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Eur. J. Biochem.

124, 585-588 (1982) 0FEBS 1982

A Rapid Method for Acid Hydrolysis of Protein with a Mixture of Trifluoroacetic Acid and Hydrochloric Acid
Akira TSUGITA and Jean-Jacques SCHEFFLER European 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 was found to be efficient for hydrolysis of a hydrophobic peptide bond. The proposed condition, a 2: 1 (by vol.) mixture of concentrated hydrochloric acid and trifluoroacetic acid at 166 "C for 25 min was observed to be equivalent to the conventional conditions (6 M HCI 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. The present method destroys tryptophan, as the conventional acid hydrolysis does.

Hydrolysis of protein into amino acids has been carried out by treatment with acid [l], alkali [2] or enzymes [3]. Acid hydrolysis with constant-boiling HCI at 110 "C for more than 24 h [4-71 has been the widely accepted standard method. We have used 6 M HCl containing 50% acetic acid in order to shorten the hydrolysis time [8].Westall and his collaborators [9,10] used 6 M HCl containing 50 "/, propionic acid especially for resin-bound peptides at elevated temperature. We have noticed that the recoveries of amino acids under the conventional hydrolysis conditions are often poor, especially for hydrophobic proteins, even after lengthy hydrolysis. While sequencing hydrophobic proteins, we have encountered difficulties such as insolubility and poor accessibility of chemical reagents or enzymes to hydrophobic regions of the proteins. The difficulties were partly overcome by use of suitable organic solvents [ll]. In principle hydrolysis may be more rapidly achieved in the presence of organic acid which is accessible to the hydrophobic regions. CF3C02H was found to satisfy these requirements. CF3C02H is a strong acid, pK, 0.23, and has a high vapour pressure and low boiling point (72.5 "C). Formic acid, acetic acid and propionic acid were also tested.

of the 17 amino acids (asparagine, glutamine and tryptophan are omitted) and ammonia. Amino acid analysis was carried out with a Durrum D500 set to a sensitivity of 2.5 nmol amino acid. RESULTS AND DISCUSSION
Model Experiments with Peptides

A dipeptide Val-Glu was hydrolysed with 6 M HCI at various temperatures (Fig. I , curve A). Between 110 "C and 210 "C for 10 min, as expected, the extent of hydrolysis was a function of temperature. Hydrolysis of the same dipeptide was carried out at 160C for 25 min in mixtures of concentrated HCI and various volatile organic acids. Table 1 shows that the mixtures of CF3C02Hand HCl were the most efficient ones. When a mixture composed of CF3C02H and HCI (1 : 2) was used for hydrolysis, the rate of hydrolysis was markedly accelerated compared to 6 M HCl (Fig. 1, curve B). To find a time sufficient for hydrolysis, dipeptides composed of valine and isoleucine, Val-Val, Val-Ile, Ile-Val and Ile-Ile, were hydrolysed at 167 "C for various times. These peptides are

MATERIALS AND METHODS

CF3C02Hwas sequanal grade from Pierce. Concentrated HCI, formic acid and n-propionic acid were purest grades available from Fluka Chemical. Myoglobin (sperm whale), egg white lysozyme (hen), and Val-Val were from Serva Feinbiochemica. Val-Ile and Ile-Val were from Biomol. Ile-Ile was donated by Dr Shimonishi (Protein Research Inst., Osaka University). Lipoprotein from the outer cell membrane of Proteus mirahilis was donated by Dr Plapp (Dept of Microbiology, University of Kaiserslautei-n). Tobacco mosaic virus coat protein was prepared by the standard method [12]. Ferramido chloromycin was donated by Dr Shimi (Ain Shams University, Cairo) [13]. Amino acid mixture was the standard H of Pierce which contains equal molar amounts
Abbreviation. 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 with

