Identification of reactive cysteines in a protein using arsenic labeling and collision-induced dissociation tandem mass spectrometry

Trivalent arsenicals have high affinity for thiols (such as free cysteines) in proteins. We describe here the use of this property to develop a collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) technique for the identification of reactive cysteines in proteins. A trivalent arsenic species, dimethylarsinous acid (DMA (III)), with a residue mass (103.9607) and mass defect distinct from the normal 20 amino acids, was used to selectively label reactive cysteine residues in proteins. The CID fragment ions of the arsenic-labeled sequences shifted away from the more abundant normal fragments that would otherwise overlap with the ions of interest. Along with the internal and immonium ions, the arsenic-labeled fragment ions served as MS/MS signatures for identification of the binding sites and for assessment of the relative reactivity of individual cysteine residues in a protein. Using this method, we have identified two highly reactive binding sites in rat hemoglobin (Hb): Cys-13alpha and Cys-125beta. Cys-13alpha was bound to DMA (III) in the Hb of rats fed with arsenic, and this binding was responsible for arsenic accumulation in rat blood, while Cys-125beta was found to bind to glutathione in rat blood. This study revealed the relative reactivity of the cysteines in rat Hb in the following decreasing order: Cys-13alpha > Cys-111alpha > Cys-104alpha and Cys-13alpha > Cys-125beta > Cys-93beta. Arsenic-labeling is easy and fast for identification of active binding sites without enzymatic digestion and acid hydrolysis, and useful for characterization and identification of metal binding sites in other proteins.     

Ligand-induced conformational changes in spin label-modified human hemoglobins and chains and their carboxypeptidase A-digested derivatives.

The reactive sulfhydryls of human adult and fetal hemoglobin and the single sulfhydryl of isolated gamma chains have been spin labeled with N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) iodoacetamide. Similar electron paramagnetic spectral differences between oxy- and deoxy-modified hemoglobins were observed for both these hemoglobins and for the isolated chains, indicating that ligand-induced conformational changes occur in isolated hemoglobin subunits as well as intact hemoglobin tetramers. Ligand induced changes in the reactivity of p-hydroxymercuribenzoate with the sulfhydryl groups of both intact hemoglobins and isolated subunits, observed by McDonald and Noble (1974) J. Biol. Chem. 249, 3161-3165), led them to draw a similar conclusion. Following carboxypeptidase A digestion of these modified hemoglobins and gamma chains, a procedure which specifically removes the two C-terminal residues of the beta or gamma chains, spectral differences between the liganded and unliganded spin-labeled derivatives still persisted. However, the magnitude of this difference was not only more reduced in the case of the hemoglobins than in that of the subunits but the spectra of both the oxy and deoxy derivatives of the hemoglobins were characteristic of the oxy derivative of a cooperative tetrameric hemoglobin. These findings support the premise that the COOH-terminal end of the beta or gamma chain contributes, although possibly to different extents, to the spectral differences exhibited by both the spin-labeled hemoglobins and chains.     

The local electrostatic environment determines cysteine reactivity of tubulin

Of the 20 cysteines of rat brain tubulin, some react rapidly with sulfhydryl reagents, and some react slowly. The fast reacting cysteines cannot be distinguished with [14C]iodoacetamide, N-[(14)C]ethylmaleimide, or IAEDANS ([5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid]), since modification to mole ratios 1 cysteine/dimer always leads to labeling of 6-7 cysteine residues. These have been identified as Cys-305alpha, Cys-315alpha, Cys-316alpha, Cys-347alpha, Cys-376alpha, Cys-241beta, and Cys-356beta by mass spectroscopy and sequencing. This lack of specificity can be ascribed to reagents that are too reactive; only with the relatively inactive chloroacetamide could we identify Cys-347alpha as the most reactive cysteine of tubulin. Using the 3.5-A electron diffraction structure, it could be shown that the reactive cysteines were within 6.5 A of positively charged arginines and lysines or the positive edges of aromatic rings, presumably promoting dissociation of the thiol to the thiolate anion. By the same reasoning the inactivity of a number of less reactive cysteines could be ascribed to inhibition of modification by negatively charged local environments, even with some surface-exposed cysteines. We conclude that the local electrostatic environment of cysteine is an important, although not necessarily the only, determinant of its reactivity.     

