Biochem. J. (1970) 118, 733-739Printed in Great Britain

733

The Reversible Reaction of Protein Amino Groups with exo-cis-3,6-Endoxo-A4-tetrahydrophthalic anhydride

THE REACTION WITH LYSOZYME

By M. RILEY AD R. N. PERHAMDepartment of Biochemi8try, Univer8ity of Cambridge, Cambridge CB2 IQW, U.K.

(Received 16 March 1970)

1. The reaction of exo-ci8-3,6-endoxo-A4-tetrahydrophthalic anhydride withamino groups ofmodel compounds and lysozyme is described. 2. Reaction with thee-amino group of Na-acetyl-L-lysine amide gives rise to two diastereoisomericproducts; at acidpH the free amino group is liberated with anchimeric assistance bythe neighbouring protonated carboxyl group with a half-time of 4-5h at pH 3.0 and25°C. 3. The amino groups of lysozyme can be completely blocked, with total loss

of enzymic activity. Dialysis at pH 3.0 results in complete recovery of the nativeprimary and tertiary structure of lysozyme and complete return of catalyticactivity. 4. The specificity of reaction of this and other anhydrides with aminogroups in proteins is discussed.

It has been recognized for some time that amidelinkages situated in close proximity to a carboxylgroup can be hydrolysed by a mechanism thatinvolves intramolecular catalysis by that carboxylgroup (Leach & Lindley, 1953). The most favour-able situation for such catalysis is that in which theamide and carboxyl functions are eclipsed andincapable of free rotation (for review see Kirby &Lancaster, 1970), and in recent years this principlehas been utilized as a means of reversibly blockingprotein amino groups. Thus maleic anhydride(Butler, Harris, Hartley & Leberman, 1967, 1969),2-methylmaleic (citraconic) anhydrideanddimethyl-maleic anhydride (Dixon & Perham, 1968) havebeen shown to react with protein amino groups togive the corresponding dicarboxylic acid mono-amides in which the protonated species of thefavourably situated carboxyl group anchimericallyassists the hydrolysis of the amide bond. Hence themaleyl, citroconyl and dimethylmaleyl groups,though stable at neutral and alkaline pH, may beremoved at acid pH.These arihydrides have a great many potential

uses, e.g. in sequence work, as a method of in-hibiting tryptic cleavage at lysine residues, as ameans of purifying lysine-containing peptides bydiagonal electrophoretic techniques (Perham &Jones, 1967; Perham, 1967) and in effecting thedisaggregation of multimeric proteins (Gibbons &Perham, 1970). To accommodate these adequatelyit would be desirable to have an extension to thisrange of anhydrides, since the conditions necessaryfor the removal of maleyl groups may be too severe

for some purposes, whereas dimethylmaleyl (andpossibly citraconyl) groups may be too labile forothers. Further, these three reagents possessactivated olefinic bonds and the irreversible nucleo-philic addition of protein thiol groups is a distinctpossibility, especially in proteins that possessreactive cysteine residues. For these. reasons welooked for an anhydride that would give derivativesmore labile than maleyl groups but less labile thancitraconyl groups, and preferably an anhydridewith no activated olefinic bonds.

It is known that mono-p-methoxyphenyl estersof ETPC* are approximately five times as labileas those of maleic acid (Bruice & Pandit, 1960a,b)and it was therefore expected that ETPA (I) wouldpossess the properties we sought. The presentpaper describes the reaction of this reagent withmodel compounds and with lysozyme (EC 3.2.1.17).

MATERIALS AND METHODSReagents and enzymes. Maleic anhydride was purchased

from Emanuel Ltd., Alperton, Middx., U.K., and purified

* Abbreviations: ETPA, exo-cie-3,6-endoxo-A4-tetra-hydrophthalic anhydride: ETPC, exo-cis-3,6-endoxo-A4-tetrahydrophthalic acid; ETP, eXo-ci8-3,6-endoxo-A4-tetrahydrophthaloyl.

vCO0

(I)

M. RILEY AND R. N. PERHAM

further by recrystallization from chloroform. Furan was

obtained from BDH (Chemicals) Ltd., Poole, Dorset, U.K.,and was purified by distillation immediately before use.