CF3COdf:HCI ( I : 2) (curve B ) at various temperatures

known to be resistant to acid hydrolysis [5,14]. Hydrolysis of 50 min or longer resulted in more than 93 y/o recovery for three valine-containing peptides, while the maximuin recovery for Ile-Ile was 90 %. Further incubation resulting in a considerable amount of alloisoleucine [5]. The rate of allo-

isoleucine formation was observed to be 3'x for 25-min hydrolysis and 5 7 ; for 50 min. Mixtures of acetic or propionic acid and HC1 also produced alloisoleucine to the same extent or more. These experiments suggest the optimum hydrolysis time to be more than 25 min.
Stahrlitj of Amino Ac id\

100

v, ._

An ainino acid standard mixture wa\ heated in various acid mixtures between 130 C and 21 0 'C for 25 min and 50 min
75
~~~

72

h v,

Acid mixture
50

Composition
~

Recovery
1"

0
c

!J;v
/

a c ,
I

w"
25 / /

Formic acid: HCI Acctic acid: HCI Trifluoroacetic acid: HCI

1:l 1.2 1:l 1.2


2:l

Propionic acid: HCI

1:l 1.2 1:l 1:2

85 95 97 100 85 100 100 90 Y 7

Table 2. RW(JLWJ, o / umino uc,id.s yfirr incuhuiioia bciih vurious ucid rnivrurcs ui elevutd fenapf'rutures Recoveries arc expressed as a percentage of the value of alanine taken as 100%. All the acid mixtures contained 0.005 : ,<; phenol Acid mixture Temperature Time Recovery of
-~ -

_ -

Thr

Ser

Glu

C
HCI CF3COzH(2 1)

mln

0 ,

_ _

.~

-~

~~

~-

~~

~~

130 150 170


190

25
50 25 50 25 50 25 50 25 50
-

210

98 98 96 99 95 90 n4 77 67 57
-

103 103 100 100 89 80 63 44 29


19
.

1 on

108 101 104 95 99 Y 6 1 I4 91 114


_

HCI propionic d a d ( I ' 1)

130
1 50

170
1 90

210
_ ~ _ -_ HCI CHSCOzH(1 1)
.~
~~~ ~ ~

25 50 25 50 25 50 25 50 25 50
-~

130
150

170
190

210

25 50 25 50 25 50 25 50 25 50

106 104 101 109 107 87 89 56 64 20 _ _ 81 81 89 87 99 94 80 44 67 41

84 93 81 87 84 78
69

13 20 5
. -

100 101 96 103 102 109 108 104 93


110
~~

94 89 91 86 87 76 63 55 28 26 _ 30 41 28 36 25 53 33 32 26 26 68 54 57 38 26 53 23 30 43 39

