(Received for publication, October 23, 1996, and in revised form, January 9, 1997)
From the Department of Pharmaceutical Chemistry, The
University of Kansas, Lawrence, Kansas 66047 and the
§ Pharmaceutical R&D, Genentech, Inc.,
South San Francisco, California 94080
Metal-catalyzed oxidation of proteins represents
an important pathway of post-translational modification. We utilized
human growth hormone (hGH), a protein with a well defined metal-binding site, to study the detailed mechanism of metal-catalyzed oxidation by
ascorbate/Cu(II)/O2. Particularly His18
and His21 within the metal-binding site were oxidized,
predominantly to 2-oxo-His with the incorporated oxygen originating
from molecular oxygen, based on amino acid analysis, tryptic mapping,
mass spectrometry, isotopic labeling, and 1H NMR. The
anaerobic reduction of a hGH/Cu(II) mixture by ascorbate generated a
hGH-Cu(I) complex with NMR spectral features different from those of
native hGH and hGH/Cu(II). The anaerobic reaction of this hGH-Cu(I)
complex with hydrogen peroxide resulted in the oxidation of
His18 and His21, suggesting that a fraction of
Cu(I) was bound at the metal-binding site of hGH. Site-specific
oxidation of hGH required an intact metal-binding site and could
largely (about 80%) be inhibited by the presence of 28% (v/v)
1-propanol which appears (i) to perturb the metal-binding site and (ii)
to interact with a reactive oxygen species formed at the perturbed
metal-binding site. The inhibition by
1-propanol-d7
(CD3CD2CD2OH) was significantly
lower than that by 1-propanol-h7 with
[residual hGH]1-propanol-h7/[residual hGH]1-propanol-d7 = 1.95 at 30% (v/v) 1-propanol,
reflecting a kinetic isotope effect close to that for the reaction of a
hydroxyl radical with C
-H/D bonds of methanol,
suggesting the involvement of a hydroxyl radical-like species in the
oxidation of His.
In physiological compartments, provided with suitable reducing agents, transition metals are able to catalyze the activation of O2 to various reactive oxygen species (ROS)1 which readily attack biomolecules. The metal-catalyzed oxidation (MCO) of proteins plays an important role during oxidative stress and aging where modified proteins may accumulate in tissue (1-9). In addition, MCO also presents a stability problem for the biotechnological manufacturing of proteins (10, 11).
A unique feature often observed during MCO of proteins is that only a few amino acid residues within a certain domain of the given protein are modified (2, 12). Such site specificity is attributed to the generation of ROS at specific metal-binding sites, where the highly reactive ROS attack labile functional groups nearby rather than diffuse into the bulk medium. It has been shown that most of the enzymes labile to MCO require metals for activity (13). The oxidation of these enzymes is less sensitive to the inhibition by exogenous ROS scavengers than would be expected on the basis of known rate constants for the reactions of specific ROS with these scavengers. However, one problem associated with all these studies is that neither the oxidizing species nor the intermediary protein-transition metal complexes formed during MCO of proteins have been well characterized. This is most pertinent to copper-catalyzed oxidation (i.e. to the frequently used ascorbate/Cu(II)/O2 system) involving the Cu(II)/Cu(I) redox couple (2). Here, Cu(II) will most probably require a different geometry and different ligands within the protein ligand sphere than Cu(I). An initial reduction of Cu(II), bound to a metal-binding site, to Cu(I) may change the geometry of the protein-metal complex, and possibly even release Cu(I) from the metal-binding site. On the other hand, the metal may be retained when (i) the protein is flexible enough to adapt to the new geometrical requirements of the reduced metal, or (ii) re-oxidation of the metal by molecular oxygen or reduction products of molecular oxygen such as superoxide or hydrogen peroxide occurs faster than the release of the reduced metal from the metal-binding site.
In a recent example for electron transfer coupled ligand dynamics in the model complex Cu(I/II)(TTCN)2, we could show that oxidation of tetrahedral [Cu(I)(TTCN)2]+ potentially led to the loss of a ligand to yield [Cu(II)(TTCN)(H2O)3]2+ before geometrical reorganization around the Cu(II) atom allowed the addition of TTCN to give the final product, octahedral [Cu(II)(TTCN)2]2+.2 Analogous mechanisms may lead to kinetic complexities during protein oxidation which may be reflected in oxidation yields and kinetics as well as the nature of the oxidation products.
One amino acid particularly affected by MCO is histidine (His) (15-17). There is as yet no detailed mechanism available for His oxidation in proteins, except a crystallographic study which showed the feasibility of metal-catalyzed processes within the metal-binding domain of glutamine synthetase (18). However, such mechanisms and reaction stoichiometries are important to recognize, particularly in light of the fact that His oxidation products such as 2-oxo-His have been proposed as general markers for oxidative stress in vivo (19).
