Preferential Release of Aspartic Acid During the Hydrolysis of Proteins

Partridge, S. M.; Davis, H. F.

doi:10.1038/165062a0

Cited by 145 publications

(41 citation statements)

References 4 publications

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“…This effect was known as early as 1950 during the hydrolysis of protein in acetic acid or oxalic acid solution at boiling temperature [1]. Same effect is also found in hot diluted hydrochloric acid, but not in strong acid at room temperature [2].…”

Section: Introductionmentioning

confidence: 71%

Rapid Online Non-Enzymatic Protein Digestion Analysis with High Pressure Superheated ESI-MS

Chen

Kinoshita

Noda

et al. 2015

J. Am. Soc. Mass Spectrom.

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Abstract. Recently, we reported a new ESI ion source that could electrospray the super-heated aqueous solution with liquid temperature much higher than the normal boiling point (J. Am. Soc. Mass Spectrom. 25, 1862-1869). The boiling of liquid was prevented by pressurizing the ion source to a pressure greater than atmospheric pressure. The maximum operating pressure in our previous prototype was 11atm, and the highest achievable temperature was 180°C. In this paper, a more compact prototype that can operate up to 27atm and 250°C liquid temperatures is constructed, and reproducible MS acquisition can be extended to electrospray temperatures that have never before been tested. Here, we apply this super-heated ESI source to the rapid online protein digestion MS. The sample solution is rapidly heated when flowing through a heated ESI capillary, and the digestion products are ionized by ESI in situ when the solution emerges from the tip of the heated capillary. With weak acid such as formic acid as solution, the thermally accelerated digestion (acid hydrolysis) has the selective cleavage at the aspartate (Asp, D) residue sites. The residence time of liquid within the active heating region is about 20s. The online operation eliminates the need to transfer the sample from the digestion reactor, and the output of the digestive reaction can be monitored and manipulated by the solution flow rate and heater temperature in a near real-time basis.

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Section: Introductionmentioning

confidence: 71%

Rapid Online Non-Enzymatic Protein Digestion Analysis with High Pressure Superheated ESI-MS

Chen

Kinoshita

Noda

et al. 2015

J. Am. Soc. Mass Spectrom.

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“…The observation that Asp is the only amino acid released when proteins are heated in weak acids has long been known [95], and results of hydrolysis in dilute HCl at pH 4 of cytochrome c, wool proteins and egg yolk apovitellenins suggest that the majority of Asp bonds are cleaved within 2 h. This cleavage is dependent on the pH, as this affects the proton donor ability of the Asp sidechain carboxy group. Asp hydrolysis accounts for approx.…”

Section: Aspartate Residuesmentioning

confidence: 99%

The denaturation and degradation of stable enzymes at high temperatures

Daniel

Dines

Petach³

1996

Biochemical Journal

277

175

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Now that enzymes are available that are stable above 100 mC it is possible to investigate conformational stability at this temperature, and also the effect of high-temperature degradative reactions in functioning enzymes and the inter-relationship between degradation and denaturation. The conformational stability of proteins depends upon stabilizing forces arising from a large number of weak interactions, which are opposed by an almost equally large destabilizing force due mostly to conformational entropy. The difference between these, the net free energy of stabilization, is relatively small, equivalent to a few interactions. The enhanced stability of very stable proteins can be achieved by an additional stabilizing force which is again equivalent to only a few stabilizing interactions. There is currently no strong evidence that any particular interaction (e.g. hydrogen bonds, hydrophobic interactions) plays a more important role in

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“…The method was based on the preferential hydrolysis of the peptide bonds of aspartyl residues in dilute acid [18]. Indeed, the peptide 143 -159 contains two Asn residues in positions 146 and 152 (see Table 2).…”

Section: Identification O J T H E Amino Acid Alkyluted By Cluc3pdad'mentioning

confidence: 99%

Properties of the Charge‐Transfer Transition Observed in Glyceraldehyde‐3‐phosphate Dehydrogenase from Sturgeon Muscle Alkylated by 3‐Chloroacetylpyridine — adenine Dinucleotide

Branlant

Tritsch

Eiler

et al. 1982

European Journal of Biochemistry

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An absorption band at 340 nm is shown to be formed concomitantly with the covalent bond between the affinity label 3-chloroacetylpyridine -adenine dinucleotide (clac3PdAD ') and glyceraldehyde-3-phosphate from sturgeon. This band corresponds to a charge-transfer transition. Its intensity depends upon the pH and the ionic strength but is almost independent of the nature of the anions present in the medium. The pH dependence shows an inflexion point at pH 7.1. This result suggests the participation of a residue with a pK, of 7.1 within the active site of the enzyme in the formation of this transition. Using various techniques, the amino acid alkylated by clac3PdAD+ is shown to be the essential Cys-149, thus excluding the participation of this residue in the formation of the charge-transfer transition. On the other hand, the modification of Cys-153 seems not to affect this charge-transfer band. Other possible donors are proposed, such as the invariant His-176 or Tyr-317 residues. These amino acids might be implicated in the formation of the Racker band.In a previous paper, 3-chloroacetylpyridine -adenine dinucleotide was shown to inactivate glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus and sturgeon muscle via an affinity label mechanism [I]. The labeling of both glyceraldehyde-3-phosphate dehydrogenases was demonstrated to be irreversible, with a covalent incorporation of one mole of inactivator/mole enzyme subunit [I].The pH dependence of the inactivation rate and the results on thiol titration under denaturating conditions were rather indicative of a modification of a cysteine residue [l]. More surprising were the results on thiol titration under native conditions which showed the accessibility of one Cys residue per subunit toward 5,5'-dithiobis(2-nitrobenzoate).This result suggested that the modification of a Cys, presumably Cys-349, could lead to a partial unfolding of the structure of the protein. As a consequence, other thiol residues would then become accessible to thiol reagents.Results presented in this paper show that an absorption band at 340 nm is formed concomitantly with the covalent bond between 3-chloroacetylpyridine -adenine dinucleotide and glyceraldehyde-3-phosphate dehydrogenase from sturgeon. The properties of this new absorption band raised the question of the true nature of the amino acid alkylated by clac3PdADC. For this reason using various techniques but in particular high-performance liquid chromatography (HPLC), we have determined the nature of the modified amino acid.Abbreviations. ac'PdAD ', ac3PdADH, 3-acetylpyridine -adenine dinucleotide, oxidised and reduced forms respectively; clac3PdAD+, clac3PdADH, 3-chloroacetylpyridine -adenine dinucleotide, oxidised and reduced forms respectively; alkylated enzyme, ~-glycerdldehyde-3-phosphate dehydrogenase fully alkylated by clac3PdAD+ ; Tes, 2-{ [2-hydroxy-I ,I -bis(hydroxymeth yl)ethyl]amino}ethanesulfonic acid ; HPLC, high-performance liquid chromatography.Enzymes. D-Glyceraldehyde-3-phosphate dehydrogenase or ...

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Preferential Release of Aspartic Acid During the Hydrolysis of Proteins

Cited by 145 publications

References 4 publications

Rapid Online Non-Enzymatic Protein Digestion Analysis with High Pressure Superheated ESI-MS

Rapid Online Non-Enzymatic Protein Digestion Analysis with High Pressure Superheated ESI-MS

The denaturation and degradation of stable enzymes at high temperatures

Properties of the Charge‐Transfer Transition Observed in Glyceraldehyde‐3‐phosphate Dehydrogenase from Sturgeon Muscle Alkylated by 3‐Chloroacetylpyridine — adenine Dinucleotide

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