`
`Review
`Stability of Protein Pharmaceuticals
`
`Mark C. Manning, 1•2 Kamlesh Patel, 1 and Ronald T. Borchardt1
`
`Recombinant DNA technology has now made it possible to produce proteins for pharmaceutical
`applications. Consequently, proteins produced via biotechnology now comprise a significant portion
`of the drugs currently underdevelopment. Isolation, purification, formulation, and delivery of proteins
`represent significant challenges to pharmaceutical scientists, as proteins possess unique chemical and
`physical properties. These properties pose difficult stability problems. A summary of both chemical
`and physical decomposition pathways for proteins is given. Chemical instability can include proteo(cid:173)
`lysis, deamidation, oxidation, racemization, and ~-elimination. Physical instability refers to processes
`such as aggregation, precipitation, denaturation, and adsorption to surfaces. Current methodology to
`stabilize proteins is presented, including additives, excipients, chemical modification, and the use of
`site-directed mutagenesis to produce a more stable protein species.
`KEY WORDS: protein stability; biotechnology; mutagenesis; denaturation.
`
`INTRODUCTION
`
`With the recent advances in recombinant DNA technol(cid:173)
`ogy, the commercial production of proteins for pharmaceu(cid:173)
`tical purposes has become feasible (1,2). As a result, the
`preparation of proteins as medicinal agents has become an
`integral part of the pharmaceutical industry. Currently, there
`are more than 150 recombinant proteins in Phase I clinical
`trials or beyond, and almost a dozen have received FDA
`approval. Unfortunately, proteins possess chemical and
`physical properties which present unique difficulties in the
`purification, separation, storage, and delivery of these ma(cid:173)
`terials. Therefore, formulation of proteins differ greatly from
`that of rigid small organic molecules. Future pharmaceutical
`scientists will need to be properly trained to address the
`various aspects of protein instability. An introduction to
`these concepts is presented below, with the view that under(cid:173)
`standing protein stability at a molecular level is essential to
`solving many of their formulation problems.
`Degradation pathways for proteins can be separated
`into two distinct classes, involving chemical instability and
`physical instability. First, chemical instability can be defined
`as any process which involves modification of the protein via
`bond formation or cleavage, yielding a new chemical entity.
`Second, physical instability does not involve covalent mod(cid:173)
`ification of the protein. Rather, it refers to changes in the
`higher order structure (secondary and above). These include
`denaturation, adsorption to surfaces, aggregation, and pre-
`
`1 Department of Pharmaceutical Chemistry, The University of Kan(cid:173)
`sas, Lawrence, Kansas 66045.
`2 To whom correspondence should be addressed.
`3 Unless otherwise noted, all amino acids listed are L-enantiomers of
`the 20 common amino acids and are referred to by their three-letter
`abbreviations.
`
`cipitation. A summary of the current understanding of each
`of these processes is presented and illustrated by well(cid:173)
`characterized systems. Finally, approaches for retarding or
`inhibiting these processes and, thereby, increasing protein
`stability is presented.
`
`CHEMICAL INST ABILITY
`
`A variety of chemical reactions is known to affect pro(cid:173)
`teins (Fig. 1). These reactions can involve hydrolysis, in(cid:173)
`cluding both cleavage of peptide bonds as well as deamida(cid:173)
`tion of Asn and Gln side chains. 3 Hydrolysis at Asp-X sites
`is particularly accelerated. Oxidation of Cys can lead to di(cid:173)
`sulfide bond formation and exchange, whereas oxidation of
`Met and other amino acids may inactivate or alter the activ(cid:173)
`ity of a protein. Other decomposition reactions include beta(cid:173)
`elimination and racemization.
`
`Deamidation
`
`In the deamidation reaction, the side chain amide link(cid:173)
`age in a Gln or Asn residue is hydrolyzed to form a free
`carboxylic acid. Over the past two decades many investiga(cid:173)
`tors have observed altered forms of proteins which have
`been attributed to deamidation. Such a list contains
`lysozyme (3), bovine growth hormone (bGH) (growth hor(cid:173)
`mone is also known as somatotropin) (4), human growth
`hormone (hGH) (5,6), insulin (7 ,8), cx-crystaJlin (9), cy(cid:173)
`tochrome c (10), ')'-immunoglobulin (11), epidermal growth
`factor (EGF) (12), hemoglobin (13), triosephosphate isomer(cid:173)
`ase (TIM) (14,15), neocarzinostatin (16), prolactin (17), gas(cid:173)
`trin releasing peptide (18), and adrenocorticotropic hormone
`(ACTH) (19,20), suggesting that ·in vitro deamidation is a
`common phenomenon.