2,4,6-Trinitrobenzenesulphonic acid was from EastmanKodak Ltd., Kirkby, Lanes., U.K.; solutions, made up inglass-distilled water, were stored in the dark at 40C andwere discarded after 24h. Guanidine hydrochloride was

prepared from guanidine carbonate as described byNozaki & Tanford (1967).

NO-Acetyl-L-lysine amide acetate (lot S-1614) was

purchased from Cyclo Chemical Corp., Los Angeles,Calif., U.S.A. Paper electrophoresis at pH16.5 gave a

major spot with mobility +0.86 relative to aspartic acid(Offord, 1966) corresponding to the desired compound,together with a trace impurity neutral at pH16.5. It was

used without further purification and was determined byhydrolysis and amino acid analysis.ETPA was synthesized from maleic anhydride and

furan as described by Diels & Alder (1929) and recrystal-lized once from chloroform, m.p. (uncorr.) 115-116°C(decomp.). Its structure and purity were confirmed by i.r.and n.m.r. spectroscopy. The product of this Diels-Alderreaction is now known to be the exo-cis isomer rather thanthe endo-cis isomer (Woodward & Baer, 1948).Lysozyme (hen's-egg white, thrice recrystallized, lot

no. 582) was purchased from Seravac Laboratories Ltd.,Maidenhead, Berks., U. K. Trypsin (twice recrystallized)was from Worthington Biochemical Corp., Freehold, N.J.,U.S.A.

Cellulose acetate electrophoresis. Electrophoresis ofproteins was carried out on Gelman Sepraphore III stripsin 40mM-sodium barbitone buffer, pH 8.5, at 12V/cm for75min. Protein was stained with 0.25% (w/v) PonceauS in 3% (w/v) trichloroacetic acid-3% (w/v) sulpho-salicylic acid.

High-voltage paper electrophoresis. Paper electrophore-sis was carried out as described by Perham (1967). Aminoacids and their ETP derivatives were detected on paper

by staining with ninhydrin-cadmium acetate (Heilmann,Barrollier & Watzke, 1957) or by the chlorine test ofRydon & Smith (1952). ETP derivatives could also bedetected by spraying the paper with 1% (w/v) KMnO4,when they appeared as yellow spots on a pink background.Arginine and its derivatives were specifically stained bymeans of the Sakaguchi reaction (Jepson & Smith, 1953).The electrophoretic mobilities of amino acids and their

derivatives were measured relative to aspartic acid(defined as -1.0) (Offord, 1966). At pH16.5 the position ofneutral amino acids was taken as the zero; at pH 8.9mobilities were measured from the origin.Amino acidanalysis. Samplesofproteinwerehydrolysed

with 6M-HCI in sealed evacuated tubes at 105°C for 24 hand the HC1 was removed in vacuo over NaOH pellets.Amino acid analysis was carried out on a Beckmanmodel 120C automatic analyser. The values for serineand threonine were corrected by 6 and 3% respectivelyfor losses during hydrolysis (Perham, 1967). No cor-

rections were made for incomplete release of valine andisoleucine.

Assay of protein amino groUps. Free amino groups inlysozyme and its derivatives were assayed with 2,4,6-trinitrobenzenesulphonic acid by the method of Habeeb(1966). Before the assays the protein solutions were

dialysed against 0.02M-sodium borate buffer, pH8.6, to

remove NH4HCO3, which interferes with this assay.Assuming complete reaction with the amino groups ofunmodified lysozyme, a value of 1.24 x 104 for the molarextinction coefficient of the trinitrophenylamino group at340nm was determined in trial experiments and usedthroughout.

Reduction and carboxymethylation of proteins. A 15mgportion of protein was dissolved in 1 ml of 0.2 M-tris-HClbuffer, pH 8.4, containing 7M-guanidine hydrochloride.Then 0.1 ml of dithiothreitol (65 mg/ml) was added, andthe tube was flushed with N2 and left at room temperature(200C) for 2h. Then 0.1 ml of sodium iodoacetate (235 mg/ml, neutralized with M-NaOH) was added, and the tubewas flushed again with N2 and left in the dark for 30minat 2000. The alkylation was stopped by adding 0.5 ml of2-mercaptoethanol and the protein was dialysed againstseveral changes of 0.5% (w/v) NH4HCO3.