Genetic variation in mouse beta globin cysteine content modifies glutathione metabolism: implications for the use of mouse models

Allelic variation in the mouse beta globin gene complex (Hbb) produces structurally different beta globins in different mouse strains. Like humans, mice with HbbS alleles produce a single beta globin with one reactive cysteine (beta Cys93). In contrast, mice with HbbD alleles produce two structurally different beta globins, each containing an additional cysteine (beta Cys13). beta Cys93 forms mixed disulfides with glutathione and plays a pivotal role in the activities of hemoglobin, glutathione, and nitric oxide. Similar roles for mouse beta Cys13 have not been described. We used capillary electrophoresis to compare reduced glutathione (GSH), glutathione disulfide (GSSG), and S-glutathionyl hemoglobin levels in erythrocytes from inbred C57BL/6J (homozygous HbbS/S) and 129S1/SvImJ (homozygous HbbD/D) mice and their homozygous and heterozygous B6129S/F2J hybrid offspring. S-glutathionyl hemoglobin was nearly undetectable in inbred or hybrid mice with only monocysteinyl beta globins (HbbS/S) but represented up to 10% of total hemoglobin in mice with polycysteinyl beta globins (HbbS/D or HbbD/D). The stepwise increase in beta globin sulfhydryl group concentration in HbbS/S, HbbS/D, and HbbD/D F2 mice was associated with increasing hemoglobin-bound glutathione and decreasing free glutathione (GSH + GSSG) concentrations. Total erythrocyte glutathione (GSH + GSSG + hemoglobin-bound) was not significantly different between groups. In vitro studies showed that beta Cys13 in mouse HbbD beta globins was more susceptible to disulfide exchange with GSSG than beta Cys93. We conclude that reactive beta globin sulfhydryl group concentration is genetically determined in mice, and that polycysteinyl beta globins markedly influence intraerythrocyte glutathione distribution between free and hemoglobin-bound compartments. Although Hbb heterozygosity and polycysteinyl beta globins are common in wild mouse populations, all common human beta globins contain only one reactive cysteine, and homozygosity is the norm. These fundamental differences in mouse and human beta globin genetics have important implications for the study of mouse biology and for the use of some mouse strains as models for humans.     

The reactivity and oxidation pathway of cysteine 232 in recombinant human alpha 1-antitrypsin

Oxidative damage to the sulfur-containing amino acids, methionine and cysteine, is a major concern in biotechnology and medicine. alpha1-Antitrypsin, which is a metastable and conformationally flexible protein that belongs to the serpin family of protease inhibitors, contains nine methionines and a single cysteine in its primary sequence. Although it is known that methionine oxidation in the protein active site results in a loss of biological activity, there is little specific knowledge regarding the reactivity of its unpaired thiol, Cys-232. In this study, the thiol-modifying reagent NBD-Cl (7-chloro-4-nitrobenz-2-oxa-1,3-diazole) was used to label peroxide-modified alpha1-antitrypsin and demonstrate that the Cys-232 in vitro oxidation pathway begins with a stable sulfenic acid intermediate and is followed by the formation of sulfinic and cysteic acid in successive steps. pH-dependent reactivity with hydrogen peroxide showed that Cys-232 has a pK(a) of 6.86 +/- 0.05, a value that is more than 1.5 pH units lower than that of a typical protein thiol. pH-induced conformational changes in the region surrounding Cys-232 were also examined and indicate that mildly acidic conditions induce a conformation that enhances Cys-232 reactivity. In summary, this work provides new insights into alpha1-antitrypsin reactivity in oxidizing environments and shows that a unique structural environment renders its unpaired thiol, Cys-232, its most reactive amino acid.     

