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    Eur. J. Biochem. 124, 585-588 (1982)0 EBS 1982

    A Rapid Method for Acid Hydrolysis of Protein with a Mixture of Trifluoroacetic Acidand Hydrochloric AcidAkira TSUGITA and Jean-Jacques SCHEFFLEREuropean Molecular Biology Laboratory, Heidelberg(Received October 7, 1981/January 12, 1982)

    Proteins have regions which resist hydrolysis with mineral acid. The presence of a strong organic acid wasfound to be efficient for hydrolysis of a hydrophobic peptide bond. The proposed condit ion, a 2 : 1 (by vol.) mixtureof concentrated hydrochloric acid and trifluoroacetic acid at 166 "C for 25 min was observed to be equivalent to theconventional conditions (6 M HC I at 110"C for more than 24 h) without significant decomposition of amino acids.The method was shown to be superior to the conventional conditions, especially for hydrophobic proteins. Thepresent method destroys tryptophan, as the conventional acid hydrolysis does.

    Hydrolysis of protein into amino acids has been carriedout by treatment with acid [l], alkali [2] or enzymes [3]. Acidhydrolysis with constant-boiling HCI at 110"C for more than24 h [4-71 has been the widely accepted standard method.We have used 6 M HCl containing 50% acetic acid in orderto shorten the hydrolysis time [8].Westall and his collaborators[9,10] used 6 M HCl containing 50 "/,propionic acid especiallyfor resin-bound peptides at elevated temperature. We havenoticed that the recoveries of amino acids under the con-ventional hydrolysis conditions are often poor, especially forhydrophobic proteins, even after lengthy hydrolysis. Whilesequencing hydrophobic proteins, we have encountereddifficulties such as insolubility and poor accessibility ofchemical reagents or enzymes to hydrophobic regions of theproteins. The difficulties were partly overcome by use ofsuitable organic solvents [ l l] . In principle hydrolysis may bemore rapidly achieved in the presence of organic acid which isaccessible to the hydrophobic regions. CF3C02H was foundto satisfy these requirements. CF3C02H is a strong acid,pK, 0.23, and has a high vapour pressure and low boilingpoint (72.5 "C). Formic acid, acetic acid and propionic acidwere also tested.

    of the 17 amino acids (asparagine, glutamine and tryptophanare omitted) and ammonia. Amino acid analysis was carriedout with a Durrum D500 set to a sensitivity of 2.5 nmolamino acid.

    RESULTS AND DISCUSSIONModel Experiments with Peptides

    A dipeptide Val-Glu was hydrolysed with 6 M HCI atvarious temperatures (Fig. I , curve A). Between 110 "C and210 "C for 10 min, as expected, the extent of hydrolysis was afunction of temperature. Hydrolysis of the same dipeptidewas carried out at 160C for 25 min in mixtures of concen-trated HCI and various volatile organic acids. Table 1 showsthat the mixturesofCF 3C 02 H nd HCl were the most efficientones. When a mixture composed of CF3C02H and HCI (1 :2)was used for hydrolysis, the rate of hydrolysis was markedlyaccelerated compared to 6 M HCl (Fig. 1, curve B). To find atime sufficient for hydrolysis, dipeptides composed of valineand isoleucine, Val-Val, Val-Ile, Ile-Val and Ile-Ile, werehydrolysed at 167 "C for various times. These peptides are

    MATERIALS AND METHODSCF3C02Hwas sequanal grade from Pierce. ConcentratedHCI, formic acid and n-propionic acid were purest gradesavailable from Fluka Chemical. Myoglobin (sperm whale),egg white lysozyme (hen), and Val-Val were from Serva Fein-biochemica. Val-Ile and Ile-Val were from Biomol. Ile-Ilewas donated by Dr Shimonishi (Protein Research Inst.,Osaka University). Lipoprotein from the outer cell membraneof Proteus mirahilis was donated by Dr Plapp (Dept ofMicrobiology, University of Kaiserslautei-n). Tobacco mosaicvirus coat protein was prepared by the standard method [12].Ferramido chloromycin was donated by Dr Shimi (AinShams University, Cairo) [13]. Amino acid mixture was thestandard H of Pierce which contains equal molar amountsAbbreviation. CF3COzH, trifluoroacetic acid.Enzyme. Lysozynie (EC 3.2.1.17).