In this work, we report a detailed mechanistic investigation of the
ascorbate/Cu(II)/O2-induced oxidation of His in a selected model protein, human growth hormone (hGH), with the particular objective to identify the nature of the oxidizing species and the
structural requirements for MCO of hGH. Ascorbate/Cu(II)/O2 has not only been used extensively as a non-enzymatic model oxidizing system but is of physiological relevance to conditions of oxidative stress. hGH was chosen because of its well characterized metal-binding site which shows high affinity for divalent metal ions, such as Zn(II)
and Co(II) (20). This site consists of His18 and
His21 from helix I, and Glu174 from helix IV. The
primary sequence of hGH is displayed in Fig. 1 (21).
Recombinant hGH (lyophilized form) and its
synthetic tryptic fragment T3 (Ala-His-Arg) were provided by Genentech,
Inc. (South San Francisco, CA). The protein was exchanged into a
desired buffer by ultrafiltration using Microcon-10 microconcentrators
from Amicon, Inc. (Beverly, MA). Typically 10 mg of hGH were dissolved
in 500 µl of 20 mM phosphate buffer, pH 7.4, the solution
transferred into the microconcentrator and centrifuged for 20 min at
12,000 rpm in a tabletop centrifuge. Subsequently, the protein was
washed three times with 250 µl of 20 mM phosphate buffer,
pH 7.4. The protein was finally taken up in a total volume of 4.5 ml of
20 mM phosphate buffer, pH 7.4, and the concentration of
hGH in the stock solution determined by UV absorption at 277 nm where
A2770.1% = 0.82 cm1 (22), [MWhGH = 22,125] (21). The
Met14 sulfoxide derivative T2ox of the tryptic fragment T2 was
obtained synthetically: first, an amount of about 1.5 nmol of authentic T2 was obtained by tryptic digestion of hGH (conditions see below), purification by reversed-phase HPLC (conditions see below), and lyophilization. The fragment was redissolved in 100 µl of neutral water and reacted for 12 h with 10 mM hydrogen
peroxide. The only product of this reaction was T2ox which was purified
by reversed-phase HPLC and characterized by MALDI-TOF mass
spectrometry. Sequencing grade trypsin was obtained from Promega
(Madison, WI); 1-propanol-d7 (CD3CD2CD2OH) and
18O-labeled water (95-98% atom pure 18O) were
from Cambridge Isotope Laboratories, Inc. (Andover, MA). All other
reagents were of the highest grade commercially available.
Unless specified otherwise, the oxidation of hGH by the ascorbate/Cu(II)/O2 system was carried out under the following conditions (referred to as "standard conditions"): 200-µl solutions containing 18 µM hGH, 10 µM CuCl2, 100 µM ascorbate, and 10 mM sodium phosphate buffer, pH 7.4, were incubated at 25 °C in 2-ml vials. In this way sufficient oxygen was available in the headspace of the vial to replenish oxygen in the reaction solution consumed through the oxidation of ascorbate. The reagents were sequentially added in the following order: water, hGH (in phosphate buffer), CuCl2, and ascorbate. The reaction was started by the addition of ascorbate, although switching the order of CuCl2 and ascorbate did not affect the reaction kinetics or final yields (data not shown). All solutions were made with doubly distilled deionized water. The phosphate buffer stock solution was treated with Chelex 100 resin (Bio-Rad; 5 g/100 ml) to minimize metal contamination. Stock solutions of CuCl2 and ascorbate were freshly prepared prior to the reactions.
Atmospheric oxygen was excluded in some reactions. Since purging inert gas through the protein-containing solutions and even freeze-thaw cycles would cause foaming and denaturation of the protein, a special piece of glassware was designed to saturate the protein reaction mixture with N2. The glassware of an H-shape was made of two small top-end-open glass tubes joined in the middle. The reaction mixture was placed in one side of the device and H2O in the other (solution surfaces below the joint). The two openings were then fitted with rubber septa, and two hypodermic needles were used as the N2 gas inlet (needle immersed in H2O) and outlet (needle above the reaction solution), respectively. Before the inlet, N2 gas was purified from residual oxygen contamination by an OxiClear disposable gas purifier (LabClear, Oakland, CA). This whole setting enabled the efficient removal of O2 without protein denaturation or solvent evaporation. Other reagents, purged with N2 separately, were added through the rubber septa using a gas tight syringe.
Tryptic digestion of hGH samples was performed at an initial dose of trypsin: hGH = 1:50 (w/w) in 10 mM phosphate buffer, pH 7.4, at 37 °C. A second equivalent dose was added after 2 h, and the samples were incubated for another 4 h. The digest was then quenched by cooling to 4 °C.
Chromatographic AnalysisIntact hGH was monitored by weak anion exchange chromatography (AXC), reversed-phase HPLC (RP-HPLC), and size exclusion chromatography with UV detection at 214, 230, or 278 nm. Tryptic maps were analyzed by RP-HPLC and cation exchange chromatography (CXC). Table I summarizes the detailed information for the employed modes of chromatography.
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The quantitative analysis of ascorbate was performed by capillary electrophoresis equipped with an electrochemical detector. The capillary electrophoresis system employed a 50 µm x 70-cm fused silica capillary (Polymicrotechnologies; Phoenix, AZ), operating at 30 kV and 0.5 nA with 10 mM MES, pH 5.5, buffer as the mobile phase. The power supply was from Spellman High Voltage Corp. (Plainview, NY), and electrochemical detection was accomplished with an amperometric detector (model LC-4B; Bioanalytical Systems; West Lafayette, IN) employing a 33-µm carbon fiber electrode operating at 800 mV (versus Ag/AgCl). Prior to capillary electrophoresis with electrochemical detection analysis, the reaction samples were quenched by EDTA and filtered by Microcon-10 micro-concentrators to remove protein components.