Role of cysteine residues in glutathione synthetase from Escherichia coli B. Chemical modification and oligonucleotide site-directed mutagenesis

Escherichia coli B glutathione synthetase is composed of four identical subunits; each subunit contains 4 cysteine residues (Cys-122, -195, -222, and -289). We constructed seven different mutant enzymes containing 3, 2, or no cysteine residues/subunit by replacement of cysteine codons with those of alanine in the gsh II gene using site-directed mutagenesis. Three mutant enzymes, Ala289, Ala222/289, Cys-free (Ala122/195/222/289), in which cysteine at residue 289 was replaced with alanine, were not inactivated by 5,5'-dithiobis(2-nitrobenzoate) (DTNB), while the other four mutants retaining Cys-289 were inactivated at the wild-type rate. From these selective inactivations of mutant enzymes by DTNB, the sulfhydryl group modified by DTNB was unambiguously identified as Cys-289. In this way, Cys-289 was found to be also a target of modification with 2-nitrothiocyanobenzoate and N-ethylmaleimide, while Cys-195 was of p-chloromercuribenzoate. These results suggest that both Cys-195 and Cys-289 were not essential for the activity of the glutathione synthetase, but chemical modification of either one of the two sulfhydryl groups resulted in complete loss of the activity. Replacement of Cys-122 to Ala-122 enhanced the reactivity of Cys-289 with sulfhydryl reagents.     

Different metabolizing ability of thiol reactants in human and rat blood: biochemical and pharmacological implications

The effect of oxidants, electrophiles, and NO donors in rat or human erythrocytes was analyzed to investigate the influence of protein sulfhydryl groups on the metabolism of these thiol reactants. Oxidant-evoked alterations in thiolic homeostasis were significantly different in the two models; large amounts of glutathione protein mixed disulfides were produced in rat but not in human erythrocytes by treatment with hydroperoxides or diamide. The disappearance of all forms of glutathione (reduced, disulfide, protein mixed disulfide) was induced by menadione only in human erythrocytes. The treatment of rat red blood cells with electrophiles produced glutathione S-conjugates to a much lower extent than in human red blood cells; GSH was only minimally depleted in rat red blood cells. The NO donor S-nitrosocysteine induced a rapid transnitrosation reaction with hemoglobin in rat erythrocytes producing high levels of S-nitrosohemoglobin; this reaction in human red blood cells was negligible. All drugs were cleared more rapidly in rat than in human erythrocytes. Unlike human Hb, rat hemoglobin contains three families of protein SH groups; one of these located at position beta125 is directly implicated in the metabolism of thiol reactants. This is thought to influence significantly the biochemical, pharmacological, and toxicological effects of some drugs.     

Mapping the binding site of colchicinoids on beta -tubulin. 2-Chloroacetyl-2-demethylthiocolchicine covalently reacts predominantly with cysteine 239 and secondarily with cysteine 354

2-Chloroacetyl-2-demethylthiocolchicine (2CTC) and 3-chloroacetyl-3-demethylthiocolchicine (3CTC) resemble colchicine in binding to tubulin and react covalently with beta-tubulin, forming adducts with cysteine residues 239 and 354. The adducts at Cys-239 are less stable than those at Cys-354 during formic acid digestion. Extrapolating to zero time, the Cys-239 to Cys-354 adduct ratio is 77:23 for 2CTC and 27:73 for 3CTC. Using energy minimization modeling to dock colchicinoids into the electron crystallographic model of beta-tubulin in protofilaments (Nogales, E. , Wolf, S. G., and Downing, K. H. (1998) Nature 391, 199-203), we found two potential binding sites. At one, entirely encompassed within beta-tubulin, the C2- and C3-oxygen atoms of 2CTC and 3CTC overlapped poorly with those of colchicine and thiocolchicine, but distances from the reactive carbon atoms of the analogs to the sulfur atoms of the cysteine residues were qualitatively consistent with reactivity. The other potential binding site was located at the alpha/beta interface. Here, the oxygen atoms of the analogs overlapped well with those of colchicine, but relative distances of the reactive carbons to the cysteine sulfur atoms did not correlate with the observed reactivity. A significant conformational change must occur in the colchicine binding site of tubulin in the transition from the unpolymerized to the polymerized state.     