97 102 99 98 89 81 69 58 31 29
~~~

94 99 93 95 88 62 55 24 40 4
~~-

96 I01 96 100 96 99 90 98 90 93 ~ 97 97 95 97 100 92 104 100

nn

94
_ _ _ _ ~

91 107 89 73 72 47 59 11 40
11

95 86 108 75 94 105 71 110 118 121

- _ 69 93 71 87 89 63 80 40 42 20

91 88 104 88 102 106 75 100 94 95

5x7
Table 3. Amino wid coniposificni o/'nijofi/ohin ~ J d(j%rcnt J nw//~id~ Values are expressed as relative values based on alanine = 17. Recovery was calculated from values of alanine Amino acid Residues obtained
~-

fiom requence

CF?COzH HCI (1 2)
-

propionic acid HCI (1 1)


~~

6 M HCI
~

25min

50min 8.2 4.6 5.3 19.0 4.0 11.2 17.0 7.7 1.7 8.7 18.3 2.0 6.0 12.0 19.3 4.0
-

15min
8.0 3.9 5.1 17.9 3.9 11.6 17.0 3.9 2.1 4.1 12.3 0.7 2.0 9.3 13.3 3.5
~~

30min 8.4 4.4 4.6 19.3 3.6 11.7 17.0 6.7 2.0 6.5 16.4 1.5 3.6 11.1 17.3 3.9
_

24 h

72 h

Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe His Lys Ar&
~

8 5 6 19 4 11 17 8 2 9 18 2 6 12 19 4
_~
~

8.0 4.8 5.6 18.9 4.1 11.0 17.0 7.0 1.8 7.7 17.7 2.0 5.6 11.9 19.1 4.1
~

8.4 8.1 4.9 4.8 5.8 5.7 19.5 18.9 3.9 3.9 11.611.0 17.0 17.0 4.8 7.3 1.8 1.9 5.3 7.7 15.3 16.3 2.0 2.0 4.6 4.9 10.731.8 17.1 19.0 3.5 4.1
. .
~

~-

Recovery
( <Y")

92

100 166 C

73

86 160 C

84

92 107 C

Tcmperature

in order to investigate side reactions (Table 2). The mixtures of HCI and CF3C02H were found to give the least side reactions among the mixtures. At higher temperatures than 18O'-C, even the HCliCF3C02H mixture yielded poor recoveries of certain amino acids. However, between 160 "C and 170 ' C recoveries were obtained comparable to 6 M HCI at 110 "C for 24 h. At 170 "C CF3C02H : HCI (1 : 2), for example, gave the following results: (a) decomposition of serine and threonine were observed (see Table 2), as has been observed for the 6 M HCI method [15]; (b) oxidation of methionine (5 - 20 %) depended on the quality of the vacuum and cystine was almost completely oxidized (70- 90 (c) alloisoleucine was formed by racemization as in the 6 M HCI hydrolysis [6,15], and (d) tryptophan was decomposed and derivatiration of tyrosine also depended on the vacuum. The addition of 0 . 2 x phenol inhibited the derivatization [16]. Because we observed that the addition of larger amounts of phenol affects the analysis of other amino acids, experiments were made to find the optimum amount. The amino acid standard mixture (2.5 nmol) was incubated at 167C with 300 pl of CF3C02H: HCI (1 :2) containing freshly distilled phenol, for 25 min and 50 min. Addition of only 0.001 phenol (by vol.) resulted in a better than 957< recovery of tyrosine while no addition gave only 71 Yg. 0.001 phenol corresponds to about 10-times molar excess over tyrosine. No serious effect on the other amino acid recoveries was observed in the presence of 0.001 and 0.03 phenol. In later experiments we generally used 0.005 %phenol. From consideration of the side reactions, we chose a hydrolysis temperature below 170 -C.

x);

Table 4. Amirio acid conzposition of proteins hq' the present and conventionul metlzorls The 25-min, 50-min and 75-min hydrolyses were with C F J C O ~ H : HCI ( I :2); the 24-h and 72-h bydrolyses were with 6 M HCI at 107 ' C . Values are based on the values for alanine, except for tobacco mosaic virus protein coat protein which is based on the value for glutamic acid Amino acid Amount present in
~~

~-

_~

~~

-~

~_

~. ~-

. _-

- ~ - .-

Iysozyme

tobacco mosaic virus protein hydrolysis for


~~~ ~

ferramido chloromycin

lipoprotein

from sequence

from sequence

hydrolysis for
~ ~

by hydrolysis for
~-- -~
~~ ~ ~

25 min 21 Asp Thr 7 Ser 10 Glu 5 Pro 2 G'Y 12 Ala 12 cys/2 8 Val 6 Met 2 Ile 6 Leu 8 T Yr 3 Phe 3 His 1 Lys 6 Arg 11 _ -. - Recovery (y,;)
"