NMR AnalysisThe 500 MHz 1H NMR spectra were recorded on a Bruker AM-500 instrument. Typically 5 mg of hGH were exchanged into 400-800 µl of 10 mM sodium phosphate buffer in D2O, pD 7.4, by ultrafiltration using Microcon-10 microconcentrators (see "Materials") (the introduction of protons by 10 mM sodium phosphate into D2O can be considered negligible). When 1-propanol-d7 was used as a co-solvent (40%, v/v), it was added after the ultrafiltration. Before the addition of 1-propanol-d7, the pD was measured to be 7.4. The final concentrations of hGH in all NMR samples were 0.2-0.4 mM. The samples were then kept overnight at room temperature to ensure the exchange of amide protons with D2O before NMR analysis.
Mass SpectrometryMolecular weights of the purified tryptic
fragments of hGH were analyzed by electrospray ionization (ESI) and/or
matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)
mass spectrometry (MS). ESI-MS experiments were performed on an
Autospec-Q tandem hybrid mass spectrometer (VG Analytical Ltd.,
Manchester, United Kingdom) equipped with an OPUS data system.
MALDI-TOF mass spectra were obtained on a Hewlett Packard model G2025A
which exhibited an accuracy of typically ± 1 atomic mass unit for
peptides with molecular masses below 3 kDa. The matrices for MALDI-TOF
measurements (supplied by Hewlett Packard, Atlanta, GA) were
3,5-dimethoxy-4-hydroxycinnamic acid for peptides of mass >1 kDa and
-cyano-4-hydroxycinnamic acid for peptides of mass <1 kDa.
A Shimadzu spectrofluorometer (RF 5000U) was used to monitor the fluorescent behavior of the tryptophan residue after the oxidative modification of hGH (standard conditions). The excitation wavelength was set at 298 nm, and the emission spectrum was recorded between 310 and 400 nm. The presence of dityrosine, one potential oxidation product of tyrosine, was also examined by fluorescence spectroscopy with an excitation wavelength at 325 nm and emission spectra recorded between 400 and 500 nm (23). Circular dichroism (CD) measurements of hGH were performed on an AVIV model 60 DS spectrometer (Lakewood, NJ) essentially according to a published procedure (21).
Amino Acid AnalysisFor the preparation of oxidized hGH for amino acid analysis, hGH (19 µM) was oxidized in 1 ml of solution (standard conditions). After a reaction time of 100 min, 100 µl of 0.1 mM EDTA were added, maintaining pH 7.4. The solution was divided into two aliquots of 550 µl, both of which were concentrated by centrifugation in a Microcon-10 microconcentrator and washed twice with 250 µl of 20 mM sodium phosphate buffer, pH 7.4. Both protein samples were then redissolved into 250 µl of 20 mM sodium phosphate buffer, pH 7.4, and two 100-µl aliquots of each sample lyophilized in small vials. Assuming an approximately 30% loss of material as a result of centrifugation in the microconcentrators, each aliquot contains about 3.3 nmol of protein. The protein samples were hydrolyzed with 6 M HCl at 110 °C for 20 h. Subsequent amino acid analysis was performed by Commonwealth Biotechnologies, Inc., Richmond, VA.
Product Characterization for the Oxidation of hGH by Ascorbate/Cu(II)/O2
Chemical CharacterizationDuring the oxidation of 18 µM hGH by 10 µM CuCl2 and 100 µM ascorbate in air saturated 10 mM phosphate
buffer at pH 7.4, AXC, pH 5.8, but not RP-HPLC revealed the rapid
degradation of the hGH main peak paralleled by the appearance of two
new more acidic peaks, implying the modification of some basic amino
acid residues. Fig. 2 superimposes representative AXC
chromatograms recorded immediately after the start (2 min) and at the
end of a reaction (i.e. after complete consumption of
ascorbate). The two major product peaks are indicated as peak 1 and
peak 2. Each peak may contain more than one degradation product as
suggested by the peak shoulders. The time profiles for their formation
as well as for the consumption of hGH are shown in Fig.
3.
Prior to the characterization of peak 1 and peak 2 in Fig. 2, the
chemical modifications of a complete reaction mixture were characterized by tryptic mapping (see Fig. 1 for the primary sequence of hGH and the assignment of the tryptic fragments). Most of the tryptic fragments are well separated by RP-HPLC; complimentary tryptic
mapping by CXC was used to monitor the T3 fragment, which is highly
positively charged and elutes closely to the solvent front during the
RP-HPLC analysis. Representative tryptic maps of native and oxidized
hGH are displayed in Fig. 4 for RP-HPLC and Fig.
5 for CXC. Peak assignment in the RP-HPLC chromatogram was achieved by fraction collection and off-line analysis by ESI or
MALDI-TOF MS. The identities of the respective peaks together with the
theoretical and measured molecular masses are displayed in Table
II. The retention behavior of the T3 fragment during CXC
was confirmed by comparison to a synthetic standard.