Preparation of peptide 'maps'. Reduced and carboxy-methylated proteins were digested with 1% (w/v) trypsininO.5% (w/v) NH4HCO3, pH8.0, at 37°Cfor2h. The bufferwas then removed by freeze-drying. Peptide 'maps' wereprepared as described by Gibbons & Perham (1970) andstained with ninhydrin-cadmium acetate (Heilmann et al.1957).

Reaction of ETPA with amino acids. Solutions ofc-DNP-L-lysine hydrochloride, L-arginine hydrochloride,glycine, and NO-acetyl-L-lysine amide acetate in water(10-20mM) were adjusted to pH9.0 and allowed to reactwith ETPA at 200C, with stirring, the pH being maintainedat 8-9 by addition of 0.2M-NaOH. The anhydride wasadded in several portions; 1-2 equiv. was generallyrequired for complete reaction as judged by paperelectrophoresis at pH16.5 and pH18.9.

Kinetics of hydrolysis of ETP-acetyl-lysine amide. Asolution of Na-acetyl-lysine amide (12mm) in water wasallowed to react quantitatively with ETPA as describedabove and was then diluted with water to a final concen-tration of 5.6mM. Hydrolysis was initiated by mixing1.5 ml of this solution with 3.5 ml of the appropriate buffersolution and incubating at 250C. The buffers used were:sodium oxalate, 0.15M with respect to oxalate (pH2.0);sodium citrate, 0.15M with respect to citrate (pH3.0,pH14.0 and pH15.0); sodium phosphate, 0.15M with respectto phosphate (pH 6.0). After various time-intervals0.3ml samples of the hydrolysis mixture were removedand hydrolysis was terminated by addition of 4.7ml of0.15M-sodium borate buffer, pH8.6. Free amino groupswere then assayed by adding 1.Oml of 2,4,6-trinitroben-zenesulphonic acid (1.5mg/ml in glass-distilled water),incubating at 370C for 75min and reading the extinctionat 335nm against a blank made up with 0.3ml of water.At pH2.0, pH3.0 and pH4.0, the extinction at 'infinitetime' was obtained by direct observation, but at pH5.0and pH 6.0 where the rate of hydrolysis is very low it wasobtained by extrapolation (Freedman & Radda, 1968) byusing the relation:

AEb2 AE.a AEc2/E, - (LEa +AE,)

where AE, AEb and AE, are the extinctions at timesta, tb and t, respectively and tb - ta = t, - tb. The resultswere plotted in accordance with the first-order rate law:k.b,. *t = 2.303 [log (AE'5 - AE03S) - log (AE' 5 -E335)]Assay of Iysozyme and its derivatives. Solutions of

734 1970

REVERSIBLE BLOCKING OF AMINO GROUPSlysozyme or its derivatives in 0.5% (w/v) NH4HCO3 werediluted just before assay with 0.1 X-sodium phosphatebuffer, pH 6.24, and their enzymic activities were deter-mined by the MicrococCus ty8odeikticu8 assay method ofShugar (1952).

Reaction of ETPA with 1ysozyme. Lysozyme dissolvedin 0.2M-sodium borate buffer, pH8.8, at 12mg/ml wasallowed to react at 2°C with a 12-fold molar excess ofETPA over protein amino groups. The ETPA was addedin several lots with continuous stirring and the pH wasmaintained at 8.5-9 with m-NaOH. The reagent dissolvedslowly during the reaction. Lysozyme is precipitated onthe first addition of ETPA but redissolves within 5min.When the reaction was complete the modified protein waspurified by dialysis against 0.5% (w/v) NH4HCO3, pH8,at 40C.For unblocking, ETP-lysozyme was dialysed against

two changes of 0.1 M-sodium citrate buffer, pH 3.0, at25°C for 30h. The protein was precipitated within 15minbut gradually redissolved and was all back in solutionafter 23 h. The sodium citrate was finally removed bydialysis against 0.5% (w/v) NH4HCO3 at 20C.