    Temperature ("C)Fig.1. Hydrolysis o j Val-Glu with 6 M HC1 (curve A ) and withCF3COd f :HC I ( I : 2) (curve B ) at various temperatures

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    known to be resistant to acid hydrolysis [5,14]. Hydrolysis of50 min o r longer resulted in mo re tha n 93 y/o recovery forthree valine-containing peptides, while the maximuin re-covery for Ile-Ile was 90 %. Fur ther inc ubation resulting in aconsiderable amoun t of alloisoleucine [5] . The rate of allo-100

    -75

    v,v,._-P72h 50L0

    c,c

    Iw" 25/

    I- //

    isoleucine formation was observed to be 3'x for 25-minhydrolysis and 5 7 for 50 m in. Mixtures of acetic or propionicacid and HC1 also produced alloisoleucine t o the sa me extentor more. These experiments suggest the optimum hydrolysist ime to be more than 25 min.Stahrlitj of Amino Ac id\

    An ainino acid standard mixture wa\ heated in variousacid mixture s between 130 C an d 21 0 'C fo r 25 min and 50 min

    ~

    Acid mixture Comp osition Recovery!J;v

    Form ic acid: HCI 1 : l1 . 2Acctic acid : HCI 1 : l1 . 2Trif luoroacetic aci d: HC I 2 : l1 : l1 . 21 : 2Propionic acid: HCI 1 : l

    ~

    1"

    8595971008510010 090Y7

    Table 2. R W ( J L W J ,/ umino uc,id.s y f i r r incuhuiioia bciih vurious ucid rnivrurcs ui e l e v u t d fenapf'ruturesRecoveries arc expressed as a percentage of the value of alanine taken as 100%. All the acid mixtures contained 0.005 :

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    5x7Table 3. Amino wid oniposificni o/ 'n i jo f i /oh in ~ J J(j%rcnt nw / / ~ i d ~Values ar e expressed as relative values based on a lan ine = 17 . Recoverywas calculated from values of alanineAm ino Residues obtainedacid - ~- -f iom CF?COzH HCI propionic 6 M HCIre- (1 2) acid HCI ( 1 1)quence - - ~ ~

    25min 50min 1 5 m i n 30min 24 h 72 hA sp 8 8.0 8.2 8.0 8 .4 8.4 8.1T h r 5 4.8 4.6 3.9 4.4 4.9 4.8Ser 6 5.6 5.3 5.1 4.6 5.8 5.7Glu 19 18.9 19.0 17.9 19.3 19.5 18.9Pro 4 4.1 4.0 3.9 3.6 3.9 3.9Gly 11 11.0 11.2 11.6 11.7 1 1 . 6 1 1 . 0Ala 17 17.0 17.0 17.0 17.0 17.0 17.0Val 8 7.0 7.7 3.9 6.7 4.8 7.3Met 2 1.8 1. 7 2.1 2.0 1.8 1.9Ile 9 7.7 8.7 4.1 6.5 5.3 7.7Leu 18 17.7 18.3 1 2.3 16.4 15.3 16.3Tyr 2 2.0 2.0 0.7 1.5 2.0 2.0Ph e 6 5.6 6.0 2.0 3.6 4.6 4.9His 12 11.9 12.0 9.3 11 .1 1 0 . 7 3 1 . 8Lys 19 19.1 19.3 13.3 17.3 17.1 19.0A r& 4 4.1 4.0 3.5 3.9 3.5 4.1RecoveryTcm-