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A comparison of the chromatograms in Figs. 4 and 5 reveals that a number of fragments were modified by the ascorbate/Cu(II)/O2 system, but T3 and T4 constituted the predominant targets. Table II summarizes the loss of each fragment and the yields of oxidation products. Two oxidation resistant fragments, T12 and T13, were chosen as the internal standards. Each oxidation product was quantified by comparing its peak area with the original peak area of its parent fragment in the unmodified protein, assuming similar response factors.
Tryptic mapping by RP-HPLC in conjunction with off-line ESI-MS revealed several product peaks originating from T4 (labeled T4ox1 to T4ox3) with the total area of these peaks accounting for as much as 60% of the loss of T4. The major product (T4ox3) had a molecular weight corresponding to MWT4 + 16, implying the incorporation of an oxygen atom into T4. One minor product had MWT4 + 15 (T4ox1) which, considering the accuracy of our MALDI-TOF MS measurements of ± 1 atomic mass unit, might in fact correspond to MWT4 + 16. One additional minor product (T4ox2) corresponded to MWT4 + 50.
Although T3 was well separated from the other fragments by CXC (which enabled the quantification of its loss), no new product peak was observed, suggesting that the products were less positively charged and co-eluted with the other earlier eluting fragments (Fig. 5). By RP-HPLC (Fig. 4), T3 (peak number 1) was barely resolved from the solvent front even with initially low concentrations of acetonitrile. The MALDI-TOF mass spectrum of peak number 1 suggested that it contained T3 as an exclusive compound. During the oxidation reaction, the loss of peak number 1 was accompanied by the emergence of a later eluting peak (peak number 2 in Fig. 4). MALDI-TOF MS analysis of peak number 2 revealed the presence of one major component with MWT3 + 16 and two minor components with MWT3 + 38 and MWT3 + 54. Peak number 2 is thus assigned as T3ox, the product of oxygen incorporation into T3. The two minor components likely represent the potassium adducts of T3 (MWT3 + 38) and T3ox (MWT3 + 54), respectively. Based on the peak areas of peak number 1 and peak number 2 in Fig. 4 it appears that T3 is nearly quantitatively converted into T3ox.
In addition to oxidation products from T3 and T4, RP-HPLC also resolved a product peak, number 11, which had identical retention time and molecular weight as the synthetic standard of the Met14 sulfoxide variant of T2. This conversion is nearly quantitative, i.e. 10.1% loss of T2 compares to 9.7% gain of T2ox.
The fragments T17-T18-T19 and T18-T19 suffered an approximately 10% loss which is, however, not accompanied by any detectable major product. The potential oxidation sites on these fragments are Met170 and/or Glu174. The Glu174 residue is the third component of the metal-binding site in addition to the two His residues (20). Met170, although buried in the hydrophobic core, is also located close to the metal-binding site according to the crystal structure of hGH (24). Our results (see below) will provide evidence for the formation of hydroxyl radical-like species at the metal-binding site of the protein. Both Glu (25) and Met (26-30) are susceptible to the attack by hydroxyl radicals, yielding a variety of non-selective reaction products. We note that Met170 could theoretically be oxidized to Met sulfoxide by two-electron oxidants such as hydrogen peroxide, but no corresponding sulfoxide variants of T18-T19 or T17-T18-T19 were experimentally observed. The observed loss of T10c2 (chymotryptic cleavage of T10; Ser100-Lys115) is considered an experimental artifact since both fragments T10c1 and T10 did not show any loss induced by metal-catalyzed oxidation.
The two major oxidation peaks (peaks 1 and 2) of hGH, resolved by AXC (Fig. 2), were purified for individual analysis by tryptic mapping. Peak 2 contained all the original fragments except T3, suggesting that this peak represented a rather homogeneous fraction of a hGH variant only modified at T3. Peak 1 suffered significant loss of T4 (about 65%) in addition to a minor loss of T3 (about 13%).
It appears that the oxidation of hGH by the ascorbate/Cu(II)/O2 system is rather selective to amino acid residues located on fragments T3 and T4. The behavior of the oxidized protein and peptide species during AXC and CXC suggests that the modifications occur at basic amino acid residues. Both T3 and T4 contain histidine residues, His18 and His21, respectively. Theoretically those are susceptible to oxidation/hydroxylation to yield 2-oxo-histidine (2-oxo-His), characterized by a gain of 16 atomic mass units in molecular weight (19, 31-33). Amino acid analysis of oxidized hGH revealed that only His was modified to a significant extent with a loss of 1.4 ± 0.2 His residues per protein, corresponding to 47 ± 7% of the total His residues. This is consistent with the observed loss of T3 and T4. Notably His18 and His21 are components of the metal-binding site of hGH (20). A third His residue, His151, is located next to the known deamidation site of the protein and solvent accessible (21). However, the corresponding tryptic fragment, T15, was not significantly modified by ascorbate/Cu(II)/O2 (Fig. 4 and Table II).