Biochemical characterization of yeast mitochondrial Grx5 monothiol glutaredoxin

Grx5 is a yeast mitochondrial protein involved in iron-sulfur biogenesis that belongs to a recently described family of monothiolic glutaredoxin-like proteins. No member of this family has been biochemically characterized previously. Grx5 contains a conserved cysteine residue (Cys-60) and a non-conserved one (Cys-117). In this work, we have purified wild type and mutant C60S and C117S proteins and characterized their biochemical properties. A redox potential of -175 mV was calculated for wild type Grx5. The pKa values obtained by titration of mutant proteins with iodoacetamide at different pHs were 5.0 for Cys-60 and 8.2 for Cys-117. When Grx5 was incubated with glutathione disulfide, a transient mixed disulfide was formed between glutathione and the cystein 60 of the protein because of its low pKa. Binding of glutathione to Cys-60 promoted a decrease in the Cys-117 pKa value that triggered the formation of a disulfide bond between both cysteine residues of the protein, indicating that Cys-117 plays an essential role in the catalytic mechanism of Grx5. The disulfide bond in Grx5 could be reduced by GSH but at a rate at least 20 times slower than that observed for the reduction of glutaredoxin 1 from E. coli, a dithiolic glutaredoxin. This slow reduction rate could suggest that GSH may not be the physiologic reducing agent of Grx5. The fact that wild type Grx5 efficiently reduced a glutathiolated protein used as a substrate indicated that Grx5 may act as a thiol reductase inside the mitochondria.     

The exceptionally high reactivity of Cys 621 is critical for electrophilic activation of the sensory nerve ion channel TRPA1

Activation of the sensory nerve ion channel TRPA1 by electrophiles is the key mechanism that initiates nociceptive signaling, and leads to defensive reflexes and avoidance behaviors, during oxidative stress in mammals. TRPA1 is rapidly activated by subtoxic levels of electrophiles, but it is unclear how TRPA1 outcompetes cellular antioxidants that protect cytosolic proteins from electrophiles. Here, using physiologically relevant exposures, we demonstrate that electrophiles react with cysteine residues on mammalian TRPA1 at rates that exceed the reactivity of typical cysteines by 6,000-fold and that also exceed the reactivity of antioxidant enzymes. We show that TRPA1 possesses a complex reactive cysteine profile in which C621 is necessary for electrophile-induced binding and activation. Modeling of deprotonation energies suggests that K620 contributes to C621 reactivity and mutation of K620 alone greatly reduces the effect of electrophiles on TRPA1. Nevertheless, binding of electrophiles to C621 is not sufficient for activation, which also depends on the function of another reactive cysteine (C665). Together, our results demonstrate that TRPA1 acts as an effective electrophilic sensor because of the exceptionally high reactivity of C621.     

O6-alkylguanine-DNA alkyltransferase: low pKa and high reactivity of cysteine 145

The active site cysteine of human O(6)-alkylguanine-DNA alkyltransferase (hAGT), Cys145, was shown to be highly reactive with model electrophiles unrelated to substrates, including 1-chloro-2,4-dinitrobenzene. The high reactivity suggested that the Cys145 thiolate anion might be stable at neutral pH. The pK(a) was estimated from plots of UV spectra (A(239)) and reactivity toward 4,4'-dithiopyridine vs pH. The estimated pK(a) for hAGT was 4-5, depending upon the method used, and near that of the extensively characterized papain Cys25. Rates of reaction with 4,4'-dithiopyridine were similar for the thiolate forms of hAGT, papain, glutathione, and the bacterial hAGT homologue Ogt (the pK(a) of the latter was 5.4). Bound Zn(2+) has previously been shown to be required for the catalytic activity of hAGT (Rasimas, J. J. et al. (2003) Biochemistry 42, 980-990). Zn(2+) was shown to be required for the low pK(a) of hAGT. The high reactivity of hAGT Cys145 is postulated to be important in normal catalytic function, in cross-linking reactions involving bis-electrophiles, and in inhibition of the DNA repair function of hAGT by electrophiles.     