`The hydrolysis of Asn and Gin residues for many pro(cid:173)
`teins and peptides has been observed under a variety of
`
`903
`
`0724-8741/89/1100-0903$06.00/0 C 1989 Plenum Publishing Corporation
`
`CFAD Exhibit 1051
`CFAD v. NPS
`IPR2015-01093
`
`
`
`904
`
`PROTEIN PHARMACEUTICALS
`
`l D~M~~
`/\
`
`CHEMICAL INSTABIUTY
`
`PHYSICAL INSTABILITY
`
`-Deamidation
`-Racemization
`-Hydrolysis
`-Oxidation
`-Beta Elimination
`·Disulfide Exchange
`
`-Denaturation
`-Aggregation
`-Precipitation
`·Adsorption
`
`Fig. 1. Summary of the chemical and physical instability processes
`observed in protein pharmaceuticals.
`
`chemical conditions and has been reviewed by Robinson and
`Rudd (21). Interestingly, it was realized that the deamidation
`of Asn residues, which occurs most often at the sequence
`Asn-Gly. was accelerated at neutral or alkaline conditions
`(22-24). The rates were also higher relative to the hydrolysis
`of the amino acid Asn itself (21). An explanation is that
`deamidation is believed to proceed through a five-membered
`cyclic imide intermediate formed by intramolecular attack of
`the succeeding peptide nitrogen at the side chain carbonyl
`carbon of the Asn residue (see Fig. 2) (25). Subsequently, the
`cyclic imide spontaneously hydrolyzes to give a mixture of
`peptides in which the polypeptide backbone is attached via
`an a-carboxyl linkage (Asp) or is attached via a 13-carboxyl
`linkage (iso-Asp) (23). Similarly, Gln can also undergo de(cid:173)
`amidation via formation of a six membered ring (23). Most of
`the information on the mechanism and rate of deamidation of
`
`0
`I f-NH1
`f 12
`
`f
`
`-NH-Oi-u-NH-CH- ~-
`0
`0
`
`L- Asparaainyl pep1idc
`
`N'~ slow
`"f ~
`°'r-L r
`u
`N-- Oi-c- .= D • Cyclic imido
`I
`-NH-Of-r
`,,
`~
`+Hptj/ L· C)'Clie imidc~~p
`+H~ D-Aspmyl peptide
`~aO
`rn1t--N~hi-c-
`-NH-&-c-o·
`~
`
`D-isoAspanyl pepddo
`
`0
`
`R
`
`A
`0
`
`•
`0
`
`0
`~-o·
`bt1
`- NH-&-C-NH-&.- C-
`
`R
`
`l
`0
`
`L·Aspmyl pep1ide
`Fig. 2. Pathways for spontaneous deamidation, isomerization, and
`racemization for aspartyl and asparaginyl peptides.
`
`L·isoAspanyl pcp1idc
`
`Manning, Patel, and Borchardt
`
`Asn residues has been obtained from studies on short model
`peptides (26,27). Clear evidence for a deamidation mecha(cid:173)
`nism involving the cyclic imide intermediate has been ob(cid:173)
`tained by Geiger and Clarke (26). In their study, deamidation
`of a hexapeptide sequence based on residues 22-27 of ACTH
`(Val-Tyr-Pro-Asn-Gly-Ala) was studied at 37°C and pH 7.4.
`Evidence from these studies supporting cyclic imide forma(cid:173)
`tion include the appearance of iso-Asp, Asp, and cyclic im(cid:173)
`ide peptides upon deamidation and a ratio of the iso-Asp to
`Asp peptide formed in the deamidation of this hexapeptide
`(2.8:1) is the same as that found when purified cyclic imide is
`hydrolyzed (3.1:1). If there is a significant amount of direct
`solvent hydrolysis of the amide linkage occurring, the pro(cid:173)
`portion of Asp peptide relative to iso-Asp peptide in the
`deamidation of a hexapeptide would have increased, which
`is not the case. The presence of iso-Asp products from the
`incubations of proteins and peptides implies cyclic imide for(cid:173)
`mation as an intermediate in deamidation reaction (28-32).
