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CLEAVAGE AT ASPARTYL--PROLYLBONDS

145

overlap CNBr-produced fragments and could serve as new start points for sequential Edman degradation. Acknowledgment We thank Professor Y. Pocker for many helpful discussions regarding the mechanism of hydroxylaminecleavage of Asn-Glybonds.

[15l C l e a v a g e at A s p a r t y l - P r o l y l B o n d s B y MICHAEL LANDON

The particular acid lability of the peptide bond linking aspartyl residues to prolyl residues in proteins was first noticed when primary structure analysis was performed on proteins containing these bonds. Substantial cleavage was occurring at such bonds during the procedures employed in the production and isolation of the peptide fragments required for protein amino acid sequence determination. 1 Work on the primary structure of bovine glutamate dehydrogenase, which contains two aspartyl-prolyl bonds, demonstrated that the bonds were being broken during the preparation of tryptic peptidesf tryptic peptides from the maleylated protein, 3 peptides produced by cyanogen bromide cleavage,~ and peptic peptides. 5 In view of the known acid lability of peptide bonds involving aspartyl residues, 6 it was not surprising to have found splitting occurring at such bonds during the acidic conditions employed in hydrolysis with pepsin and in chemical cleavage using cyanogen bromide. Howover, two other observations were less expected. First, that the milder conditions, albeit still acidic, that were used at that time for peptide purification (and indeed are still used) should cause splitting and, second and more important, that cleavage under all conditions was substantially confined to Asp-Pro bonds. The lability of Asp-Pro bonds was, therefore, initially seen as anomalous cleavage under the acid conditions routinely used in specific peptide bond scission and subsequent peptide separation procedures. The apparent high specificity of the reaction and the yields obtained, even D. Piszkiewicz, M. Landon, and E. L. Smith, Biochem. Biophys. Res. Commun. 40, 1173 (1970). M. Landon, M. D. Melamed, and E. L. Smith, J. Biol. Chem. 246, 2360 (1971). W. J. Brattin and E. L. Smith, J. Biol. Chem. 246, 2400 (1971). 4T. J. Langley and E. L. Smith, J. Biol. Chem. 246, 3789 (1971). 5D. Piszkiewicz, M. Landon, and E. L. Smith, J. Biol. Chem. 248, 3067 (1973). J. Schultz, this series Vol. 11 [28].

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SELECTIVE CLEAVAGE BY CHEMICAL METHODS

[15]

under conditions that were appropriate for the intended procedure and not necessarily for the anomalous cleavage that was observed, suggested that the lability of peptide bonds linking aspartyl residues to prolyl residues would lend itself to a specific chemical cleavage procedure. The limitation of specific chemical cleavage at Asp-Pro bonds is the rarity of these bonds, but this is also an inherent advantage of the method. The mechanism of selective cleavage at Asp-Pro bonds has been discussed by Piszkiewicz e t al. 1 Selective hydrolysis of aspartyl peptide bonds occurs under mildly acidic conditions for periods of 4 to 24 hr at elevated temperatures. 6 Mechanistically this involves intramolecular catalysis by aspartate carboxylate anion displacement of the protonated nitrogen of the peptide bond. The properties of proline are clearly influential with regard to the greatly increased lability of the bond linking an aspartyl residue to a prolyl residue. Piszkiewicz e t al. ~ suggested that in peptide linkage there is a greater basicity of the prolyl nitrogen compared with that of other amino acid residues. This greater basicity would, by analogy with the hydrolysis of anilides, 7 enhance the rate of hydrolysis of the peptide bond by increasing protonation of the leaving group. It is pertinent to note that specific cleavage at bonds preceding prolyl residues in the peptide antibiotic alamethicin was obtained under acid conditions (12 M HCI, 37 °, 30 min).S Piszkiewicz e t al. ~ also commented on other factors that might influence the reaction.

Procedure

The protein should be dissolved at a concentration in the range 0.510 mg/ml in the appropriate acid solution. Table I lists the various acid solutions that have been used for specific cleavage at Asp-Pro bonds. The final choice of conditions will depend on the particular protein and will have to be determined by trial procedures. In general, the temperature should not exceed 40 ° and the duration of the procedure can be as long as 120 hr in the milder conditions, but should not be extended beyond 48 hr in the case of the more vigorous conditions (a formic acid concentration above 70%), where significant nonspecific peptide bond splitting may occur if the exposure to acid is prolonged. The extent of cleavage can be determined by procedures involving the separation and quantitation of peptide products or by quantitative end-group analysis.