RESULTS

Reaction of ETPA with amino acid8. WhenETPA was allowed to react with the two opticallyactive amino acids, E-DNP-L-lysine and L-arginine,and samples of the resulting reaction mixture weresubjected to electrophoresis at pH6.5 and pH 8.9, itwas observed that each amino acid gave rise to tworeaction products, which separated at both pHvalues, migrating towards the anode (Table 1). Ineach case the slower-moving component was presentin slightly greater amount than the faster-movingone. However, on reaction of ETPA with glycineor with N-acetyl-L-lysine amide only a singleproduct could be detected by paper electrophoresisat pH 6.5 and 8.9. The mobilities of the productsproduced by reaction of ETPA with these modelcompounds support the assumption that ETPAacylated the free amino groups to give the corres-ponding dicarboxylic acid monoamides. The twoproducts arising from reaction with e-DNP-lysineand with arginine are presumably the two diastereo-isomers that would be expected to result from reac-tion of ETPA with an optically active amino acid.Glycine, being optically inactive, can give rise to buta single product, and with Na-acetyl-lysine amideit would appear that the two diastereoisomericproducts are sufficiently similar to be unresolvableby paper electrophoresis.

Kinetic of hydroly8i8 of ETP-Na-acetyl-L-lysineamide. As a model for the c-amino groups of pro-teins we chose Na-acetyl-L-lysine amide. In apreliminary experiment a solution ofthis compoundthat had quantitatively reacted with ETPA wasadjusted to pH 2 with M-hydrochloric acid. The pHwas maintained at 2-3 by periodically addingM-hydrochloric acid, and samples were removed at

Table 1. Electrophoretic mobilitie at pH 6.5 of theproduct8 of reaction of ETPA with amino acids

The yields of the products from E-DNP-L-lysine weredetermined by elution with 0.1 M-potassium phosphatebuffer, pH6.0, and by reading the extinction at 360nmagainst a paper blank. The relative yields of those fromL-arginine were judged (++ and +) by visual inspection.

Amino acidE-DNP-L-lysine

L-Arginine

GlycineNa-Acetyl-L-lysine

No. of Mobilityproducts (pH 6.5)

2 -0.50-0.73

2 -0.27-0.54

1 -1.201 -0.50

Yield (%)57.642.4+++

various times and applied to paper for electrophore-sis at pH 6.5. Hydrolysis of the derivative wasessentially complete after approx. 30h at pH2-3and 200C, indicating the half-life under theseconditions to be about 4-6h. Finally, after about48h at pH2-3 a sample corresponding to about1,umol of unblocked Na-acetyl-lysine amide wasapplied to paper as a 1cm band and subjected toelectrophoresis at pH 6.5. The only spot that couldbe detected was that corresponding to free Nl-acetyl-lysine amide, indicating that exposure ofthisderivative to acidpH results simply in intramolecu-lar hydrolysis of the amide bond with completeregeneration of the free amino group.The accurate determination of the rate of

hydrolysis of ETP-Na-acetyl-lysine amide as afunction of pH was performed at 25°C as describedabove. At all pH values plots of log (AE'5- AE'335)versus time were linear, indicating that the kineticsof hydrolysis were first-order. The experimentallydetermined half-times (ti) of hydrolysis are:pH2.0, 283min; pH3.0, 284min; pH4.0, 364min;pH5.0, 2350min; pH6, 21200min. A plot oflog kobs. versus pH (Fig. 1) shows that the rateconstants increase, below pH6.0, with a slope of-1.0, reaching a steady maximum at approx.pH 4.0, indicating hydrolysis via intramolecularparticipation by the protonated species of theneighbouring carboxyl group. The kinetic pK(pKapp.) value determined from this plot is 4.13-,which means that at pH 3.0 and below the rate ofintramolecular hydrolysis will be essentially maxi-mal.The results presented above had indicated that

the two diastereoiosmeric products obtained fromreaction of ETPA with N-acetyl-L-lysine amidewere sufficiently similar as to be unresolvable bypaper electrophoresis. The first-order kinetics ofhydrolysis observed here indicate that the two

Vol. 118 735

-2.0 2,4,6-trinitrobenzenesulphonic acid, consistent withthe half-time of hydrolysis of 4-5h found for modelcompounds.