50 min 18 16 16 'I6 8 6 14 1 14 0 9 12 4 8 0 2 11
~ ~ ~

25 min 18.0 15.5 13.7 16.0 8.2 6.0 14.0 0.32 12.6
-

50 min 18.0 15.3 11.3 16.0 8.1 6.1 14.0 0.18 13.6
-

25 min 2.0
-

50 min

24 h

72 h

temporary conclusion

by hydrolysis for
_ _ _ 25 50 min min
~~~ ~

75 min 6.0 2.6 2.1 7.4 2.0 4.7 6.0 4.5 1.8 2.9 4.8 1.2 1.8 1.1 3.3 3.4
_
~~ ~

24 h

72 h

temporary conclusion

20.8 20.9 6.2 5.7 8.1 6.3 5.1 4.9 2.0 1.8 12.0 12.0 12.0 12.0 5.2 3.6 4.7 5.2 1.4 1.3 4.0 5.8 5.8 8.0 2.9 2.9 3.0 3.0 1.1 1.1 6.0 5.8 10.8 11.1
-~ -~ - -

1.6
-

2.0 2.1 1.4 1.5 ~_

4.0 2.0 1.9 1.1


-

4.0 2.0 0.80 1.3


-

4.5 2.0 0.75 0.20


-

7.8 12.0 3.8 7.6


-

8.4 12.0 3.8 7.4


-

0.80 0.95 0.59 1.7


-

0.92 0.91 0.58 1.6


-

0.26 0.88 0.68 1.3


-

2.4 11.2
~ ~

2.1 11.4 100

.-

~~. -

- -~ -

2.1 1.6 4.2 2.0 0.97 0.40 0.38 0.74 0.58 1.1 ~~

2 0 2 0 0 4 2 2" 1-2 0 1 1 1 2 0 0 0
~

6.2 2.8 3.0 7.2 1.7 4.8 6.0


-

6.1

3.7 1.9 2.6 4.8 1.0 1.9 1.2 3.4 2.6


_ ~_ _~

2.7 2.5 7.0 1.7 4.8 6.0 _ 3.9 1.8 2.9 4.9 0.94 1.9 1.1 3.5 3.2
~~

6.3 3.1 3.4 7.5 1.4 4.6 6.0 _ 2.5 1.7 2.0 4.8 1.0 1.5 0.74 3.2 2.6
~ ~~

5.9 2.8 3.2 7.2 1.6 4.8 6.0


-

3.6 1.3 3.0 5.3 1.2 1.8 0.91 3.5 3.2 92

6 3 4 7 2 5 6 0 4-5 2 3 5 2 2 1 4 3-4
' J

88

100

99

81

99

57

65

89

95

100

81

Value obtained by separate hydrolysis of oxidized protein [20]

588

Hydrolysis of Proteins
Myoglobin was chose as a test protein and 5.5 pg was hydrolysed in CF3C02H/HCl (1 :2) at various temperatures for 10 and 25 min. Fig. 2 shows the extent of protein hydrolysis in which recoveries were expressed relative to alanine which is hydrolysed at an average rate [I71 and is stable under these conditions. The data indicated that a 25-min hydrolysis at 170C resulted in a recovery of 94%. Myoglobin was hydrolysed at 160 "C for 15 min with a mixture of propionic acid:HCl (1 : I), according to the conditions of [lo] and for 30 min as an additional hydrolysis time. Hydrolysis was incomplete after 15 min (73 %) and reached 86 % even after 30 min. The data in Table 3 suggest that these conditions can be improved. The hydrolysis temperature was studied further between 150 "C and 170 "C to investigate the recovery of amino acids and for reducing side reactions using glucagon and myoglobin. Our conclusion is that the optimum conditions are between 165 "C and 170 "C. Table 3 lists the composition of myoglobin obtained at 166 "C. The degradation of threonine and serine was slightly greater than that found in experiments with free amino acids, but the zero-time extrapolation values [18] were almost exactly the figures expected. With the exception of isoleucine, nearly quantitative recovery was obtained for all amino acids. The low recovery of isoleucine can be explained by the presence of an Ile-Ile sequence in myoglobin. Amino acid composition results for lysozyme and several other proteins are listed in Table4. In general, the conditions were quite acceptable. Yields of valine and isoleucine after 72-h hydrolysis by the conventional method were about the same as those obtained after 25 min by the new method. On the other hand decomposition rates of serine and threonine, requiring 