Identification of in vivo disulfide conformation of TRPA1 ion channel

TRPA1 (transient receptor potential ankyrin 1) is an ion channel expressed in the termini of sensory neurons and is activated in response to a broad array of noxious exogenous and endogenous thiol-reactive compounds, making it a crucial player in chemical nociception. A number of conserved cysteine residues on the N-terminal domain of the channel have been identified as critical for sensing these electrophilic pungent chemicals, and our recent EM structure with modeled domains predicts that these cysteines form a ligand-binding pocket, allowing for the possibility of disulfide bonding between the cysteine residues. Here, we present a comprehensive mass spectrometry investigation of the in vivo disulfide bonding conformation and in vitro reactivity of 30 of the 31 cysteine residues in the TRPA1 ion channel. Four disulfide bonds were detected in the in vivo TRPA1 structure: Cys-666-Cys-622, Cys-666-Cys-463, Cys-622-Cys-609, and Cys-666-Cys-193. All of the cysteines detected were reactive to N-methylmaleimide (NMM) in vitro, with varying degrees of labeling efficiency. Comparison of the ratio of the labeling efficiency at 300 μM versus 2 mM NMM identified a number of cysteine residues that were outliers from the mean labeling ratio, suggesting that protein conformation changes rendered these cysteines either more or less protected from labeling at the higher NMM concentrations. These results indicate that the activation mechanism of TRPA1 may involve N-terminal conformation changes and disulfide bonding between critical cysteine residues.     

Regulation of plant cytosolic glyceraldehyde 3-phosphate dehydrogenase isoforms by thiol modifications

Cytosolic NAD-dependent glyceraldehyde 3-P dehydrogenase (GAPDH; GapC; EC 1.2.1.12) catalyzes the oxidation of triose phosphates during glycolysis in all organisms, but additional functions of the protein has been put forward. Because of its reactive cysteine residue in the active site, it is susceptible to protein modification and oxidation. The addition of GSSG, and much more efficiently of S-nitrosoglutathione, was shown to inactivate the enzymes from Arabidopsis thaliana (isoforms GapC1 and 2), spinach, yeast and rabbit muscle. Inactivation was fully or at least partially reversible upon addition of DTT. The incorporation of glutathione upon formation of a mixed disulfide could be shown using biotinylated glutathione ethyl ester. Furthermore, using the biotin-switch assay, nitrosylated thiol groups could be shown to occur after treatment with nitric oxide donors. Using mass spectrometry and mutant proteins with one cysteine lacking, both cysteines (Cys-155 and Cys-159) were found to occur as glutathionylated and as nitrosylated forms. In preliminary experiments, it was shown that both GapC1 and GapC2 can bind to a partial gene sequence of the NADP-dependent malate dehydrogenase (EC 1.2.1.37; At5g58330). Transiently expressed GapC-green fluorescent protein fusion proteins were localized to the nucleus in A. thaliana protoplasts. As nuclear localization and DNA binding of GAPDH had been shown in numerous systems to occur upon stress, we assume that such mechanism might be part of the signaling pathway to induce increased malate-valve capacity and possibly other protective systems upon overreduction and initial formation of reactive oxygen and nitrogen species as well as to decrease and protect metabolism at the same time by modification of essential cysteine residues.     

Ruthenium-iron hybrid hemoglobins as a model for partially liganded hemoglobin: oxygen equilibrium curves and resonance Raman spectra

The structure and function of iron(II)-ruthenium(II) hybrid hemoglobins alpha(Ru-CO)2 beta(Fe)2 and alpha(Fe)2 beta(Ru-CO)2, which can serve as models for the intermediate species of the oxygenation step in native human adult hemoglobin, were investigated by measuring oxygen equilibrium curves and the Fe(II)-N epsilon (His F8) stretching resonance Raman lines. The oxygen equilibrium properties indicated that these iron-ruthenium hybrid hemoglobins are good models for the half-liganded hemoglobin. The pH dependence of the oxygen binding properties and the resonance Raman line revealed that the quaternary and tertiary structural transition was induced by pH changes. When the pH was lowered, both the iron-ruthenium hybrid hemoglobins exhibited relatively higher cooperativity and a Raman line typical of normal deoxy structure, suggesting that their structure is stabilized at a "T-like" state. However, the oxygen affinity of alpha(Fe)2 beta(Ru-CO)2 was lower than that of alpha(Ru-CO)2 beta(Fe)2, and the transition to the "deoxy-type" Fe-N epsilon stretching Raman line of alpha(Fe2)beta(Ru-CO)2 was completed at pH 7.4, while that of the complementary counterpart still remained in an "oxy-like" state under the same condition. These observations clearly indicate that the beta-liganded hybrid has more "T"-state character than the alpha-liganded hybrid. In other words, the ligation to the alpha subunit induces more pronounced changes in the structure and function in Hb than the ligation to the beta subunit. This feature agrees with our previous observations by NMR and sulfhydryl reactivity experiments. The present results are discussed in relation to the molecular mechanism of the cooperative stepwise oxygenation in native human adult hemoglobin.     