Propertie8 of blocked, unblocked and native-3.0 - lysozyme. To check whether any irreversible

reactionshad taken place either during reaction withXo . \ETPA or during the unblocking procedure, samples_ -4.0 of fully blocked, unblocked and native lysozyme

were reduced and carboxymethylated and thensubjected to amino acid analysis after hydrolysis

-5.0 with 6M-hydrochloric acid. Under these con-2.0 3.0 4.0 5.0 6.0 ditions, any amide or ester bonds of ETPC would

pH be completely hydrolysed. Therefore, in the absenceFig. 1. First-order rate constants (kb,.) for the hydrolysis of any peculiar side-reactions, the amino acidof ETP-NO-acetyl-L-lysine amide at various pH values. compositions ofall three samples shouldbe identical.

This, indeed, was found to be the case withinexperimental error (±3%). A more sensitive

diastereoisomers are also kinetically indistinguish. criterion for regeneration of primary structure is aable. comparison of tryptic peptide 'maps'. Samples of

Reaction of ly8ozyme with ETPA and unblocking. native and unblocked lysozyme were reduced andTo test whether ETPA could be used for reversibly carboxymethylated, and the tryptic peptide 'maps'blocking protein amino groups, we chose lysozyme were compared. No differences could be seen be-as a model. The reasons for this choice were based tween the 'maps' of native and unblocked samples,on several considerations: it is a reasonably small indicating that, within the limits of this system, themonomeric protein, the primary (Canfield, 1963; primary structure had been completely regenerated.Canfield & Liu, 1965; Jolles, Jauregui-Adell, A possible side-reaction of anhydrides (see theBernier & Joll6s, 1963) and tertiary (Blake, Mair, Discussion section) is acylation of protein hydroxylNorth, Phillips & Sarma, 1967) structures of which groups, in particular those of serine and threoninehave been well studied; the six E-amino and the residues. The identity of the tryptic peptide 'maps'single oc-amino groups are well-exposed and react indicates that if such reactions had occurred theyrapidly and quantitatively with 2,4,6-trinitro. were completely reversed under the conditions usedbenzenesulphonic acid (Freedman & Radda, 1968) for unblocking. However, this still leaves unan-andshouldthereforealsoreactsmoothlywithETPA; swered the question whether in the modified proteinit is a very stable protein that will regenerate its reaction had been confined entirely to protein aminonative conformation even after complete reduction groups. Acylation ofprotein hydroxyl groups wouldand denaturation (Isemura, Takagi, Maeda & Imai, give rise to minor components of increased acidity1961), and so should be well able to tolerate the that should be detectable by electrophoresis onconditions used for removing the blocking groups. cellulose acetate strips. Samples of fully blocked,

In a preliminary experiment it was found that a unblocked and native lysozyme were thereforefivefold excess of ETPA over protein amino groups submitted to electrophoresis on strips of cellulosewas sufficient to block more than 90% of the amino acetate in 40r)m-sodium barbitone buffer, pH 8.5,groups, as judged by assay with 2,4,6-trinitro- for 75min and the strips were then stained withbenzenesulphonic acid. In all subsequent experi- Ponceau S. Native and unblocked lysozyme bothments a 12-fold excess of reagent was used to bring gave single bands migrating identically towards theabout complete blocking. In the early stages of anode; the blocked derivative gave a somewhatreaction with ETPA the protein is precipitated, smeared band migrating towards the cathode. Thispresumably as the isoelectric point is lowered by the experiment confirmed very nicely that all theintroduction of negatively charged groups, but it ETP groups introduced during the conditions usedredissolves as reaction proceeds. To remove the for quantitative blocking of amino groups wereETP groups, the fully blocked protein was dialysed removed by dialysis at pH 3.0 and 256C for 30h.at 256C against two changes of 0.1 M-sodium citrate However, there remained the possibility that thebuffer,pH3.0,for30h. Theproteinwasprecipitated smeared band given by the blocked derivative wasduring the first 30min of dialysis at pH 3.0, but was due to the presence of minor components arisingall back in solution again after about 23h. Finally from acylation ofprotein hydroxyl groups. Accord-it was dialysed against 0.5% (w/v) ammonium ingly, samples of the three proteins were treatedhydrogen carbonate, pH 8.0, at 40C. Dialysis at withM-hydroxylamine in 0.2M-N-ethylmorpholine-pH 3.0 for 30h was sufficient to regenerate more than acetic acid buffer, pH 8.2, at 250C for 2h, and the95% of the amino groups, as judged by assay with proteins were purified by exhaustive dialysis