We employed 1H NMR to confirm the degradation of His by
virtue of the characteristic chemical shifts of the imidazole C-2
protons (34, 35). As shown in Fig. 6a, the
imidazole C-2 protons of the three His residues in native hGH gave
distinct chemical shifts at 7.5-8.2 ppm at pD 7.4, comparable to the
published values (34, 35). Characteristic resonances were also observed
at = 6.47 ppm (resonance II), representing Tyr28 or
Tyr160 (35), and at
= 6.1 ppm (resonance I),
representing a presently non-assigned Tyr and/or Phe residue (34, 35).
The oxidative modification by the ascorbate/Cu(II)/O2
system led to a significant decrease of signal intensity only for the
C-2 protons of His18 and His21, but not for
that of His151 (Fig. 6b). Thus, the NMR results
corroborate the findings from both the amino acid analysis and tryptic
mapping. We note that the oxidation of hGH also yields a new weak
resonance at 6.2 ppm. Earlier we had shown that 2-oxo-His within the
peptide Gly-(2-oxo-His)-Gly-Met-Gly-Gly-Gly displays a resonance at
around 6.3 ppm, assigned to the C-4 proton of the 2-oxo-His residue
(36).
Thus, on the basis of NMR, amino acid analysis, tryptic mapping, and mass spectrometry, we conclude that 2-oxo-His at positions 18 and 21 constitutes a major oxidation product of hGH modified by ascorbate/Cu(II)/O2. Minor yields of other hydroxylated/oxidized products are observed, such as T4ox1 (MWT4 + 15) and T4ox2 (MWT4 + 50), where T4ox2 most likely corresponds to 4,5-dihydroxy-2-oxo-His (37), but its low yield precluded further characterization by NMR.
Structural CharacterizationOxidation of hGH by the
ascorbate/Cu(II)/O2 system did not significantly affect the
higher order structure of hGH. Nondenaturing size-exclusion
chromatography revealed about 23% loss of the hGH monomer paralleled
by some dimer formation, while denaturing size-exclusion chromatography
indicated only a 4% loss in monomer content. hGH has a single
tryptophan in helix II, Trp86, which constrains the
distance between helices II and IV through a hydrogen bond with
Asp169 on helix IV (24). The fluorescence property of
Trp86 is a good indicator of the integrity of the protein
tertiary structure (38, 39). With excitation at 298 nm, the emission spectrum of hGH was recorded between 310 and 400 nm. No significant change of the spectrum was observed for oxidized hGH. Fluorescence spectroscopic analysis also revealed no formation of dityrosine during
the oxidation (ex = 325 nm,
em = 400-500
nm). Far-UV circular dichroism measurements showed only minimal loss of
overall
-helical content of the oxidized hGH. In addition, the NMR
spectra of the native and oxidized hGH (Fig. 6, a and
b) were largely identical besides the changes of the
intensity of the histidine-related signals, confirming that the
integrity of the higher order structure of hGH was maintained after the
oxidation by ascorbate/Cu(II)/O2.
The Source of Oxygen in 2-Oxo-His
Although 2-oxo-His has been documented as a product from
metal-catalyzed oxidation of His (19, 31-33, 36), its mechanism of
formation as well as the source of oxygen has never been characterized in detail. In particular the source of oxygen is critical:
incorporation of oxygen from the solvent water would indicate an
intermediary imidazole cation or radical cation, generated by electron
transfer to any type of oxidant, followed by reaction with aqueous
OH; in contrast, the incorporation of oxygen from
molecular oxygen of air would indicate the involvement of
1O2, HO· (formed via three consecutive
reduction steps of O2), or possibly peroxyl radicals formed
by the addition of O2 to any type of carbon-centered radical of the His residue. Therefore, our oxidation experiment of hGH
by ascorbate/Cu(II)/16O2 was carried out in
18O-labeled water, H218O (95-98%
atom pure 18O), followed by tryptic digest in the same
solvent. By RP-HPLC separation, four fractions containing T3, T4, and
their 2-oxo-His variants (T3ox and T4ox3) were collected
and lyophilized immediately. MALDI-TOF and ESI-MS analysis revealed a
gain of 4 atomic mass units for purified fractions of T3 and T4, and
the fractions of T3ox and T4ox3 exhibited molecular weights
of MWT3 + 20 and MWT4 + 20, respectively. The
mass differences between the products and their parent peptides were
still 16 atomic mass units, indicating that the oxygen incorporation
into 2-oxo-His resulted from an interaction with a species derived from
molecular oxygen. In control experiments we verified that there was no
oxygen exchange between 2-oxo-His and the solvent during HPLC
purification and subsequent lyophilization. This was done by incubation
of 2-oxo-His(16O) of T3 and T4 in
H218O/acetonitrile mixtures of compositions and
pH identical to the eluting mobile phase during RP-HPLC analysis. The
observed increase in molecular weight of 4 atomic mass units for all
fragments obtained by tryptic digest in H218O
versus H216O is likely the result of
oxygen exchange between solvent and a tetrahedral intermediate formed
during tryptic hydrolysis.