Dopamine-derived quinones affect the structure of the redox sensor DJ-1 through modifications at Cys-106 and Cys-53

The physiological role of DJ-1, a protein involved in familial Parkinson disease is still controversial. One of the hypotheses proposed indicates a sensor role for oxidative stress, through oxidation of a conserved cysteine residue (Cys-106). The association of DJ-1 mutations with Parkinson disease suggests a loss of function, specific to dopaminergic neurons. Under oxidative conditions, highly reactive dopamine quinones (DAQs) can be produced, which can modify cysteine residues. In cellular models, DJ-1 was found covalently modified by dopamine. We analyzed the structural modifications induced on human DJ-1 by DAQs in vitro. We described the structural perturbations induced by DAQ adduct formation on each of the three cysteine residues of DJ-1 using specific mutants. Cys-53 is the most reactive residue and forms a covalent dimer also in SH-SY5Y DJ-1-transfected cells, but modification of Cys-106 induces the most severe structural perturbations; Cys-46 is not reactive. The relevance of these covalent modifications to the several functions ascribed to DJ-1 is discussed in the context of the cell response to a dopamine-derived oxidative insult.     

Interaction of triethyltin with cat hemoglobin: Identification of binding sites and effects on hemoglobin function

Binding of triethyltin to the cat hemoglobins (HbA and HbB) results in the “masking” of two of the freely reactive sulfhydryl groups (SH) within the hemoglobin tetramer. That the “masked” SH groups occur in position 13α of each α-subunit was demonstrated by the lack of labeling of cysteine 13α with [¹⁴C]N-ethylmaleimide when triethyltin is present. Studies with cat-human hybrid hemoglobins indicate that the α-subunit of the cat hemoglobins alone is involved in the formation of a complex with triethyltin. Using available data on the primary as well as three dimensional structures of animal hemoglobins, it is suggested the cysteine 13α and histidine 20α serve as axial ligands in the formation of a pentacoordinate triethyltin cat hemoglobin complex. The binding of triethyltin results in an increase in the oxygen affinity of the two cat hemoglobins.     

Analysis of protein adduction kinetics by quantitative mass spectrometry: competing adduction reactions of glutathione-S-transferase P1-1 with electrophiles

Defining the mechanisms and consequences of protein adduction is crucial to understanding the toxicity of reactive electrophiles. Application of tandem mass spectrometry and data analysis algorithms enables detection and mapping of chemical adducts at the level of amino acid sequence. Nevertheless, detection of adducts does not indicate relative reactivity of different sites. Here, we describe a method to measure the kinetics of competing adduction reactions at different sites on the same protein. Adducts are formed by electrophiles at Cys14 and Cys47 on the metabolic enzyme glutathione-S-transferase P1-1 and modification is accompanied by a loss of enzymatic activity. Relative quantitation of protein adducts was done by tagging N-termini of peptide digests with isotopically labeled phenyl isocyanate and tracking the ratio of light-tagged peptide adducts to heavy-tagged reference samples in liquid chromatography-tandem mass spectrometry analyses using a multiple reaction monitoring method. This approach was used to measure rate constants for adduction at both positions with two different model electrophiles, N-iodoacetyl-N-biotinylhexylenediamine and 1-biotinamido-4-(4'-[maleimidoethyl-cyclohexane]-carboxamido)butane. The results indicate that Cys47 was approximately two- to three-fold more reactive toward both electrophiles than was Cys14. This result was consistent with the relative reactivity of these electrophiles in a complex proteome system and with previously reported trends in reactivity of these sites. Kinetic analyses of protein modification reactions provide a means of evaluating the selectivity of reactive mediators of chemical toxicity.     