736 M. RILEY AND R. N. PERHAM 1970

REVERSIBLE BLOCKING OF AMINO GROUPS

against 0.5% (w/v) ammonium hydrogen carbonate,pH 8.0, and freeze-dried. Treatment with hydroxyl-amine under these conditions is sufficient to destroythe comparable maleyl ester linkages in tobacco-mosaic-virus protein (L. King & R. N. Perham,unpublished work), while leaving maleylaminobonds intact. However, electrophoresis of thevarious lysozymes after hydroxylamine treatmentgave exactly the same pattern as before, with theblocked derivative still producing a somewhatsmeared band migrating towards the cathode. Onthis basis it would appear unlikely that the smearingof the blocked protein is due to the presence ofETPester components, although the possibility cannotbe rigorously excluded. What emerges very clearlyfrom these experiments is that if such side reactionsoccur they are completely reversible under thedeblocking conditions.

Samples of the lysozyme solutions that had beendialysed against 0.5% (w/v) ammonium hydrogencarbonate, pH8.0, were diluted with 0.1M-sodiumphosphate buffer, pH 6.24, and assayed immediatelyby the method of Shugar (1952). Since at pH6.24the half-time of hydrolysis of ETP-amino bands isin excess of 14 days it can be safely assumed that,during the time required for dilution and assay,unblocking did not occur to any significant extent.The relative specific activities of the three proteinswere: 0.0 (blocked); 1.07 (unblocked); 1.0 (native).

DISCUSSION

The acylation of an amino group by ETPA, withresulting fission of the anhydride ring, is accom-panied by the generation of a new asymmetriccentre. If the molecule containing the amino groupitself contains a centre of asymmetry one expectsto obtain two diastereoisomeric products. Since theproducts are diastereoisomeric, the transition statesleading to them will also be diastereoisomeric andtherefore different in free energy, and hence theyshould be differentiated kinetically. In general, thedifference in free energy between diastereoisomerictransition states, and the resulting preferentialsynthesis of one diastereoisomeric product over theother is greatest when the two asymmetric centresare close together (for review see Eliel, 1962). Thetwo products obtained in unequal yield from reactionofETPA with the ec-amino group of E-DNP-L-lysineand L-arginine are therefore presumably diastereo-isomers. An entirely analogous situation is seen inthe reaction of f-phenylglutaric anhydride withL-oc-phenethylamine to give two diastereoisomericmonoamides in yields of 60% and 40% (Schwartz& Carter, 1954). In support of this conclusion it isfound that reaction of ETPA with the ac-aminogroup of glycine, a molecule with no centre ofasymmetry, gives only a single product.

The observation that the reaction of ETPA withthe E-amino group ofN'-acetyl-L-lysine amide givesrise apparently to only one product, as judged bypaper electrophoresis, indicates that in this case thetwo diastereoisomeric products are physically verysimilar if not identical. Further, the simple linearplots obtained by assuming first-order kinetics ofhydrolysis show that the two diastereoisomericproducts are also kinetically indistinguishable. Thisis entirely as expected since the two asymmetriccentres are well separated and little or no asym-metric induction should occur. From the point ofview of protein chemistry, therefore, the complica-tion of ETPA reacting with c-amino groups to givetwo products that hydrolyse at different rates doesnot arise. One possible minor complication is thatthe two products produced by reaction with theoc-amino group of a protein may have half-lives ofhydrolysis different from those arising from reactionwith E-amino groups. However, one would expectthis difference to be slight and not a major drawback.The pH-dependence of the hydrolysis of the ETP