The Importance of an Intact Metal-binding Site and Site Specificity of Oxidation
The importance of an intact metal-binding site and site specificity for the oxidation of His18 and His21 was further demonstrated by experiments with (i) individual tryptic fragments containing only His18 (T3) or His21 (T4), and (ii) with different concentrations of Cu(II) specifically selected to obtain conditions of [Cu(II)] < [hGH] and [Cu(II)] > [hGH]. Both fragments T3 (synthetic) and T4 (purified by RP-HPLC after tryptic digestion of native hGH) were resistant to oxidation under conditions identical to those applied to hGH (i.e. 18 µM peptide, 10 µM Cu(II), 100 µM ascorbate in air-saturated 10 mM phosphate buffer, pH 7.4).
At constant concentrations of ascorbate and hGH, the oxidation yields
were sensitive to different concentrations of Cu(II), [Cu(II)],
summarized in Table III. An increase of [Cu(II)] from 2 to 10 µM, i.e. under conditions of
[Cu(II)] < [hGH], resulted in a significant increase of the
consumption of hGH, expressed as hGH, paralleled by an
increase of the yields of oxidation products, quantified as peaks 1 and
2 by AXC (see Fig. 2). However, conditions of [Cu(II)] > [hGH]
(i.e. at [Cu(II)] = 25 and 50 µM) resulted
in a slight decrease of hGH consumption, paralleled by a slight
decrease of the product peaks 1 and 2, as compared with [Cu(II)] = 10 µM. The inspection of the respective ratios peak 1/
hGH and peak 2/
hGH reveals a
bell-shaped characteristic for peak 1/
hGH with low
values at 2 and 50 µM Cu(II), whereas peak 2/
hGH shows a steady decrease with increasing
[Cu(II)]. These differences are likely the result of different
kinetics for the oxidation of His18, represented by peak 2, and His21, the major oxidized amino acid of peak 1. It
appears that His18 represents an early target of hGH,
followed by His21 and, eventually, some other minor targets
such as Met14.
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The oxidation of hGH (18 µM hGH, 10 µM Cu(II), 100 µM ascorbate in air-saturated 10 mM phosphate buffer, pH 7.4) was completely inhibited by the addition of 15 µM EDTA. However, Zn(II) (added as ZnCl2) was unable to inhibit the oxidation of hGH until the ratio of [Zn(II)]:[Cu(II)] was higher than 80:1. This result suggests a strong binding of Cu(II) to the metal-binding site of hGH and is particularly interesting with regard to the fact that Zn(II) regulates storage and biological activity of hGH under physiological conditions (20, 40).
Characterization of Hydrogen Peroxide and Cu(I) as Important Intermediates
Hydrogen PeroxideThe oxidation of hGH was inhibited by >50 units/ml native catalase (18 µM hGH, 10 µM Cu(II), 100 µM ascorbate in air-saturated 10 mM phosphate buffer, pH 7.4) but not by heat-inactivated catalase, indicating that freely diffusable hydrogen peroxide plays an important role during the reaction. Cu,Zn-superoxide dismutase (at 200 units/ml) had no effect on the final yields of hGH oxidation but slowed down the reaction kinetics.
Cu(I)Three vials (I-III) with each containing an
N2-saturated solution of 18 µM hGH and 15 µM Cu(II) in 10 mM phosphate buffer, pH 7.4, were incubated under N2 for 20 min with 7.5 µM ascorbate, i.e. with a ratio of
[ascorbate]:[Cu(II)] = 1:2 to promote a stoichiometric formation of
Cu(I). Subsequently, to vial I a final concentration of 100 µM EDTA (separately saturated with N2) was
added under N2, vial II was opened and exposed to air, and
to vial III a final concentration of 100 µM hydrogen
peroxide (H2O2) (separately saturated with
N2) was added under N2. Analysis of the
reaction mixture of vial I by AXC revealed that hGH had not been
oxidized during the 20-min incubation (before the addition of EDTA),
whereas analysis by capillary electrophoresis with electrochemical
detection revealed that the reduced form of ascorbate had been
completely consumed. Analysis of the reaction mixtures of vials II and
III revealed that hGH had been oxidized, with higher yields in vial
III, where the product stoichiometries were identical to those obtained
during the oxidation of hGH by the standard system in air-saturated
solutions, i.e. showed predominant oxidation of T3 and T4.
In a separate experiment we added a final concentration of 100 µM H2O2 aerobically to a solution
containing 18 µM hGH, 10 µM Cu(II), and 10 mM phosphate buffer, pH 7.4. Here, no oxidation of hGH was
observed over a time of 150 min. These results imply the anaerobic
reduction of Cu(II) to Cu(I) via net reactions 1 and 2, where
AH, A
, and A denote ascorbate, the ascorbyl
radical anion, and dehydroascorbic acid, respectively.
![]() |
(Eq. 1) |
![]() |
(Eq. 2) |
![]() |
(Eq. 3) |
The Effect of 1-Propanol
It was reported that 1-propanol induces a small change of the
tertiary structure of hGH to a molten globular state (39). When we
exposed hGH to the ascorbate/Cu(II)/O2 system in mixtures of (i) methanol and water, and (ii) 1-propanol and water, there was no
effect of methanol up to a content of 40% (v/v). However, the extent
of oxidation followed a sigmoidal behavior with no significant change
between 0 and 20% 1-propanol (v/v), but about 80% inhibition at 28%
1-propanol (Fig. 7).