    ~ - - _~ ~ ~ - ~ _ ~- . . ~( Y" ) 92 100 73 86 84 92perature 16 6 C 160 C 107 C

    in order to investigate side reactions (Table 2). The m ixturesof HCI and CF3C02H were found to give the least side re-act ions among the mixtures. At higher temperatures than18O'-C, even the H Cl iC F3 C0 2H mixture yielded poo r re-coveries of certain amin o acids. However, between 160"C and170 'C recoveries were obtained comparable to 6 M HCI at110"C for 24 h. At 170 "C C F 3 C 0 2 H H CI (1 :2), for example,gave the following results: (a) decomposition of serine andthreonin e were observed (see Table 2), as has been observedfor the 6 M HCI method [15]; (b) oxidation of methionine(5- 0 %) depended on the quality of the vacuum and cystinewas almost completely oxidized (70- 90x);c) alloisoleucinewas formed by racemization as in the 6 M HCI hydrolysis[6,15], and (d) tryptophan was decomposed and derivati-rat ion of tyrosine also depended on the vacuum. The addit ionof 0 . 2 x phenol inhibited the derivatization [16]. Because weobserved that the addition of larger amounts of phenolaffects the analysis of other amino acids, experiments weremade to find the optimum amou nt. The am ino acid standardmixture (2.5 nmol) w as incubated a t 167 C with 300 pl ofCF3C 02H : H CI (1 :2) containing freshly distilled phenol, for25 min a nd 50 min. Addit ion of only 0.001 pheno l (by vol.)resulted in a better th an 9 57< recovery of tyrosine while noadd ition gave only 71 Y g . 0.001 phenol corresponds to about10-tim es mo lar excess over tyrosine. N o serious effect on th eother amino acid recoveries was observed in the presence of0.001 a nd 0.03 pheno l. In later experiments we generallyused 0.005 %ph enol. Fr om considerat ion of the side reactions,we chose a hydrolysis tem peratur e below 170 -C.

    Table 4. Am i r i o acid conzposition of prot eins hq' the present an d conventionul metlzorlsThe 25-min, 50-min and 75-min hydrolyses were with CFJCO~HHCI ( I : ) ; the 24-h a nd 72-h bydrolyses were with 6 M HCI at 107 ' C . Values are basedon the values for alanine, except for tobacco mosaic virus protein coat protein which is based on the value for glutamic acidAmino Amou nt present inacid ~ - ~- _~ ~ - - - - -~ ~_ ~ ~.~- ._- ~ - - - ~-.-Iysozyme tobacco mosaic virus ferramido chloromycin lipoproteinprotein

    from hydrolysis from hydrolysis by hydrolysis for tempo- by hydrolysis for tem po-sequence fo r sequence for rary rarycon-_ _ - ~ ~~ con-~ ~-- -~ ~~ -25 50 25 50 25 50 24 h 72 h clusion 25 50 75 24 h 72 h clusionmin min min min min min min min minAsp 21 20.8 20.9 18 18.0 18.0 2.0 2.0 2.1 2.1 2 6.2 6.1 6.0 6.3 5.9 6Th r 7 6.2 5.7 16 15.5 15.3 - - 0 2.8 2.7 2.6 3.1 2.8 3Se r 10 8.1 6.3 16 13.7 11.3 1.6 1.4 1.5 1.6 2 3.0 2.5 2.1 3.4 3.2 4Glu 5 5.1 4.9 'I6 16.0 16.0 - - 0 7.2 7.0 7.4 7.5 7.2 7Pro 2 2.0 1.8 8 8.2 8.1 - - - 0 1 .7 1 . 7 2.0 1.4 1.6 2G'Y 12 12.0 12.0 6 6.0 6.1 4.0 4.0 4.5 4.2 4 4.8 4.8 4.7 4.6 4.8 5Ala 12 12.0 12.0 14 14.0 14.0 2.0 2.0 2.0 2.0 2 6.0 6.0 6.0 6.0 6.0 6