25 and 50 min in the new hydrolysates, were approximately twice those in the conventional hydrolysates, requiring 24 h and 48 h. Table 4 also contains the results obtained for two hydrophobic proteins of unknown sequence. In these cases we also listed the data given by the conventional hydrolysis. Ferramidochloromycin is an extremely hydrophobic antibiotic peptide [I 31. Lipoprotein from the bacterial outer cell membrane has two moles lipid/ mole in the N-terminal region. Table 4 also lists the recovery of amino acids based on the value of alanine. It is important to note that the conventional hydrolysis gave recoveries which were much too low for hydrophobic proteins, while the new method gave more realistic results.

We propose the following method for rapid acid hydrolysis: 5 pg of peptide or protein is dissolved in 50-300 pl of a con, mixture of 2vol. concentrated HC1 and 1vol. C F ~C O Z H taining 0.005 "/, (by volume) freshly distilled phenol. The tube is sealed under a high vacuum (50 pmHg, i.e. 7 Pa) and placed in an oil bath, the temperature of which is regulated at 166 "C. Hydrolysis times are 25 min and 50 min and can be extended if necessary. After cooling down, the acid is removed on a rotary evaporator with a water vacuum pump at 65C. This rapid evaporation is needed to avoid esterification between serine and glutamic acid [19].

REFERENCES
Dreze, A. & Reith, W. S. (1956) Biochem. J . 62, 3. Macpherson, H. T. (1946) Biochem. J . 40, 470-481. Hill, R. L. & Schmidt, W. R. (1962) J . Biol. Chem. 237, 389-396. Smith, E. L. & Stockell, A. (1954) J . Biol. Chem. 207, 501 -514. Synge, R. L. M. (1945) Biochem. J . 39, 351-354. Moore, S. &Stein, H. (1963) Methods Enzymol. 6, 819-831. Noltmann, E. A,, Mahowald, T. A. & Kuly, S. A. (1962) J . B i d . Chem. 237,1146- 1154. 8. Inouye, M., Okada, Y . & Tsugita, A. (1970) J . Biol. Chem. 245, 3439 - 3454. 9. Westall, F. C., Scotchler, J. & Robinson, A. B. (1972) J . Org. Chem. 37,3363 - 3365. 10. Westall, F. C. & Hesser, H. (1974) Anal. Biochem. 61, 610-613. 11. Tsugita, A., Gregor, I., Kubota, I. & v. d. Broek, R. (1979) Cytochrome Oxidase (King, T. E. et al., eds) pp. 67-77, Elsevier/North Holland, Biomedical Press, Amsterdam. 12. Tsugita, A. & Fraenkel-Conrat, H. (1960) Proc. Nut/ Acad. Sci. USA, 46,636-642. 13. Shimi, I. R. & Shoukry, S. (1976) J . Anfibiot. Ser. A . 19, 110-114. 14. Desnuelle, P. & Casel, A. (1948) Biochinz. Biophys. Acfu, 2, 64-75. 15. Dustin, J. P., Schram, E., Moore, S. & Bigwood, E. J. (1953) Bull. Soc. Chim. Biol. 35, 1137-1147. 16. Sanger, F. & Thompson, E. 0. P. (1963) Biochim. Biophys. Acta, 71, 463 - 490. 17. Muramatu, M., Hirohata, R., Kanda, Y., Shibuya, S., Fujii, S., Nagamatsu, A. & Ono, T. (1963) Hoppe-Seyler's 2.Physiol. Chem. 332,263 - 270. 18. Hirs, C . H. W., Stein, W. H. & Moore, S. (1954) J. Bid. Chem. 211, 941 - 950. 19. Ikawa, M. & Snell, E. E. (1961) J . Bid. Chem. 236, 1955-1959. 20. Hirs, C. H. W. (1956) J . Biol. Chem. 219, 611 -621. 1. 2. 3. 4. 5. 6. 7.

A. Tsugita and J.-J. Schemer, Europaisches Laboratorium fur Molekularbiologie, Postfach 10 22 09, D-6900 Heidelberg, Federal Republic of Germany