This significant change in inhibition within a narrow range of
1-propanol content suggests the involvement of a structural transition
of the protein. Indeed, the sigmoidal inhibition curve of hGH oxidation
approximately paralleled the variation of protein tertiary structure
characterized by fluorescence spectroscopy (39). On the other hand, the
lack of any effect of comparable concentrations of methanol shows that
it was not merely the presence of an alcoholic functional group in the
solvent which caused the observed protection. Fig. 6f shows
the 1H NMR spectrum of native hGH in D2O in the
presence of 40% 1-propanol-d7 (CD3CD2CD2OH) (v/v; pD = 7.4, measured before the addition of 1-propanol-d7).
The three resonances in the range = 7.6-7.8 ppm were assigned to
the three His residues in the following way. A sample of hGH was
exposed to ascorbate/Cu(II)/O2 in the absence of 1-propanol
to oxidize His18 and His21. Oxidized hGH is
characterized through the lack of the characteristic resonances of
His18 and His21 in the NMR spectrum (Fig.
6b). Subsequently, the sample containing oxidized hGH was
lyophilized and redissolved in aqueous (D2O) solution, pD
7.4, containing 40% (v/v) 1-propanol-d7. The
NMR spectrum of this sample was identical to Fig. 6f except
that it did not show the two resonances in the region
= 7.60-7.65
ppm. Therefore, in Fig. 6f the two resonances around
= 7.60-7.65 ppm can be assigned to His18 and
His21 in some order, whereas the resonance around 7.8 ppm
can be assigned to His151, as labeled. In particular the
resonance of His18 is significantly shifted in the presence
of 40% (v/v) 1-propanol-d7 as compared with
native hGH in the absence of 1-propanol. A nearly similar upfield shift
(to
= 7.64 ppm) of the His18 resonance is achieved in
the absence of 1-propanol by an increase of the pD to pD 8.5, shown in
the NMR spectrum in Fig. 6g where the broad peak around
= 7.65 ppm contains the resonances of both His18 and
His151 (35). At pD 8.5 all His residues of hGH present
their imidazole side chains in the neutral form (35) and the salt
bridge between His18 of helix I and Asp171 of
helix IV is lost, resulting in a change of the relative positions of
helix I and helix IV (35). By analogy this suggests that 1-propanol
perturbs the interactions of helix I and helix IV through the weakening
of hydrophobic interactions and, therefore, perturbs the metal-binding
site. This hypothesis derives further support from the 1H
NMR spectra of Fig. 6, a, f, and g, in the
6.0-6.6 ppm region. Native hGH shows resonances I and II in the
absence of 1-propanol at pD 7.4 (Fig. 6a) and pD 8.5 (Fig.
6g), whereas both resonances are absent in the presence of
40% 1-propanol at pD 7.4 (Fig. 6f). Both Tyr28
and Tyr160, represented by resonance II, are located in the
contact area of helices I and IV, respectively (35). Resonance I
represents a presently unidentified Tyr or Phe residue (34, 35) several of which are located in the contact area of helices I and IV such as,
e.g. Phe25, Phe31, and
Phe166 (35).
At pH 8.5 in the absence of 1-propanol (18 µM hGH, 10 µM Cu(II), 100 µM ascorbate in air-saturated 10 mM phosphate buffer), hGH was oxidized to an extent comparable to that at pH 7.4 in the absence of 1-propanol (data not shown). Thus, the mere lack of a salt bridge between His18 and Asp171 (see above) is not sufficient for an inhibition of oxidation, suggesting that the protection exerted by 1-propanol requires (i) additional structural modifications of the metal-binding site and/or (ii) other additional parameters. If 1-propanol perturbs the metal-binding site by weakening hydrophobic interactions between helix I and helix IV, 1-propanol has to insert between the helices where it may locate close to the perturbed metal-binding site. A close proximity would enable 1-propanol to scavenge ROS generated at the metal. To test this possibility we performed the oxidation of hGH (standard conditions) in the additional presence of (i) 30% (v/v) 1-propanol-h7 or (ii) 30% (v/v) 1-propanol-d7 (CD3CD2CD2OH). A content of 30% (v/v) 1-propanol-h7 exerted maximum protection with about 77 ± 7% native hGH left after oxidation (Fig. 7). In contrast, the inhibition by 30% (v/v) 1-propanol-d7 was significantly less efficient, with only 39 ± 5% native hGH left after oxidation (data not shown). This product isotope effect, expressed as [residual hGH]1-propanol-h7/[residual hGH]1-propanol-d7 = 1.95, likely reflects a kinetic isotope effect with 1-propanol-d7 being a less efficient scavenger of ROS. Thus, part of the inhibition by 1-propanol may be due to the scavenging of ROS, generated within a perturbed metal-binding site, and the product isotope effect may serve as a basis for mechanistic considerations.
The exposure of hGH to
ascorbate/Cu(II)/O2 leads to a rather selective oxidation
of His18 and His21 which are part of the
metal-binding site of hGH. Hydrogen peroxide represents an important
intermediate which appears to exist freely diffusable in solution based
on the fact that added catalase inhibited the oxidation of hGH.