`The Fourier transform infrared photoacoustic spectroscopic
`measurements (FTIR-PAS) have also provided direct evi(cid:173)
`dence for the formation of a cyclic imide in peptides with
`Asn-Gly sequences induced by heating in the dry state (33).
`Recently, we have shown that both Asp- and iso(cid:173)
`Asp-hexapeptides are formed upon deamidation of Val(cid:173)
`Tyr-Pro-Asn-Gly-Ala, ACTHn-27 (Asn-hexapeptide), in the
`pH range of 5 to 12 at 37°C (34,35). This further confirms the
`formation of a cyclic imide intermediate in the deamidation
`process at neutral and alkaline pH's. In the pH range 7 to 12,
`buffer concentration had significant effect on the rate of de(cid:173)
`amidation, indicating general acid-base catalysis. No buffer
`catalysis was observed at pH 5, 6, and 6 . .5. The ratio of
`iso-Asp- and Asp-hexapeptides was independent of buffer
`concentration at all pWs and was approximately 4:1. At
`acidic pH's (pH 1-2), the deamidation was much slower than
`at alkaline pH, and only Asp-hexapeptide was produced
`upon deamidation. Although iso-Asp-hexapeptide was not
`detected at acidic pH, one new product (Val-Tyr-Pro-Asp)
`was observed by HPLC. These results suggest that at acidic
`pH, the probable mechanism of deamidation is direct hydrol(cid:173)
`ysis of the amide side chain of Asn, to form the Asp(cid:173)
`hexapeptide, which further degrades in acidic media via pep(cid:173)
`tide bond cleavage at the Asp-Gly bond. Reactions at pH 3
`and 4 were very slow at 37°C (degradation of Asn(cid:173)
`hexapeptide was not detected for 60 days).
`By comparison, when deamidation experiments were
`carried out with ACTH (l-39), the separation of Asp- and
`iso-Asp products could not be achieved by either isoelectric
`focusing or cation-exchange HPLC (20). However, these
`techniques did separate native ACTH from the deamidated
`ACTHs (Asp- and iso-Asp-ACTH). The rate constants for
`the deamidation of both ACTH and Asn-hexapeptide
`27
`(ACTH22
`) at pH 2.0, 7.0, and 9.6 at 37°C were similar.
`-
`Formation of the iso-Asp product upon deamidation of
`ACTH at pH 7.0 and 9.6 was verified by the protein car(cid:173)
`boxymethyltransferase (PCM)-catalyzed methylation of de(cid:173)
`amidated ACTH. No such methylation was observed when
`ACTH was incubated at pH 2.0, 37°C. These data indicate
`the involvement of cyclic imide intermediate at neutral and
`alkaline pH but not at pH 2.0.
`Since the formation of a cyclic imide involves partici(cid:173)
`pation of the succeeding amino acid, the size and physico-
`
`
`
`Stability of Protein Pharmaceuticals
`
`chemical properties of neighboring amino acid side chain is
`expected to play an important role in the rate of formation of
`cyclic imide. Evidence in support of this conclusion comes
`from studying the rates of cyclic imide formation in peptides
`containing Asn (26) or Asp 13-benzyl esters (36-45). For ex(cid:173)
`ample, the rate of cyclic imide formation at pH 7 .4 was ap(cid:173)
`proximately 50 times slower in the Asn-Leu-hexapeptide
`than the Asn-Gly-hexapeptide due to steric hinderance by
`the Leu side chain (26).
`Robinson and co-workers (46-49) investigated the non(cid:173)
`enzymatic deamidation of Asn residues in synthetic pen(cid:173)
`tapeptides, and the effects of amino acid sequence, pH, tem(cid:173)
`perature, buffer species, and ionic strength. Using synthetic
`pentapeptides, it has been shown that deamldation is favored
`by increased pH, temperature, and ionic strength (46,47).
`These studies showed the importance of primary sequence
`around the Asn residue, but did not investigate the formation
`of either iso-Asp or cyclic imide. Similar results are obtained
`for cytochrome c (50). Similarly, the rate of deamidation of
`human TIM was facilitated by high temperatures, and was
`also found to be dependent on the presence of substrate and
`specific buffers (14). Unlike the hydrolysis of peptides con(cid:173)
`taining esters of Asp, where the cyclic imide intermediate
`can be trapped (22,24), cyclic imide formation during the
`deamidation of Asn peptides is the rate determining step
`(26,27).