derivative of N'-acetyl-lysine amide clearly indi-cates that the protonated form ofthe carboxyl groupis the active species in catalysis. The kineticallydetermined pK is 4.13 and the rate of hydrolysisshould therefore be maximal below pH 3.0. Theobservation that essentially all the amino groups oflysozyme are regenerated after approx. 30h dialysisatpH 3.0 and 250C is consistent with the assumptionthat the ETP groups are released under these con-ditions with a half-life of 4-5h, in agreement withthe data for model compounds, indicating that theinsolubility of the modified protein does not signifi-cantly affect the rate of hydrolysis.ETP-amino groups are, then, a good deal more

labile than maleylamino groups, which have ahalf-life of 1Ih at pH 3.5 and 370C (Butler et al.1969), conditions that might conceivably causehydrolysis of some labile bonds in proteins. On theother hand, they are significantly more stable thancitraconyl groups. Thus the two isomeric productsobtained by reaction of citraconic anhydride withthe ac-amino group of arginine hydrolyse at pH2.0and 200C with half-lives of 1-2h (Dixon & Perham,1968). Of more relevance, however, is the situationthat obtains with c-amino groups, and it seems thatin this case citraconyl groups are somewhat morelabile. For example, it has been shown (I. Gibbons,unpublished work) that the isomeric productsresulting from the reaction of citraconic anhydridewith the E-amino group of N'-acetyl-L-lysine amideboth hydrolyse with a half-life of approx. 20min atpH2.0 and 200C. Above this pH the two isomersdiverge considerably in their rates of hydrolysis.Presumably at pH 2.0 both forms are completelyprotonated and the rate constants become identical.Thus the stability of the ETP-amino group falls

Vol. 118 737

M. RILEY AND R. N. PERHAM

conveniently between those of the maleylamino andcitraconylamino groups, which may have someusefulness in the chemical modification of peptidesand proteins.The occurrence of reaction at residues other than

lysine in the use of these various anhydrides callsfor some discussion. One possible side-reaction isacylation ofthe hydroxyl groups of serine, threonineand tyrosine residues. Substantial acylation ofserine and threonine residues has been observedduring maleylation of tobacco-mosaic-virus protein(L. King & R. N. Perham, unpublished work) andof immunoglobulins (Freedman, Grossberg &Pressman, 1968). Thiol esters could also be pro-duced, but would presumably break down spon-taneously at the pH of the reaction. The work ofThanassi & Bruice (1966) on the hydrolysis ofmonohydrogen phthalate esters has shown that themechanism of hydrolysis and the values of theassociated rate constants are dependent on thePKa of the conjugate acid of the leaving group. Ifthis pKa is greater than approx. 13.5 the protonatedcarboxyl group is the active catalytic species; if it isless than approx. 13.5 the unprotonated carboxylgroup is involved in catalysis. Although themechanisms of hydrolysis of monoesters of ETPC,maleic acid and citraconic acid have not beeninvestigated as fully, the available evidence (Bruice& Pandit, 1960a,b; Pekkarinen, 1954) indicatesthat for these reagents also the same generalphenomenon is true. Now the pKa of the hydroxylgroup of N-acetylserine amide is 13.6 (Bruice, Fife,Bruno & Brandon, 1962) and it is found that therate constant for the hydrolysis of O-phthaloyl-N-acetylserine amide is independent ofpH betweenpH 2.0 and pH4.3, indicating the expected partici-pation by both the protonated and the unprotonatedcarboxyl groups (Thanassi & Bruice, 1966). Finally,it should be noted that the first-order rate constantfor the hydrolysis of phenyl hydrogen phthalate is104-106 times that for the hydrolysis of methylhydrogen phthalate but the rate constant for thehydrolysis of phthalamic acid is about the same asthat for methyl hydrogen phthalate hydrolysis(Bender, Chow & Chloupek, 1958; Thanassi &Bruice, 1966). The implications of this with respectto the specificity of reaction of these variousanhydrides with protein amino groups are thereforeas follows. Any tyrosine esters that formed wouldbe unstable and would break down rapidly atneutral and alkaline pH. Any serine or threonineesters that were produced would certainly be labileat acid pH and would be completely cleaved underthe conditions used to liberate the free amino groups,as found in the experiments reported above. Theywould also possibly be labile at alkaline pH,although the rate of hydrolysis would be very muchlower (several thousand-fold) thanthecorresponding

