr (20 mg/8 ml). Two dimensional patterns can then be obtained by reelectrophoresis at an angle of 90, to original direction (Lonsdale-Eccles et al., 198 I). The highly reproducible electrophoretic patterns may be obtained (Richard et al., 1985; Mahboub et al., 1986a; Mahboub et al., 1986b). The extent of CNBr cleavage is about 98% (Mahboub et al., 1986). We would like to take an example of the application of chemical cleavage procedures to the sequence analogy investigation by peptide mapping. Richard e/ a/. (1985) have used BNPS-skatole for the cleavage of tryptophanyl bonds of the SDS-PAGE system in a comparative study of the triplet proteins of ox neurofilaments. The triplet proteins subunits possess apparent molecular weights of 210, 160 and 70 kdalton. 210 kdalton, “H” as heaviest subunit as Middle or “M” subunit and the 70 kdalton as the lightest or “L” subunit (Shaw et al., 1984). Geisler CJ~ al. (1985) have recently established the complete amino acid sequence of “L” subunit and Geisler, Fischer et al. (1984) have reported the partial amino acid sequence of “M” subunit and Geisler, Fischer et ai. (1985) have performed the partial amino acid sequence of “l-l” subunit. Therefore, in the “L” subunit, the only tryptophanyl residue is positioned at Trp-278 and the first Trp in the “M” subunit is positioned at Trp-291 and the first Trp in the “H” subunit is not established yet. The cleavage of the unique Trp residue in “L” subunit on the SDS-PAGE system by BNPS-Skatole yields two fragments of 30.5 and 40.5 kdalton MW {see Fig. 1). This result is in excellent agreement with the work of Geisler et ul. (1985). Thus the 30.5 kdalton fragment corresponds to the N-terminal part and the 40.5 kdalton fragment to the C-terminal part. Furthermore, the 30.5 kdalton fragment is also characterized among the cleavage fragments of “M” subunit (Trp-291) and of the “H” subunit (Trp-X) su~esting that the N-terminal part of the three neurofilament proteins subunits is very similar, at least with one tryptophan residue located at the same place. Therefore, we have shown that chemical cleavage of the tryptophanyl residues by BNPS-Skatole is a valuable tool and this technique may be applied to be a probe for the position of Trp in proteins with high MW and with unknown sequence. The extent of cleavage is about 61% (Mahboub et al.. 1986) and in our experimental conditions, the presence of aqueous phenol as the scavenger reagent, there are no side reactions resulting from the partial cleavage of tyrosyl or histidyl bonds by brominating agent. Ma~~ub et al. (1986) have reported the application of this technique at analytical scale with 25 pg of starting material with Coomassie Blue staining or silver staining with the CNBr cleavage at methionyl bonds, the four other chemical cleavage methods described can be usually performed with good yields (98% for CNBr, 61% for BNPS-Skatole, 60-75% for partial acid hydrolysis (Sonderegger rf ul., 1982) and 5475% for the nitro-thiocyano-benzoic acid (Mahboub er al., 1986). Because of their specificity, the SDS-PAGE systems, after chemical cleavage may be applied on the

preparative scale in order to generate polypeptides which are good immunogens for the preparation of specific antibodies against initial proteins since these antibodies possess less cross-reactions. The use of immunoblots and immunohistochemistry in combination with chemical cleavage methods are considered below. Owing to its easiness of use and its high resolution power, the SDS-PAGE system may be applied to the preparative scale in order to isolate chemical cleavage-generated polypeptides which may be used as for the preparation of antisera specific of different part of a Iarge protein. We would like to take two examples in order to illustrate the immunological applications. Geisler et al. (1983) have reported that neurofilament subunits possess two different functional parts: the N-terminal part, with an analogy of sequence common to the triplet neurofilament subunits and very similar to inte~ediate filament subunits and the C-terminal part which are very likely side arms involved in interaction with other neurofilaments or organelles. Since the three neurofilament subunits are related proteins with homologies and differences, the production of antibodies against precise parts of the proteins can give tools 01 interest for a better understanding of their roles in the different aspects of the neurofilament physiology. With that in mind, Richard et al. (1985) Mahboub. Hemon et al. (1986) we have elaborated antisera against two characteristic CNBr-generated polypeptides located in the C-terminal part of “H” and “M” subunits proteins and with molecular weights of 13.5 and 85 kdalton respectively. Results obtained with these antibodies by immunoblotting techniques revealed the similarities between C-terminal part “H” and “M” ox neurofilament proteins and also between C-terminal “H” proteins from ox and pig, whereas these antibodies showed that C-terminal part of “M” proteins from ox and pig share few common antigenic determinants. Thus, these results show that neurofilaments from two related higher vertebrate species (ox and pig) possess significant differences ol structure. The comparison of labelling between antisera against neurofilaments, antisera against cleavage generated fragments and monoclonal antibodies against neurofilament is investigated in our laboratory. First results show that antisera against chemical cleavage- generated polypeptides corresponding to the side arms of neurofilaments label very intensely and very specifically certain populations of ncurofilaments. Thus, it is very likely that this approach of production of antisera against generated polypeptides which is rapid, cheap and precise will be able to compete with monoclonal antibodies as far as the determination of protein structure and function arc concerned. The second example is chosen because the very interesting works reported recently by Mather and Taylor-Papadimitriou (1985). These authors have reported that the monoclonal antibodies, electrophoretically transferred from pol~crylamide gels. retain their ability to bind specific antigens. These authors have demonstrated for two groups of antibodies, directed to a large MW glycoprotcin of human milk fat globule and human interferon a-1.