`The rates of deamidation of Asn residues in proteins are
`influenced by the secondary and tertiary structures of pro(cid:173)
`teins. Clarke has shown that Asp and Asn residues in native
`proteins generally exist in conformations where the peptide
`bond nitrogen atom cannot approach the side chain carbonyl
`carbon without large scale conformational changes (51).
`Therefore, certain proteins will not undergo deamidation un(cid:173)
`less they have been denatured. Cyclic imide could only be
`formed in vitro at Asn67 of bovine pancreatic ribonuclease in
`the unfolded state (52). While in the native structure, this
`residue is poorly positioned for cyclic imide formation. Sim(cid:173)
`ilarly, it has been shown that urea (a strong denaturant)
`accelerates the deamidation of bGH, hGH, and prolactin,
`presumably by unfolding the protein (5). Tertiary structure
`appears to be the principle determinant for the de(cid:173)
`amidation of trypsin (53). The study also showed that adja(cid:173)
`cent Ser residues aid in the formation of the cyclic imide
`intermediate, consistent with earlier studies on small p·eptide
`systems (36,37). Recently, Lura and Schirch (54) have
`shown that the mechanism of deamidation of Val-Asn(cid:173)
`Gly-Ala and N-acetyl-Val-Asn-Gly-Ala varies according to
`the conformation of the peptide backbone. Above pH 9.0,
`both peptides have similar conformations and thus deami(cid:173)
`date by the same mechanism to give mixture of Asp and
`iso-Asp peptides. However, at pH 7.0, while the N-acetyl
`peptide yielded a mixture of Asp and iso-Asp peptides, the
`non-acetylated peptide gave no detectable amounts of these
`products, but rather yielded a cyclic peptide believed to be
`formed by nucleophilic attack of the amide of the Asn resi(cid:173)
`due by the terminal amino group.
`It is well known that peptides of Asp esters undergo
`intramolecular cyclization, under both acidic and basic con(cid:173)
`ditions, leading to a cyclic imide derivative (39,55). How(cid:173)
`ever, no reports are available showing the formation of cy(cid:173)
`clic imide from Asn peptides in acidic media. There are few
`
`905
`
`examples in the literature which at least indicate that cyclic
`imide is not involved in the deamidation reaction under
`acidic conditions. For example, insulin (7 ,8), neocarzinosta(cid:173)
`tin (16), and ribonuclease A (56), when incubated in acidic
`media, yield only Asp-containing products from the deami(cid:173)
`dation of Asn residues. Similar results were obtained by
`Meinwald and co-workers (27), where Ac-Asn-Gly-NHMe
`produced only Ac-Asp-Gly-NHMe and the analogous iso(cid:173)
`Asn produced only the iso-Asp-containing peptide after 1
`day in IM HCl.
`It has been postulated that deamidation may play a cen(cid:173)
`tral role as a timer in protein turnover and in aging (21).
`However, for pharmaceutical preparations, the major con(cid:173)
`cern is the change in protein function upon deamidation. In
`a few cases, the deamidation of specific Asn residues has
`been linked to the changes in the protein function, for ex(cid:173)
`ample, deamidation at two Asn-Gly sequences in TIM re(cid:173)
`sulted in subunit dissociation (15). Deamidation at an Asn(cid:173)
`Gly site in a hemoglobin mutant (Hb providence) changed its
`oxygen affinity (57), and deamidation at an Asn-Asp site in
`hGH altered its proteolytic cleavage properties (58). Re(cid:173)
`cently, deamidation was shown as one of the major chemical
`processes responsible for irreversible enzyme inactivation of
`lysozyme (59) and ribonuclease (60) at 100°C. Deamidation
`was also responsible for the decrease in biological activity
`for porcine ACTH (62) and slower rate of refolding after
`deamidation for ribonuclease (63,64).
`With small peptides, the iso-Asp and Asp peptides are
`separable by chromatographic or electrophoretic methods
`(65-67). However, with larger proteins similar methodology
`has not been successful. Chromatofocusing (68) and HPLC
`(20,29) have been used for separating the native protein from
`the product mixture, but these techniques do not separate
`the iso-Asp-peptide from the Asp-peptide. However, there
`are several indirect ways of showing the presence of iso-Asp
`residues in proteins. These include (i) NMR methods to dis(cid:173)
`tinguish Asp and iso-Asp (27 ,54,69); (ii) Leu aminopeptidase
`digestion, since this enzyme will not cleave an iso-Asp pep(cid:173)
`tide bond (67,70); (iii) tryptic peptide mapping and amino
`acid sequencing (71); and (iv) use of PCM, which is known to
`methylate selectively the free a-carboxy group of iso-Asp
`peptides (72). Recently, Johnson and co-workers have
`shown the use of this enzyme as a powerful analytical tool
`for estimating minimum levels of protein deamidation (73).
