Hydrolysis peptides and proteins

SH Chiou, KT Wang. Peptide and protein hydrolysis by microwave irradiation. J Chromatogr 491 424-431, 1989. 

Part 1 of this chapter is intended to provide background material for the analytical procedures described later in this chapter for amino acids and peptides, but it also provides a broad survey of the topic that can be read in isolation from the analytical context. The derivatisation of amino acids is the basis of many of the sensitive analytical amino-acid assay procedures in current use and this chapter covers the normal profile of reactions of the amino and carboxy groups, knowledge of which is an essential prerequisite for appreciating the analytical context. Reactions of peptides are also covered here (e.g. peptide and protein hydrolysis is covered in Section 4.4.7), though the coverage is restricted in scope because parts of this topic are discussed in Chapter 5, where it is relevant to sequence-determination procedures (see also Barrett, 1985). 

The sequence of each different peptide or protein is important for understanding the activity of peptides and proteins and for enabling their independent synthesis, since the natural ones may be difficult to obtain in small quantities. To obtain the sequence, the numbers of each type of amino acid are determined by breaking down the protein into its individual amino acids using concentrated acid (hydrolysis). For example, hydrolysis of the tetrapeptide shown in Figure 45.3 would give one unit of glycine, two units of alanine, and one unit of phenylalanine. Of course, information as to which amino acid was linked to which others is lost. 

Peptidases are enzymes that catalyse the hydrolysis of peptide bonds - the bonds between amino acids that are found in peptides and proteins. The terms protease , proteinase and proteolytic enzyme are synonymous, but strictly speaking can only be applied to peptidases that hydrolase bonds in proteins. Because there are many peptidases that act only on peptides, the term peptidase is recommended. Peptidases are included in subclass 3.4 of enzyme nomenclature [1,5]. 

Selective cleavage of peptides and proteins is an important procedure in biochemistry and molecular biology. The half-life for the uncatalyzed hydrolysis of amide bonds is 350 500 years at room temperature and pH 4 8. Clearly, efficient methods of cleavage are needed. Despite their great catalytic power and selectivity to sequence, proteinases have some disadvantages. Peptides 420,423,424,426 an(j proteins428 429 can be hydrolytically cleaved near histidine and methionine residues with several palladium(II) aqua complexes, often with catalytic turnover. 

Peptide hydrolases (peptidases or proteases, i.e., enzymes hydrolyzing peptide bonds in peptides and proteins, see Chapt. 2) have received particular attention among hydrolases. As already described in Chapt. 2, peptidases are divided into exopeptidases (EC 3.4.11 -19), which cleave one or a few amino acids from the N- or C-terminus, and endopeptidas-es (proteinases, EC 3.4.21-99), which act internally in polypeptide chains [2], The presentation of enzymatic mechanisms of hydrolysis in the following sections will begin with peptidases and continue with other hydrolases such as esterases. 

Proteins are fundamentally polymers of a-amino acids linked by amide linkages (see Section 13.1). It is a pity that biochemists refer to these amide linkages as peptide bonds remember, a peptide is a small protein (less than about 40 amino acid residues), whereas a peptide bond is an amide. Therefore, peptides and proteins may be hydrolysed to their constituent amino acids by either acid or base hydrolysis. The amide bond is quite resistant to hydrolytic conditions (see above), an important feature for natural proteins. 

Tyrosine (9-sulfate is stable under alkaline conditions, thus allowing for its quantification by amino acid analysis upon alkaline hydrolysis [0.2 M Ba(OH)2, 110 °C, 24 h] of sulfated tyrosine peptides and proteins.[6 331 Conversely, more than 95% of the ester is hydrolyzed after five minutes in 1M hydrogen chloride at 100 °C. Despite this pronounced acid lability, sulfated tyrosine peptides are sufficiently stable to short exposures of TFA134 35 or aqueous TFA[36 as required in peptide synthesis for removal of add-labile protecting groups. 