b M, I

160 +135 3125

1

70

-46

-44.5 -40.5

40.5

-34 30.5

70 K

-30.5

160

K

210

K

Fig. 1. BNPS-Skatole Cleavage of 70 kdalton (“L” subunit), 160 kdalton (“M” subunit) and 210 kdalton ox neurofilament triplet proteins. M,: Molecular ratio expressed in kdalton. The (“H” subunit) Mono-dimensional SDS-PAGE system.

1079

1081

Sequence homotogy analysis of proteins by chemical cleavages

Immunoreactive antibody fragments produ~~I by protease digestion could be also be identified in this way on western blots, thus permitting the development of optimal conditions for digestion, without the need for extensive pu~~~ation procedures. Furthermore, the enzymatic protease digestion may be considerably improved by replacing the protease digest by chemical cleavages. The chemically cleaved resulting polypeptides possess usually more higher MW and therefore more suitable for immunological investigations (our unpublished works).

The chemical cleavage methods applied directly in SDS-PAGE systems should have wide applications in the analysis of sequence homology of proteins that contain methionine, tryptophan residues as well as Asp-Pro, Asn-Gty and X-cysteinyl bonds. The prep arative scab in SDS-PAGE after chemical cleavage is about 3-5 mg of starting materials per one gel and the analytical scale is about 2540 pg of starting materials (Mahboub et al., 1986). Towbin et al. (1979) have reported the separation of protein mixtures by PAGE system followed by electrophoretic transfer to nitrocellulose with subsequent characterization of the resolved mixture by antibody probes. Conventionally, after first dimcnsion running on SDS-isocratic polyacrylamide gels, the separated protein bands are then submitted to a second electrophorsis at right-angles to the plane of the SDS-PAGE in order to transfer them to nitrocellulose paper to which they become strongly bound. The nitrocellulose can thus be incubated with monoclonal or polyclonat antibodies which bind to proteins bearing the relevant antigenic determinants which can be detected directly if labelled with an isotope or indirectly using a conjugated second antibody. Such procedures permit the unequivocal detection of a minor constituent in biological sample or the characterization of component recognized by a monoclonal antibody (Mather and TaylorPapadimitriou, 1985). By combining and modifying the techniques described by Cleveland et al. (l977), the chemical cleavage procedures are the very valuabte tools for the analysis of sequence homology of proteins and also these techniques are suitable for generating polypeptides adequate for immunological investigations. REFERENCES

Anderson N. L, and Anderson N. G. (1978) 2 dimensional anaiysis of serum and tissue proteins: Multiple gradient slab-gel elcctrophoresis. Am&t. &&em. 85; 341-354. Bahrman N. and Thiellement H. (198% Sensitive nitra-bluetetrazolium staining of pro&s in high resolutian 2 dimensional gel electrophoresis. Efectrophoresis 6, 357-358. Banner W. M. and Laskey R. A. (1974) A film detection method for tritium labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochcm. 46, 83-88. Bornstein P. (lQ69) The nature of hydroxylamine sensitive bond in collagen Biochem biuphJx Res. Commun. 36, 957-964.

Bravo R. and Celis J. E. (1980) A search for differential polypeptide synthesis throughout the cell cycle of Hela

cells. J. cell, Bid.

84, 795-802.