`In their work, they monitored the increase in methylation for
`aldolase, bovine serum albumin, cytochrome c, lysozyme,
`ovalbumin, ribonuclease A, and TIM upon incubation at pH
`11, finding evidence that iso-Asp is formed upon deamida(cid:173)
`tion.
`
`Oxidation
`
`The side chains of His, Met, Cys, Trp, and Tyr residues
`in proteins are potential oxidation sites. Even atmospheric
`oxygen can oxidize Met residues. Oxidation has been ob(cid:173)
`served in many peptide hormones during their isolation (74-
`77), synthesis (78), and storage (79). Since the thioether
`group of Met is a weak nucleophile and is not protonated at
`low pH, it can be selectively oxidized by certain reagents
`under acidic conditions (80). For example, hydrogen perox(cid:173)
`ide can modify indole, sulfhydryl, disulfide, imidazole, phe-
`
`
`
`906
`
`Manning, Patel, and Borchardt
`
`nol and thioether groups of proteins at neutral or slightly
`alkaline conditions, but under acidic conditions the primary
`reaction is the oxidation of Met to Met sulfoxide (81). In
`addition to hydrogen peroxide, a variety of other reagents
`have been used to oxidize Met to Met sulfoxide. These in(cid:173)
`clude periodate, iodine, dimethylsulfoxide, a dye-sensitized
`photooxidation, chloramine-T, and N-chlorosuccinamide
`(82,83). To oxidize Met to Met sulfone, more drastic condi(cid:173)
`tions and reagents are needed, e.g., 95% performic acid. The
`structures of the oxidation products of Met, i.e. Met sulfox(cid:173)
`ide and Met sulfone, are shown in Fig. 3.
`Oxidation of Met residues to their corresponding sulfox(cid:173)
`ides is associated with loss of biological activity for many
`peptide hormones [e.g., corticotropin (84), a- and
`(3-melanotropins (85), parathyroid hormone (86), gastrin
`(87), calcitonin (88), and corticotropin releasing factor (77)]
`as well as nonhormonal peptides and proteins (81). It has
`been shown that E.coli ribosomal protein Ll2 loses activity
`after oxidation of Met residues to Met sulfoxide and that the
`activity can be restored by incubating the protein with high
`concentrations of ~-mercaptoethanol (89). Restoration of bi(cid:173)
`ological activity was found to coincide with the reduction of
`Met sulfoxide to Met (89). Alpha-1-proteinase inhibitor pro(cid:173)
`tein, which is a major serum inhibitor of elastase activity,
`loses its ability to inactivate elastase when chemically oxi(cid:173)
`dized (90,91). Oxidation by hydrogen peroxide of a single
`Met residue in subtilisin at pH 8.8 occurs concurrently with
`changes in kinetic parameters of the enzyme, although it
`does not abolish enzymatic activity (92). Similar results were
`obtained with a disulfoxide derivative of a-chymotrypsin
`(93,94), and trypsin (95). In many cases, such as parathyroid
`hormone (86), ribonuclease $-peptide (96), ribonuclease
`
`CH3 I s
`I
`CHz
`I
`jHz
`R- c-~-CH -
`a
`0
`
`A' a._) H ..... 2__,02..__
`
`c~
`
`I s-o
`I
`c~
`I
`12
`
`R-C-N-4-0i - R'
`II
`0
`Sulfoxlde
`
`CH3 I
`o-s-o
`
`I r CHa
`
`I
`R- C-~-Q-1- R'
`0
`0
`
`Su If one
`Fig. 3. Mechanism of oxidation of Met-containing peptide under (a)
`mild and (b) strong conditions.
`
`(97), and lysozyme (98), reduction of Met sulfoxide by thiols
`results in the recovery of nearly full biological activity.