Among the analytical methods presently used for the characterization of natural and synthetic peptides and proteins, the primary value of amino acid analysis is the determination of absolute peptide and protein content in solids and solutions and the quantitation of their amino acid composition and stoichiometry. It involves two steps, i.e. complete hydrolysis of peptides and proteins, followed by photometric determination of the released amino adds. The steps are laborious and time-consuming, and there is a continuous need for improvement of the techniques to increase precision and sensitivity. 

Table 2 Add Hydrolysis of Peptides and Proteins Containing Unstable Amino Add Residues1131417-19 ...

The lability of peptides and proteins to acidic conditions was first reported in 1920 by Dakin,12031 who found that acid hydrolysis of peptides or proteins that contain consecutive N-alkyl amino acids leads to the formation of piperazine-2,5-diones (DKP) this side reaction lowered their yield during amino acid analysis. For example, the piperazine-2,5-dione c[-Hyp-Pro-] was isolated from the hydrolyzate of gelatine. 

Biological amide hydrolysis, as in the hydrolysis of peptides and proteins, is catalyzed by the proteolytic enzymes. These reactions will be discussed in Chapter 25. 

Whilst metal-N(peptide) bond formation inhibits hydrolysis of the peptide bond, coordination to O(peptide) has the opposite effect. These differences in reactivity can be readily demonstrated and put to practical use with the inert Co111 complexes. One of the first examples was the reaction of [Co(trien)(H20)(OH)]2+ with peptides to give hydrolysis of the peptide bond at the N-terminal end. The proposed mechanism involving nucleophilic attack by hydroxide at the peptide carbon is shown in Scheme 7.110 Similar selective hydrolyses of N-terminal peptide bonds have since been demonstrated with other Co111 amine complexes and the reaction has been examined as a method for determining the N-terminal amino acid residue in peptides and proteins.1"112... 

The amide bonds in peptides and proteins can be hydrolyzed in strong acid or base Treatment of a peptide or protein under either of these conditions yields a mixture of the constituent amino acids. Neither acid- nor base-catalyzed hydrolysis of a protein leads to ideal results because both tend to destroy some constituent ammo acids. Acid-catalyzed hydrolysis destroys tryptophan and cysteine, causes some loss of serine and threonine, and converts asparagine and glutamine to aspartic acid and glutamic acid, respectively. Base-catalyzed hydrolysis leads to destruction of serine, threonine, cysteine, and cystine and also results in racemization of the free amino acids. Because acid-catalyzed hydrolysis is less destructive, it is often the method of choice. The hydrolysis procedure consists of dissolving the protein sample in aqueous acid, usually 6 M HC1, and heating the solution in a sealed, evacuated vial at 100°C for 12 to 24 hours. 

A Tsugita, T Uchida, HW Mewes, T Ataka. Rapid vapor-phase acid (hydrochloric acid and trifluoroacetic acid) hydrolysis of peptide and protein. J Biochem 102 1593-1597, 1987. 

WG Engelhart. Microwave hydrolysis of peptides and proteins for amino acid analysis. Am Biotech Lab 8(15) 30-35, 1990. 

K Muramoto, H Kamiya. Recovery of tryptophan in peptides and proteins by high-temperature and short-term acid hydrolysis in the presence of phenol. Anal Biochem 189 223-230, 1990. 

B Penke, R. Ferenczi, K Kovacs. A new acid hydrolysis method for determining tryptophan in peptides and proteins. Anal Biochem 60 45-50, 1974. 

As mentioned above, the rectal route is very attractive for systemic delivery of peptide and protein drugs, but rectal administration of peptides often results in very low bioavailability due to not only poor membrane penetration characteristics (transport barrier) but also due to hydrolysis of peptides by digestive enzymes of the GI tract (enzymatic barrier). Of these two barriers, the latter is of greater importance for certain unstable small peptides, as these peptides, unless they have been degraded by various proteases, can be transported across the intestinal membrane. Therefore, the use of protease inhibitors is one of the most promising approaches to overcome the delivery problems of these peptides and proteins. Many compounds have been used as protease inhibitors for improving the stability of various peptides and proteins. These include aprotinin, trypsin inhibitors, bacitracin, puromycin, bestatin, and bile salts such as NaCC and are frequently used with absorption enhancers for improvement in rectal absorption. 

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