Brown W. E. and Howard G. C. (1980) Amino acid compostion of proteins eluted from polyacrylamide gets: back8rotmd eons~deratio~s. &u&. &ctrem. 181, 294298. Cleveland D. W.. Fischer S. Cr., Kirschner M. W. and Laemmli U. K. (19771 Peotide maopine. bv limited nroteolysis in SDS and analysis by gel &&phoresis. /. biol. Chem. 252, 1302-l 106. Debuire B., Han K. K., Dautrevaux M., Biserte G.. Regnouf F. and Kassab R. (1977) Amino acid sequence of CNBr fragment containing the 2 tryptopbany~ residues of Lobster arginine-kinase. J. Biochem. 8f, 61 f-619. Degani Y. and Patchornik A. (1974) Cyanylation of SHgroups by nitrothiocyanobenzoic acid: high yield modification and cleavage of peptides at cysteine residues. Biochemistry 13, l-l 1. Dennison C., Lindner W. H. and Phillips N. C. K. (1982) Nonuniform field gel eIectrophor&s. Ana&t. Binc!rern. X20, t2- 18. Detke S. and Keller J. M. (1982) Comparison OF proteins present in Hela cells interphase nucleoskeletons and metaphase chromose scaffolds. J. biol. Chem. 251, 3905-391

I.

Duncan R. and Hersey J. W. B. (1984) Evaluation of isoelectric focusing running conditions during 2 dimensional isoelectric focusing~SDS-PAGE. An&t. Biwhmm. 138, t44155. Fairbanks G.. Levinthal C. and Reeder R. H. (1965) Analysis of “C-labelled proteins by disc electrophoresis. Biochem.

biophys.

Res. Cammun.

20, 393-399.

Fontana A. (1972) Modification of tryptophanyl residues with BNPS-Skatole. Meth. Enzymol. 25, 419.423. Geisler N., Fischer S., Vandekerckbove J., Plessmann U. and Weber K. (13843 Hybrid character of a large Nfprotein (MI: intermediate filament type sequence followed by a long and acidic C-terminal extension. EMBD 1. 3, 2701-2706. Geisler N., Kaufmann E., Fischer S., Plessmann U. and Weber K. (1983) Nf architecture combines structural principles of intermediate filaments with C-terminal extension increasing in size between triplet proteins. EMBU J. 2, 1295%1382. Geisler N., Fischer S., Vandekerckhove J., Van Damme J_” Plessman U. and Weber K. (1985) Protein chemical characterization of Nf-H, the largest mammalian Nf component; intermediate filament type sequences followed by a unique C-terminal extension. EM&U J. 4, 57-63. Geisler N., Plessmann LXand Weber K. fi985) Complete amino acid sequence of major mammahan Nf protein (Neurofii~ment-lightest subunit: Nf-L). FEBS Leti. 182, 475478, Gross E. and Witkop B. (1961) Selective cleavage of methionyl peptide bonds in RNAase with CNBr. J. Am. C&VI. Sot, 83, 3510-151 I. Han K. K., Richard C. and Biserte G. (1983) Current developments in chemical cleavage of proteins. Inl. J. Biuchem. IS, 875-884. Houston L. L. (1971) Amino acid analysis of stained bands from polyacrylamide gels. Analyf. Biochem, 44, 81..-88. Hunziker P, E., Hughes G. J. and Wilson K. J. (1980) Peptide fragmentation suitable for solid phase sequencing: use of NBS and BNPS-Skatole. Bkhem. .I, t87.

515-519.

Jaurcgu~-Ade~I J, and Marti J. (1975) Acidic cleavage of Asp-Pro bond and the limitation of the reaction. An&r. B&hem.

69, 468473.

Kowit J. D. and Maloney J. (1982) Protein cleavage by boiling in SDS prior to electropboresis. Anal.vt. Rio&em. 123, 8693.

1082

KIA-KI HAN er al.

Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of bacteriophage T4. Nature, Lond. 227, 680-685. Lam K. S. and Kasper C. B. (1980) Sequence homology analysis of a heterogeneous protein population by chemical and enzymic digestion using a 2 dimensional SDSPAGE systems. Analyt. Biochem. 108, 220-226. Landon M. (1977) Cleavage at Asp-Pro bonds. Me/h. Enzymol. 47, 145-149. Lischwe M. A. and Ochs D. (1982) A new method for partial peptide mapping using N-chloro-succinimide:’ urea and peptide silver staining in SDS-PAGE. Anaiyt. Biochem. 127, 453457. Lischwe M. A. and Sung M. T. (1977) Use of NCS-urea for the selective cleavage of tryptophanyl peptide bonds in proteins. J. biol. Chem. 252, 4976-4980. Lonsdale-Eccles J. D.. Lynley A. M. and Dale B. A. (1981) CNBr cleavage of proteins in SDS-PAGE. Biochem. J. 197, 591-597. Mahboub S., Hemon B.. Defossez A., Delacourte A. and Han K. K. (1986) Biochemical and immunological studies after CNBr cleavage. Comp. Biochem. Ph.vsio/. In press. Mahboub S., Richard C., Delacourte A. and Han K. K. (1986) Applications of chemical cleavage procedures to the peptide mapping of Nf triplet proteins bands in SDS-PAGE. Analyr. Biochem. 154, 171 -182. Manabe T., Oda 0. and Okuyama T. (1982) Amino acid microanalysis of proteins extracted from spots of fixed stained 2 dimensional gels. J. Chromatogr. 241, 361 370. Mather S. and Taylor-Papadimitriou (1985) Monoclonal antibodies, electrophoretically transferred from polyacrylamide gels. retain their ability to bind specific antigens. Biochem. biophys. Res. Commun. 133, 102C-1025. Merril C. R., Dunau M. I. and Goldmann D. (1980) A rapid sensitive silver stain for polypeptides in polyacrylamide gels. At&y/. Biochem. 110, 201- 207. Merril C. R., Goldmann D., Sedmann S. A. and Ebert M. H. (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Sciencr 211, 1437-1438. Nikodem V. and Fresco J. R. (1979) Protein fingerprinting by SDS-PAGE after partial fragmentation with CNBr. Anuiyl. Biochem. 97, 382-386. Oakley B. R., Kirsch D. R. and Morris N. R. (1980) A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Analyr. Biochem. 105, 361-363. Ochs D., McConkey E. H. and Sammons D. W. (1981) Silver stains for proteins in polyacrylamide gels: a comparison of 6 methods. Electrophoresis 2, 304307. O’Farrell P. H. (1975) High resolution of 2 dimensional electrophoresis of proteins. J. hiol. Chem. 250,4007-4021.

Richard C., Mahboub S., Delacourte A., Hemon B. and Han K. K. (1985) Comparative studies of neurofilament triplet proteins: peptide mapping by chemical cleavage. Comp. Biochem. Physiol. 8OB, 7077112. Ritthenhouse J. and Marcus F. (1984) Peptide mapping by PAGE after cleavage at Asp-Pro peptide bonds in SDS containing buffers. Anal.vf. Biochem. 138, 442-448. Sammons D. A., Adams L. D. and Nishizawa E. E. (1981) Silver staining techniques of proteins. Eleclrophorrsis 2. 135-141. Saris C. J. M., Eenbergen J. Van, Jenks B. G. and Bloemers H. P. J. (1983) Hydroxylamine cleavage of proteins in polyacrylamide gels. Anulyt. Biochem. 132, 5467. Shaw G., Debus E. and Weber K. (1984) The immunological relatedness of neurofilament proteins of higher vertebrates, Eur. J. cell. Biol. 34, 130-136. Sonderegger P., Jaussi R., Gehring H.. Brunschweiler K. and Christen P. (1982) Peptide mapping of protein bands from PAGE by chemical cleavage in gel pieces and re-electrophoresis. Ana~J~r. Biochem. 122,298.130 I. Sreekrishna K.. Jones C. E.. Guetzow K. A.. Prasad M. R. and Joshi V. C. (1980) A modified method for amino acid analysis with ninhydrin of Coomassie stained proteins from nolvacrvlamide gel. An&t. Biochem. 103, 55 57. Stephens R. E.- (1975) High resolution preparative SDSPAGE: fluorescent visualization and electrophoretic elution concentration of protein bands An&r. Biochem. 98, 231-237. Switzer R. C., Merrill C. R. and Shifrin S. (1979) A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Analyt. Biochem. 98, 231-237. Takeda A. and Cone R. E. (1984) 2 dimensional peptide mapping by PAGE with limited proteolysis in SDS. Biochem biophys. Res. Commun. 122, 932-937. Thorpe R., Delacourte A., Ayers M., Bullock C. and Anderton B. H. (1979) The polypeptides of isolated brain 10 nm filaments and their association with polymerized tubulin. Biochem. J. 181, 275-284. Towbin H., Staehelin T. and Gordon J. (1979) Electrophoretic transfer of Proteins from polyacrylamide gels to nitrocellulose and some applications. Proc. nafn. Acad. Sri. U.S.A. 76, 4350-1354. Tsugita A., Sasada S., van den Broek R. and Schemer J. J. (1982) A new PAGE system. Eur. J. Biochem. 124, 171-176. Weber K. and Osborn M. (1969) The reliability of MW determination by SDS-PAGE J. biol. Chem. 244, 44064412. Wray W., Boulikas T., Wray V. P. and Hancok R. (1981) Silver staining of proteins in polyacrylamide gels. Ana!,~~. Biochem. 118, 1977208.