`There are also examples where protein functions are not
`affected upon Met oxidation. Active monosulfoxide deriva(cid:173)
`tives of pancreatic ribonuclease (99), a-chymotrypsin (100),
`and Kunitz trypsin inhibitor (101) have been prepared using
`mild hydrogen peroxide treatment at low pH (pH 1 to 3).
`Similarly, EGF (102, 103) and glucagon (104) are biologically
`active when chemically oxidized.
`It is also shown that within a given protein, the reactiv(cid:173)
`ity of Met residues towards oxidation may be different de(cid:173)
`pending upon their position. For example, in hGH, Met170
`was found to be completely resistant to oxidation by hydro(cid:173)
`gen peroxide (105). In addition, it was shown that when bio(cid:173)
`synthetic hGH is chemically oxidized at Met 14
`, it exhibits
`full biological activity and has immunoreactivity identical to
`that of authentic hGH (6). In human chorionic somatomarn(cid:173)
`motropin (hCS), Met64
`, Met16, and Met179 have markedly
`different reaction rates (105). The oxidation of Met64 and/or
`Met 179 markedly reduced both its affinity for lactogenic re(cid:173)
`ceptors and its in vitro biological potency (105).
`Determination of oxidized Met in proteins is generally a
`problem, because during conventional amino acid analysis
`Met sulfoxide is converted to Met during acid hydrolysis.
`Therefore, Met is commonly determined by using its specific
`reactions with alkyl halides (106) or cyanogen bromide (107),
`to which the sulfoxide is resistant. After alkylating the Met
`residues of the peptide, its Met sulfoxide is oxidized with
`performic acid to the acid stable sulfones; the sulfone con(cid:173)
`tent, determined by amino acid analysis, is then used to
`correct the Met estimate obtained by conventional amino
`acid analysis (99). Alternatively, Met containing peptides
`have been separated from peptides containing oxidized Met
`residues by ion-exchange chromatography (108), counter(cid:173)
`current distribution (109), HPLC (103, l IO), or affinity chro(cid:173)
`matography (111). A radioassay for non oxidized Met in pep(cid:173)
`tide hormones based on its specific reaction with
`iodo[2- 14C]acetic acid is also developed (112).
`The thiol group of Cys (RSH) can be oxidized in steps,
`successively, to a sulfenic acid (RSOH), a disulfide (RSSH),
`a sulfinic acid (RS02H), and, finally, a sulfonic acid
`(RS03H), depending upon reaction conditions. The factors
`which influence the rate of oxidation include the tempera(cid:173)
`ture, pH, and buffer medium used, the type of catalyst (e.g.,
`traces of metal ions), and the oxygen tension (113). An im..;
`portant factor is the spatial positioning of the thiol groups in
`the proteins. In those cases where contact between thiol
`groups within the molecule of the protein is hindered, or
`when the protein contains only a single thiol group, intramo(cid:173)
`lecular disulfide bonds are not formed, but sometimes, under
`favorable steric conditions, intermolecular disulfide bonds
`arise, and the protein aggregates (114). Thiol groups are ox(cid:173)
`idized not only when oxidizing agents (e.g., iodine, ferricy(cid:173)
`anide, tetrathionate, 0-iodosobenzoate, and hydrogen per(cid:173)
`oxide) are added, but also "spontaneously," by oxygen from
`the air (autooxidation). The oxidation ofthiol groups by mo(cid:173)
`lecular oxygen takes place at an appreciable rate in the pres(cid:173)
`ence of catalytic quantities of metal ions, such as iron and
`copper ions (115, 116). The speed of oxidation ofthiol groups
`is also greatly influenced by the nature of neighboring
`
`
`
`Stability of Protein Pharmaceuticals
`
`907
`
`groups. This was clearly demonstrated by Barron el al. (117)
`and also by Ovaberger and Ferraro (118). From their findings
`it appears that the rate of oxidation of dithiols is diminished
`on increasing the distance between the thiol groups in the
`molecule and also under the influence of neighboring elec(cid:173)
`tronegative groups such as carboxyl group (i.e., groups that
`raise the pKa of the thiol group). This fact indicates that the
`mercaptide ion is oxidized more easily than the undissoci(cid:173)
`ated thiol group. Thus, it is shown that usually the oxidation
`rate increases with increasing pH (119). At 90°C and pH 8.0,
`a-amylase from Bacillus was shown to undergo irreversible
`thermoinactivation due to air oxidation of the Cys residues
`along with formation of incorrect or ••misfolded" structures
`(120). Inactivation of rabbit muscle glyceraldehyde-
`3-phosphate dehydrogenase by hydrogen peroxide has been
`shown to result from sulfhydryl group modification to
`sulfenic acid (114). Various methods for quantitative deter(cid:173)
`mination of thiol and disulfide groups in proteins are de(cid:173)
`scribed by Torchinskii (121).