Specific modification of the carboxyl groups of hemoglobin S

The reactivity of the carboxyl groups of hemoglobin S to form amide bonds with glycine ethyl ester by carbodiimide-activated coupling, and the influence of this derivatization on the functional properties of the protein have been investigated. Incubation of carbonmonoxy or oxyhemoglobin S with 20 mM 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide in the presence of 100 mM [14C]glycine ethyl ester, at pH 6.0 and 23 degrees C for 1 h resulted in the modification of, on an average, three carboxyl groups of the protein. The Hill coefficient of the modified hemoglobin S was 2.7, indicating normal subunit interactions. The derivatization increased the oxygen affinity of the molecule (the P50 was lowered from 8.0 to 5.0). The derivatization also resulted in an increase in the minimum gelling concentration of hemoglobin S from 16 to 24 g/100 ml. The reaction conditions used for the derivatization of the carboxyl groups of hemoglobin S are very selective for the protein carboxyl groups; very little of the label is associated with the heme carboxyls. Tryptic peptide mapping of the modified hemoglobin S indicated that the peptide beta T5, i.e. the segment representing amino acid residues 41 to 59 of beta-chain, accounted for nearly 75% of the label associated with the globin, demonstrating the high selectivity of the derivatization. Sequence analysis of the derivatized beta T5 demonstrated that at least 65% of the label incorporated into hemoglobin S is targeted toward the carboxyl group of Glu-43(beta), identifying it as the most reactive carboxyl group in hemoglobins. The results suggest that modification of the carboxyl group of hemoglobins S, presumably the gamma-carboxyl of Glu-43(beta), reduces the propensity of deoxyhemoglobin S to polymerize.     

Comparative immunochemical studies of primate hemoglobins

The antigenic properties of a number of chromatographically purified primate hemoglobins were compared to those of normal human hemoglobin using a sensitive radioimmunochemical procedure. The degree of inhibition of the antigen-antibody reaction with heterologous hemoglobins appeared to be related to the structural similarity of these proteins to the normal human hemoglobin immunogen. With the exception of the baboon hemoglobin, the antigenicity of the hemoglobins paralleled the phylogeny of the primates. The gorilla and chimpanzee hemoglobins were antigenically identical to normal human hemoglobin, whereas the gibbon and orangutan hemoglobins were substantially more variable. Of the Old World monkey hemoglobins examined, the baboon produced lower inhibition values, suggesting a greater degree of structural dissimilarity than other Cercopithecoidea hemoglobins, which is compatible with a greater rate of evolutionary change occurring in this protein. Using the known amino acid sequences of human and other primate hemoglobins, we have attempted to identify antigenic determinant areas of the proteins.     

Amino acid sequences of hemoglobins I and II from root nodules of the non-leguminous Parasponia rigida-rhizobium symbiosis, and a correction of the sequence of hemoglobin I from Parasponia andersonii

The amino acid sequence of hemoglobins I (pI 6.15 as oxyhemoglobin) and II (pI 5.64 as oxyhemoglobin) from the nitrogen-fixing root nodules of Parasponia rigida have been determined by protein sequencing. The sequence of hemoglobin I (pI 6.16, as oxyhemoglobin) from Parasponia andersonii was re-examined and the corrected primary structure, now in agreement with that predicted from the DNA sequence, is reported. The three Parasponia hemoglobins contain 161 amino acid residues (Mr approximately equal to 18,700 including the heme) with a single cysteine residue and five methionine residues. The N-terminal serine is blocked by an acetyl group. The primary structure of the Parasponia hemoglobins is highly conserved. Hemoglobins I from the two species of Parasponia are identical; both show microheterogeneity at position 30 (Asp/Glu substitution) and hemoglobin I from P. rigida shows microheterogeneity at position 150 (Ala/Val) while hemoglobin I from P. andersonii has only an Ala at 150. P. rigida hemoglobin II shows no microheterogeneity at these positions, having Asp and Val residues respectively, and it contains a single amino acid change of a Gln for an Arg at position 85, which accounts for the 0.5 unit difference in isoelectric point observed between hemoglobins I and II. The sequence data are consistent with allelic heterogeneity at a single locus rather than different genes.