Mechanistically, the formation of H2O2 can be
formulated by Equations 4-8 where the ligand system Lx can
represent any potential ligand in our experimental system,
i.e. ascorbate, phosphate, protein, or water, or
combinations of these. Evidence for Equations 5-8 and the existence of
peroxodicopper (II) complexes (trans-µ or
µ-2:
2), here represented as
LxCu(II)(O22
)Cu(II)Lx,
has been provided (43).
![]() |
(Eq. 4) |
![]() |
(Eq. 5) |
![]() |
(Eq. 6) |
![]() |
(Eq. 7) |
![]() |
(Eq. 8) |
![]() |
(Eq. 9) |
![]() |
(Eq. 10) |
![]() |
![]() |
(Eq. 11) |
There was a marked reduction of hGH oxidation in the presence of
additional 28% (v/v) 1-propanol, i.e. under conditions
where hGH undergoes a structural transition to a molten globular state. The 1H NMR spectrum of hGH recorded in aqueous solution
containing 40% (v/v) 1-propanol suggests that the presence of
1-propanol affects the interactions between helix I and helix IV of hGH
which construct the metal-binding site. However, even at a high content of 1-propanol there is a residual level (about 23%) of metal-catalyzed oxidation of hGH, suggesting that even the molten globular state of hGH
provides a chelating entity sufficient for some binding of the metal.
This observation leads to the question whether the basis of the
inhibitory role of 1-propanol is only the perturbation of the
metal-binding site or the combination of such perturbation and
additional factors. The experimental product isotope effect of
[residual hGH]1-propanol-h7/[residual
hGH]1-propanol-d7 = 1.95 likely reflects a kinetic isotope
effect where an oxidizing species reacts less efficiently with the C-D
bond of 1-propanol-d7 as compared with the C-H
bond of 1-propanol-h7. Hence, there is a more
efficient protection by 1-propanol-h7. The fact
that a product isotope effect is measurable may indicate that the
perturbation by 1-propanol is through insertion between the hydrophobic
sites of helix I and helix IV bringing 1-propanol sufficiently close to
the yet perturbed metal-binding site to scavenge reactive oxygen species. Three experimental facts would support an assignment that this
reactive species is either a hydroxyl radical or a metal-bound equivalent, a complexed hydroxyl radical: (i) the anaerobic reaction of
hydrogen peroxide with a hGH-Cu(I) complex, most likely
[hGH-Cu(I)]mbs, is expected to proceed according to
Equations 12 and 13 (44).
![]() |
(Eq. 12) |
![]() |
(Eq. 13) |
We note that the formation of 2-oxo-His via the displayed pathway requires the net addition of HO· at C-2 of the imidazole ring whereas ESR investigations on the addition of radiation chemically produced freely diffusable hydroxyl radicals to imidazole derivatives not complexed to transition metals have provided evidence for the addition of HO· at C-4 and C-5 (47). A preferred net addition of HO· to C-2 of His during the metal-catalyzed oxidation of hGH could be the result of metal-binding, the geometry of the metal-binding site, and the fact that a complexed rather than a free hydroxyl radical is the actual reactive oxygen species. In our tryptic map of hGH we identified low quantities of other oxidation products of His (T4ox1 and T4ox2) most likely representing products of the net addition of HO· at other positions of the imidazole ring such as C-4 and C-5. In this regard we note that the generation of free and/or complexed hydroxyl radicals has also been proposed for the reaction of hydrogen peroxide with Cu(I) superoxide dismutase (44, 48, 49) where 2-oxo-His was a major product of the modification of His118 (32) located at the metal-binding site.
Biological SignificanceThe binding of hGH to the hGH receptor is largely Zn2+-independent whereas binding to the human prolactin (hPRL) receptor requires Zn2+ and an intact metal-binding site of hGH (14). When each of the three amino acids involved in metal binding, i.e. His18, His21, and Glu174, was individually mutated to Ala, there was a 3,000-12,000-fold decrease of affinity of the respective hGH mutants to a recombinant protein containing the hGH-binding sequence of the hPRL receptor (hPRL binding protein, hPRLbp) (14). In contrast, these hGH mutants showed less significant change of binding to a recombinant protein containing the hGH-binding sequence of the hGH receptor (hGH-binding protein) (14). Thus, it appears that the chemical modification of a metal-binding side chain to a non-metal-binding side chain of only one amino acid of the metal-binding site is sufficient for the change of the affinity to hPRL-binding protein. We can reasonably assume that the 2-oxo tautomer of 2-oxo-His has a significantly lower affinity for metals as compared with a native imidazole ring due to the lack of the pyridine nitrogen. Therefore, we expect that the modification of His to 2-oxo-His in hGH may result in a decrease of the affinity to hPRLbp but not to hGH-binding protein, but this remains to be demonstrated in future experiments.
We thank Drs. Martha Morton and David Vander Velde for NMR analysis, Dr. Todd D. Williams for the assistance in product identification by mass spectroscopy, and Dr. James Q. Oeswein and Brian L. Miller for helpful discussions. We also thank the reviewer for valuable suggestions on the manuscript.