`The side chains of His, Tyr, Met, Cys, and Trp residues
`can also be oxidized by visible light in the presence of dyes,
`i.e., via photooxidation. The specificity for the various
`amino acid side chains is particularly determined by pH.
`Oxidation of His is a rapid reaction at neutral pH but is quite
`slow at low pH. At higher pH, Tyr is most reactive (122-
`124). while Trp and Met are the only amino acids readily
`oxidized below pH 4. More information on photooxidation is
`available in a few review articles (125-127). In many cases,
`loss of enzymatic activity following photooxidation has been
`attributed to the destruction of critical His residues. For
`example, the inactivation ofrabbit muscle aldolase (128), pig
`heart aspartate aminotransferase (129, 130), cytochrome c
`(131), renin, and yeast enolase (132) has been attributed to
`photodegradation of His residues.
`
`Proteolysis
`
`It has been established that peptide bonds of Asp resi(cid:173)
`dues are cleaved in dilute acid at a rate at least 100 times
`faster than other peptide bonds (133). Selective hydrolysis is
`usually achieved by heating for 5-18 hr at llOgC in either
`0.03 N HCI or 0.25 N acetic acid (134). The mechanism of
`hydrolysis undoubtedly involves intramolecular catalysis by
`a carboxyl group of the Asp residue. Hydrolysis can take
`place at either the N-terminal and/or C-terminal peptide
`bonds adjacent to the Asp residue. Inglis (135) has described
`the mechanism for such hydrolysis as shown in Fig. 4, where
`cleavage of the N-terminal peptide bond would proceed via
`an intermediate containing a six membered ring rather than
`via a five-membered ring as proposed for C-terminal peptide
`fission. Such peptide bond cleavage can contribute to the
`inactivation of proteins. Significant irreversible thermoinac(cid:173)
`tivation in lysozyme (59) and ribonuclease A (60) at 90-100°C
`and pH 4 was found to be due to peptide bond cleavage at
`Asp-X bonds.
`It is now well established that Asp-Pro peptide bonds
`are particularly labile and are hydrolyzed under conditions
`where other Asp peptide bonds are stable (136). For exam(cid:173)
`ple, when rabbit antibody light chain was subjected to 10%
`acetic acid-pyridine (pH 2.5) in 7 M guanidinium hydrochlo-
`
`0
`cir-~~
`I
`R-C--Nl+-GH---0--NH--fl'
`II
`II
`0
`0
`Aspartyl poplido
`
`/
`
`N·poptide bond fission
`
`J
`0
`a"'A,
`12
`I
`A-4
`o+--o-+u+-A'
`0 ~ ~
`~J-~
`
`CH,r-A-Qi
`I
`NHa-Cl+--0-NH-R'
`II
`0
`Fig. 4. Mechanism of degradation of aspartyl peptides in acidic
`media.
`
`ride (GnHCI) for 24-90 hr. selective cleavage was ob(cid:173)
`served at Asp 109-Pro 110 (137). Piszkiewicz el al. have sug(cid:173)
`gested that the hydrolytic reaction proceeds via intramolec·
`ular catalysis by carboxylate anion displacement of the
`protonated nitrogen of the peptide bond and the rate en(cid:173)
`hancement occurs due to the greater basicity of the Pro ni(cid:173)
`trogen (136). Marcus has compared the !ability of the Asp(cid:173)
`Pro bonds to the lability of other peptide bonds, in particular
`to those of Asp residues (138). In his study, a variety of
`dipeptides was heated at l 10°C in 0.015 M HCl. The con(cid:173)
`centration of amino acid released during the heat treatment
`was determined by amino acid analysis. The results indi(cid:173)
`cated that Asp-Pro bonds were 8- to 20-fold more labile than
`other Asp-X or X-Asp peptide bonds. Other peptide bonds
`that do not involve Asp were found to be stable to hydrolysis
`under these conditions.