    - -

    ~-_

    c y s / 2 8 5.2 3.6 1 0.32 0.18 1.9 0.80 0.75 0.97 2" - _ - _ - 0Val 6 4.7 5.2 14 12.6 13.6 1.1 1.3 0.20 0.40 1- 2 3.7 3.9 4.5 2.5 3.6 4-5Met 2 1.4 1.3 0 - - - 0 1 .9 1.8 1.8 1.7 1.3 2Ile 6 4.0 5.8 9 7.8 8.4 0.80 0.92 0.26 0.38 1 2.6 2.9 2.9 2.0 3.0 3L eu 8 5.8 8.0 12 12.0 12.0 0.95 0.91 0.88 0.74 1 4.8 4.9 4.8 4.8 5.3 5TY r 3 2.9 2.9 4 3.8 3.8 0.59 0.58 0.68 0.58 1 1.0 0.94 1.2 1.0 1.2 2P he 3 3.0 3.0 8 7.6 7.4 1.7 1.6 1.3 1.1 2 1.9 1.9 1.8 1.5 1.8 2- - - - - 0 1.2 1. 1 1 . 1 0.74 0.91 1is 1 1. 1 1 .1 0 -Ly s 6 6.0 5.8 2 2.4 2.1 - - - 0 3.4 3.5 3.3 3.2 3.5 4A rg 11 10.8 11.1 11 11.2 11.4 - - - 0 2.6 3.2 3.4 2.6 3.2 3 -4Recovery ( y , ; ) 88 100 99 100 81 99 57 65 89 95 100 81 92

    - - -

    _ -. - - -~~- - ~ ~ ~ ~ ~ ~~. - -~- .- ~ - ~ _ ~_ _~ ~ _ ~ ~ ~ ~" Value obtai ned by separate hydrolysis of oxidized protein [20]

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    588Hydrolysis of Proteins

    Myoglobin was chose as a test protein and 5. 5 pg washydrolysed in CF3C02H/HCl (1 :2) at various temperaturesfor 10and 25 min. Fig. 2 shows the extent of protein hydroly-sis in which recoveries were expressed relative to alanine whichis hydrolysed at an average rate [I71 and is stable under theseconditions. The data indicated that a 25-min hydrolysis at170C resulted in a recovery of 94%. Myoglobin washydrolysed at 160 "C for 15 min with a mixture of propionicacid:HCl (1 :I),ccording to the conditions of [lo] and for30 min as an additional hydrolysis time. Hydrolysis was in-complete after 15 min (73 %) and reached 86% even after30 min. The data in Table 3 suggest that these conditions canbe improved. The hydrolysis temperature was studied furtherbetween 150 "C and 170"C to investigate the recovery of aminoacids and for reducing side reactions using glucagon andmyoglobin. Our conclusion is that the optimum conditionsare between 165 "C and 170 "C. Table 3 lists the compositionof myoglobin obtained at 166 "C. The degradation of threonineand serine was slightly greater than that found in experimentswith free amino acids, but the zero-time extrapolation values[18] were almost exactly the figures expected. With the ex-ception of isoleucine, nearly quantitative recovery wasobtained for all amino acids. The low recovery of isoleucinecan be explained by the presence of an Ile-Ile sequence inmyoglobin. Amino acid composition results for lysozyme andseveral other proteins are listed in Table4. In general, theconditions were quite acceptable. Yields of valine and iso-leucine after 72-h hydrolysis by the conventional method wereabout the same as those obtained after 25 min by the newmethod. On the other hand decomposition rates of serineand threonine, requiring 25 and 50 min in the new hydroly-sates, were approximately twice those in the conventionalhydrolysates, requiring 24 h and 48 h. Table 4 also containsthe results obtained for two hydrophobic proteins of unknownsequence. In these cases we also listed the data given by theconventional hydrolysis. Ferramidochloromycin is an ex-tremely hydrophobic antibiotic peptide [I 31. Lipoproteinfrom the bacterial outer cell membrane has two moles lipid/mole in the N-terminal region. Table 4 also lists the recoveryof amino acids based on the value of alanine. It is importantto note that the conventional hydrolysis gave recoveries whichwere much too low for hydrophobic proteins, while the newmethod gave more realistic results.

    We propose the following method for rapid acid hydroly-sis: 5 pg of peptide or protein is dissolved in 50-300 pl of amixture of 2vol. concentrated HC1 and 1vol. CF~ COZ H,on-taining 0.005"/,(by volume) freshly distilled phenol. The tubeis sealed under a high vacuum (50 pmHg, i.e. 7 Pa) and placedin an oil bath, the temperature of which is regulated at 166"C.Hydrolysis times are 25 min and 50 min and can be extended ifnecessary. After cooling down, the acid is removed on a rotaryevaporator with a water vacuum pump at 65C. This rapidevaporation is needed to avoid esterification between serineand glutamic acid [19].

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