M. L. Bender and R. J. Thomas, J. Am. Chem. Soc. 83, 4183 (1961). 8j. W. Payne, R. Jakes, and B. S. Hartley, Biochem. J. 117, 757 (1970).

CLEAVAGE AT ASPARTYL--PROLYL BONDS

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147

TABLE I METHODS FOR SPECIFIC CLEAVAGE AT ASPARTYL-PROLYL BONDS

Conditions Method

Acid solution



10% (v/v) acetic acid, adjusted to pH 2.5 with pyridine 10% (v/v) acetic acid, adjusted to pH 2.5 with pyridine, in 25% (v/v) 1-propanol 10% (v/v) acetic acid, adjusted to pH 2.5 with pyridine, in 7 M guanidinium chloride 70% (v/v) formic acid 70% (v/v) formic acid, in 7 M guanidinium chloride 75% (v/v) formic acid 75% (v/v) formic acid, in 7 M guanidinium chloride 90% (v/v) formic acid

Ba Ca I) b Eb Fb Gb Hb

Temp. (°C)

Duration (hr)

40

24-120

40

24-96

40

24-96

37 37

24-72 24-48

37 37

24-48 24-48

37

24

K. J. Fraser, K. Poulsen, and E. Haber, Biochemistry 11, 4974 (1972). b j. Jauregui-Adell and J. Marti, Anal. Biochem. 69, 468 (1975). Discussion o] Applications

The first reported use of specific chemical cleavage at Asp-Pro bonds was by Poulsen et al., 9 who applied the procedure to rabbit antibody light chain. The conditions employed were 10% acetic acid, adjusted to p H 2.5 with pyridine, in 7 M guanidinium chloride, at 40 ° for 96 hr. This system gave cleavage at the single susceptible bond in yields of 90%. The effects of different conditions on the splitting of rabbit antibody light chain were investigated by Fraser et al., 1° who showed t h a t the effectiveness of the acid employed (as above) was substantially increased by the presence of 7 M guanidinium chloride. However, in the case of a carboxypeptidase inhibitor from potato, splitting was obtained in yields of 84% using acetic acid (10%, at p H 2.5) alone." More vigorous conditions were required with other proteins. M a r e milk lysozyme required 75% formic acid in 7 M guanidinium chloride, at 37 ° for 48 hr for quantitative cleavage of the single Asp-Pro bond, 12 K. Poulsen, K. J. Fraser, and E. Haber, Proc. Ncdl. Acad. Sci. U.S.A. 69, 2495 (1972). 1oK J. Fraser, K. Poulsen, and E. Haber, Biochemistry 11, 4974 (1972). 11G. M. Hass, H. Nau, K. Biermann, D. T. Grahn, L. H. Ericsson, and H. Neurath, Biochemistry 14, 1334 (1975). 1~j. Jauregui-Adell and J. Marti, Anal. Biochem. 69, 468 (1975).

148

[15]

SELECTIVE CLEAVAGE BY CHEMICAL METHODS

while w i t h t o b a c c o m o s a i c v i r u s p r o t e i n ( v u l g a r e s t r a i n ) m a x i m u m y i e l d s of o n l y 3 8 % a t t h e m o r e l a b i l e of two b o n d s were o b t a i n e d using 90% f o r m i c acid, a t 37 ° for 24 hr. 12 I n t h e l a t t e r case the exposure to acid could n o t be e x t e n d e d as nonspecific c l e a v a g e was observed. S t u d i e s on t h e p r o t e i n f r o m t h e o t h e r s t r a i n s of t o b a c c o m o s a i c v i r u s showed t h a t even s m a l l changes in o v e r a l l p r i m a r y s t r u c t u r e s i g n i f i c a n t l y c h a n g e d t h e l a b i l i t y a t A s p - P r o bonds. ~-~ V a r i a t i o n in s u s c e p t i b i l i t y to c l e a v a g e of A s p - P r o b o n d s is t h e r e f o r e a p p a r e n t . A s u m m a r y of t h e results of p r o c e d u r e s a p p l i e d to p a r t i c u l a r p r o t e i n s is given in T a b l e I I . TABLE I I APPLICATIONS OF SPECIFIC CLEAVAGE AT ASPARTYL-PROLYL BONDS