`Asp-X peptide bonds also undergo a reversible isomer(cid:173)
`ization between the Asp and the iso-Asp forms via the cyclic
`imide intermediate as shown in Fig. 3 (139.140). This reac·
`tion was first noted by Swallow and Abraham with Asp-Lys
`derived from hydrolyzates of bacitracin (139). Similar inter(cid:173)
`conversion was also shown for Val-Tyr-Pro-Asp-Gly-Ala
`(ACTH22-27), displaying a half-life of 53 days at pH 7.4 and
`37°C (26). Even storage of aqueous solutions of an Asp(cid:173)
`containing peptide can result in the formation of cyclic im.ide
`derivatives (141). The ring closure is particularly fast when
`an Asp residue is followed by Gly in the sequence (142).
`Peptide bonds formed by X-Ser and X-Thr are also labile,
`but require strong acidic conditions (e.g., 11.6 M HCl) (143).
`The mechanism involves N-0 acyl rearrangement (144).
`The time course of hydrolysis of amide peptide bonds
`
`
`
`908
`
`Manning, Patel, and Borchardt
`
`can be monitored by gel chromatography or sodium dode(cid:173)
`cylsulfate polyacrylamide gel electrophoresis (145), and
`quantitatively assessed by gel scanning densitometry (146).
`The identities of the amino acids at the new carboxyl and
`amino termini resulting from peptide chain hydrolysis can be
`determined by hydrazinolysis/HPLC (147) and dansylation/
`TLC ( 148), respectively. Identification of the position of the
`new termini in the sequences of the protein can be learned
`from amino acid analysis by HPLC of the sequential digest
`of the COOH terminus by carboxypeptidases (149) and the
`NH2 terminus by the Edman degradation procedure
`(150,151). Recently, a radioassay was used to study peptide
`bond hydrolysis at neutral pH and room temperature ( 152).
`In this work, a peptide (Phe-Phe-Phe-Gly), radiolabeled with
`14C at the a carbon of the C-terminal residue, was attached
`to resin and the release of radiolabel due to amide bond
`hydrolysis was monitored. The half life for this peptide at
`neutral pH was found to be 7 years (152).
`
`Incorrect Disulfide Formation
`Sulfhydryl groups and disulfide bonds and their interre(cid:173)
`lationships are an important factor affecting the properties of
`the majority of proteins. The interchange of disulfide bonds
`can result in incorrect pairings, leading to an altered three(cid:173)
`dimensional structure and, hence, loss of catalytic activity.
`The reaction mechanism is different in alkaline and acidic
`media (153,154). In neutral and alkaline media the reaction is
`catalyzed by thiols, which, in the form of thiolate ions, carry
`out nucleophilic attack on a sulfur atom of the disulfide (Fig.
`5). Catalytic quantities of thiols can arise by hydrolytic
`cleavage of disulfides to carry out such disulfide exchange.
`For example, lysozyme, when heated at 100°C at neutral pH,
`undergoes beta-elimination of Cys to produce free thiols,
`which cause disulfide interchange (155). Benesch and Ben(cid:173)
`esch have studied the mechanism of disulfide exchange in
`acidic media and they proposed that the exchange takes
`place through a sulfenium cation, which is formed by attack
`of a proton on the disulfide bond ( 156). The sulfenium cation
`carries out an electrophilic displacement on a sulfur atom of
`the disulfide (Fig. 6). Addition of thiols can inhibit such ex(cid:173)
`change by scavenging the sulfenium cations.
`Disulfide exchange can be prevented ifthiol scavengers,
`such as p-mercuribenzoate, N-ethylmaleimide, or copper
`ion, which catalyzes the air oxidation of thiols, are present
`(157 ,158). Zale and Klibanov recently reported that the irre(cid:173)
`versible loss of activity of ribonuclease A at 90°C and pH 8
`was significantly lower when incubated in the presence of
`above-mentioned reagents (60). Proteins with scrambled di(cid:173)
`sulfide bonds can be rearranged to yield native, catalytically
`active material by incubating the protein with small amounts
`of thiols, such as mercaptoethano) or Cys (159, 160).
`
`Racemization
`All amino acid residues except Gly are chiral at the car-
`
`R'S+ + R"S-SR"
`
`R"S+ + R'S-SR'
`
`R'S·SR" + A"S+
`
`A"S-SR' + R'S+
`
`Fig. 6. Mechanism of disulfide exchange in acidic media.
`
`bon bearing the side chain and are subject to base catalyzed
`racemization. Racemization is generally considered to pro(cid:173)
`ceed throu