tyrosine esters. Therefore to ensure that reaction isconfined entirely to amino groups the minimumquantity of anhydride that is required to blockquantitatively the amino groups should be used andthepH maintained during reaction between 8 and 9,when the amino group is a very much better nucleo-phile than is the hydroxyl group; once reaction iscomplete the modified protein should be dialysedextensively at alkaline pH to destroy any esterbonds that may have formed. With these conditionsfor the reaction ofETPA with lysozyme it is foundthat reaction is confined almost entirely, if notexclusively, to the amino groups. Finally, it maybe noted that a short treatment with hydroxylamineat alkaline pH can also be used selectively to cleavelabile ester bonds as a convenient alternative toprolonged dialysis at alkaline pH.

Maleic anhydride, citraconic anhydride anddimethylmaleic anhydride (and the correspondingacids) allpossess activated olefinic bonds, andnucleo -philic addition to protein thiol groups could con-ceivably occur when using these reagents. It isknown that maleic acid can alkylate simple thiolcompounds, e.g. cysteine and GSH, under mildconditions (Morgan & Friedmann, 1938), and it hasbeen found (Gibbons & Perham, 1970) that reactionof rabbit muscle aldolase with an amount of citra-conic anhydride sufficient to block quantitativelythe amino groups gives rise to significant loss of thethiol groups in this protein. For the purposes of'diagonal' electrophoretic techniques these difficul-ties can be circumvented by prior carboxymethy-lation ofthe protein thiol residues. For enzymologi-cal studies, however, this cannot be resorted to andthe use of these anhydrides might be restricted. Inthe experiments reported in this paper, the questionof alkylation of thiol residues did not arise sincelysozyme contains only four disulphide bridges.However, the double bond in ETPA is not activatedby conjugation with carbonyl groups and additionto thiol residues is very unlikely. Further, with thecorresponding saturated derivative of ETPA,exo-cis-3,6-endoxo-hexahydrophthalic anhydride,the possibility of alkylation of thiol groups does notexist, and preliminary experiments (M. Riley,unpublished work) indicate that the reaction of thiscompound with amino groups and the kinetics ofhydrolysis of the resulting amides are indistinguish-able from the results with ETPA.Although modification of the amino groups of

lysozyme with ETPA causes complete loss ofactivity, exposure of the blocked lysozyme to theunblocking conditions is sufficient to regenerate fullenzymic activity, so that not only the primarystructure but also the tertiary structure is fullyregained after reaction and unblocking. It is knownthat modification of the amino groups of lysozymewith O-methylisourea (Parsons et al. 1969) and with

738 1970

Vol. 118 REVERSIBLE BLOCKING OF AMINO GROUPS 739

methyl picolinimidate (Benisek & Richards, 1968)does not result in total loss of activity and that, incontrast with earlier findings (Fraenkel-Conrat,1950), acetylation of lysozyme amino groups causesloss of only 60% of the activity with a shift in thepH optimum ofthe enzyme to a lowerpH (YamasakiHayashi & Funatsu, 1968). Further, fully acety-lated lysozyme still retains full activity towardsglycol-chitin as a substrate (Yamasaki et al. 1968).The complete loss of activity (at pH 6.24) inlysozyme that has fully reacted with ETPA maytherefore be due to one of several causes: perhapsthe enzyme is unfolded owing to repulsion betweenthe introduced negative charges or perhaps only thepH optimum of the reaction catalysed is shifted.In any event one should be aware of the dangerin being too dogmatic in interpreting the effectof chemical modification of particular residueson enzymic activity.

We thank Dr A. J. Kirby for valuable discussion andthe Science Research Council for the award of a scholar-ship to M. R.

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