Percentage cleavage after hours of reaction Protein Rabbit antibody light chain b Rabbit antibody light chain b Rabbit antibody light chain b Rabbit antibody light chain c Rabbit antibody light chain e Carboxypeptidase inhibitor from potatoS Mare milk lysozymeg Mare milk lysozymea Mare milk lysozymeg Mare milk lysozymeg Mare milk lysozymeg Tobacco mosaic virus protein g'h Tobacco mosaic virus proteing 'h Tobacco mosaic virus protein g'h Rubredoxin from Desulfovibrio vulgaris i

Method a 24 A B C Cd Cd A Ca D E F G D G H D

15 18 30

35 30 70 12

48

72

27 24 56

24 26 28 29 44 68 87 64 70 84

30 40 35 80 100

96 120

Protein concentration (mg/ml) 0.5-5 0.5-5 0.5-5 8-16 16 5

75

19 38 40

a The letter refers to the method in Table I. b K. J. Fraser, K. Poulsen, and E. Haber, Biochemistry 11, 4974 (1972). c j. Jaton. Biochem. J. 141, 1 (1974). d Procedure carried out at 37 °. J. Jaton, Biochem. J. 141, 15 (1974). / G. M. Hass, H. Nau, K. Biemann, D. T. Grahn, L. H. Ericsson, and H. Neurath, Biochemistry 14, 1334 (1975). o j. Jauregui-Adell and J. Marti, Anal. Biochem. 69, 468 (1975). h Vulgare strain: figures are for cleavage at the more labile Asp-Pro bond; cleavage at the other Asp-Pro bond occurred only in Method H and did not exceed 10%. i M. Bruschi, J. Bonicel, G. Bovier-Lapierre, and P. Couchoud, Biochim. Biophys. Acta 434, 4 (1976). Ahnost equal cleavage occurred at each of the two Asp-Pro bonds.

[161

PItTHALYLATION OF AMINO GROUPS

149

Summary Specific cleavage at Asp-Pro bonds of a protein can be effeeted by exposure to acid at moderate temperature for periods up to 120 hr. The effectiveness of cleavage is usually increased by incorporating a denaturing agent in the acid solution. Some Asp-Pro bonds are resistant to cleavage probably because of retention of protein folding even under the most vigorous conditions that have been employed, and under these circumstances some nonspecific peptide bond cleavage may occur, particularly if the exposure is prolonged. It is very likely that cleavage at Asp-Pro bonds will occur when acid procedures are employed in peptide bond scission or peptide separation and purification. With the larger peptides produced by cyanogen bromide cleavage it has been observed that partial cleavage at Asp-Pro near the termini of the authentic peptide may yield fragments that copurify with the larger component, thus complicating subsequent sequence determinations.IS The relative paucity of Asp-Pro bonds in proteins is both a limitation and an advantage of this specific cleavage procedure. The limitation is that many proteins do not contain any Asp-Pro bonds but as the Asp-Pro bond is never abundant the number of fragments obtained by application of the procedure can be advantageously small, which will simplify subsequent fraetionation processes. l~K. Titani, M. A. Hermodson, L. H. Ericsson, K. A. Walsh, and H. Neurath, Biochemistry 11, 2427 (1972).

[16] P h t h a l y l a t i o n o f A m i n o G r o u p s

B y J.-F. FECHt}REand R. BERTRAND The reversible chemical modification of amino groups of proteins and peptides by acylating reagents is a widely used technique in protein chemistry because of its vast range of applications in both active-site and sequence studies. Comparative studies performed on lysozyme1 and myoglobin2 have shown that maleic anhydrideS,~ and citraconic anhydride ~,6 are the most satisfactory reagents for such modifications, and A. F. S. A. Habeeb and M. Z. Atassi, Biochemistry 9, 4939 (1970). R. P. Singhal and M. Z. Atassi, Biochemistry 10, 1756 (1971). 3p. j. G. Butler, J. I. Harris, B. S. Hartley, and R. Leberman, Biochem. J. 112, 679 (1969). 4p. j. G. Butler and B. S. ttartley, this series Vol. 25, p. 191. 5 H. B. F. Dixon and R. N. Perham, Biochem. J. 109, 312 (1968). "M. Z. Atassi and A. F. S. A. Habeeb, this series Vol. 25, p. 546.

Cleavage at aspartyl-prolyl bonds.

[15] CLEAVAGE AT ASPARTYL--PROLYLBONDS 145 overlap CNBr-produced fragments and could serve as new start points for sequential Edman degradation. Ac...
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