C-terminal Lys Residue Research Articles

A Collision-Induced Dissociation Cleavable Isobaric Tag for Peptide Fragment Ion-Based Quantification in Proteomics.

Quantifying peptides based on unique peptide fragment ions avoids the issue of ratio distortion that is commonly observed for reporter ion-based quantification approaches. Herein, we present a collision-induced dissociation-cleavable, isobaric acetyl-isoleucine-proline-glycine (Ac-IPG) tag, which conserves the merits of quantifying peptides based on unique fragments while reducing the complexity of the b-ion series compared to conventional fragment ion-based quantification methods thus facilitating data processing. Multiplex labeling is based on selective N-terminal dimethylation followed by derivatization of the ε-amino group of the C-terminal Lys residue of LysC peptides with isobaric Ac-IPG tags having complementary isotope distributions on Pro-Gly and Ac-Ile. Upon fragmentation between Ile and Pro, the resulting y ions, with the neutral loss of Ac-Ile, can be distinguished between the different labeling channels based on different numbers of isotope labels on the Pro-Gly part and thus contain the information for relative quantification, while b ions of different labeling channels have the same m/z values. The proteome quantification capability of this method was demonstrated by triplex labeling of a yeast proteome spiked with bovine serum albumin (BSA) over a 10-fold dynamic range. With the yeast proteins as the background, BSA was detected at ratios of 1.14:5.06:9.78 when spiked at 1:5:10 ratios. The raw mass data is available on the ProteomeXchange with the identifier PXD 018790.

TRPV1 SUMOylation regulates nociceptive signaling in models of inflammatory pain

Although TRPV1 channels represent a key player of noxious heat sensation, the precise mechanisms for thermal hyperalgesia remain unknown. We report here that conditional knockout of deSUMOylation enzyme, SENP1, in mouse dorsal root ganglion (DRG) neurons exacerbated thermal hyperalgesia in both carrageenan- and Complete Freund’s adjuvant-induced inflammation models. TRPV1 is SUMOylated at a C-terminal Lys residue (K822), which specifically enhances the channel sensitivity to stimulation by heat, but not capsaicin, protons or voltage. TRPV1 SUMOylation is decreased by SENP1 but upregulated upon peripheral inflammation. More importantly, the reduced ability of TRPV1 knockout mice to develop inflammatory thermal hyperalgesia was rescued by viral infection of lumbar 3/4 DRG neurons of wild-type TRPV1, but not its SUMOylation-deficient mutant, K822R. These data suggest that TRPV1 SUMOylation is essential for the development of inflammatory thermal hyperalgesia, through a mechanism that involves sensitization of the channel response specifically to thermal stimulation.

Kinetically Trapped Liquid-State Conformers of a Sodiated Model Peptide Observed in the Gas Phase.

We investigate the peptide AcPheAla5LysH+, a model system for studying helix formation in the gas phase, in order to fully understand the forces that stabilize the helical structure. In particular, we address the question of whether the local fixation of the positive charge at the peptide's C-terminus is a prerequisite for forming helices by replacing the protonated C-terminal Lys residue by Ala and a sodium cation. The combination of gas-phase vibrational spectroscopy of cryogenically cooled ions with molecular simulations based on density-functional theory (DFT) allows for detailed structure elucidation. For sodiated AcPheAla6, we find globular rather than helical structures, as the mobile positive charge strongly interacts with the peptide backbone and disrupts secondary structure formation. Interestingly, the global minimum structure from simulation is not present in the experiment. We interpret that this is due to high barriers involved in rearranging the peptide-cation interaction that ultimately result in kinetically trapped structures being observed in the experiment.

Extension of Light-Harvesting Ability of Photosynthetic Light-Harvesting Complex 2 (LH2) through Ultrafast Energy Transfer from Covalently Attached Artificial Chromophores.

Introducing appropriate artificial components into natural biological systems could enrich the original functionality. To expand the available wavelength range of photosynthetic bacterial light-harvesting complex 2 (LH2 from Rhodopseudomonas acidophila 10050), artificial fluorescent dye (Alexa Fluor 647: A647) was covalently attached to N- and C-terminal Lys residues in LH2 α-polypeptides with a molar ratio of A647/LH2 ≃ 9/1. Fluorescence and transient absorption spectroscopies revealed that intracomplex energy transfer from A647 to intrinsic chromophores of LH2 (B850) occurs in a multiexponential manner, with time constants varying from 440 fs to 23 ps through direct and B800-mediated indirect pathways. Kinetic analyses suggested that B800 chromophores mediate faster energy transfer, and the mechanism was interpretable in terms of Förster theory. This study demonstrates that a simple attachment of external chromophores with a flexible linkage can enhance the light harvesting activity of LH2 without affecting inherent functions of energy transfer, and can achieve energy transfer in the subpicosecond range. Addition of external chromophores, thus, represents a useful methodology for construction of advanced hybrid light-harvesting systems that afford solar energy in the broad spectrum.

Visualizing pHLIP Insertion in Plasmamembrane and Endosomal Membrane

The pH-(Low) Insertion Peptide (pHLIP) has potential as a tumor-targeting drug carrier. At neutral pH, pHLIP has affinity for the surface of a lipid bilayer, whereas under slightly acidic conditions (e.g. pH ∼ 6) pHLIP inserts into the membrane, forming a transmembrane helix. Since many solid tumors are more acidic than healthy tissues, pHLIP may be used to translocate chemotherapeutic agents selectively into cancer cells. Knowledge of the exact location of pHLIP insertion in cells can guide the rational design of delivery constructs. We envision two scenarios for pHLIP insertion in cells: First, pHLIP may directly insert into the plasmamembrane; alternatively, cells may internalize pHLIP molecules via endocytosis, and subsequently pHLIP may insert into the endosomal membrane. In this study, several fluorescently self-quenched pHLIP constructs were synthesized to visualize to what extend these two scenarios are occurring in cells at pH 7.3 and 6.2. In these self-quenched pHLIP constructs, a rhodamine dye (TAMRA or Alexa Fluor 568) is attached to a C-terminal Lys residue, with the quencher QSY-9 conjugated to an adjacent Cys via a cleavable disulfide linker (or a stable thio-ether bond). Upon insertion, pHLIP would translocate its C-terminus into the cell cytoplasm, where cleavage of the disulfide linkage and release of the quencher QSY9 can take place. In turn, the pHLIP construct would become more fluorescent.

A modulator domain controlling thermal stability in the Group II chaperonins of Archaea

Archaeal Group II chaperonins (Cpns) are strongly conserved, considering that their growth temperatures range from 23 to 122 °C. The C-terminal 15–25 residues are hypervariable, and highly charged in thermophilic species. Our hypothesis is that the C-terminal is a key determinant of stabilization of the Cpn complex. The C-terminus of the Cpn from the hyperthermophile Pyrococcus furiosus was mutated to test this hypothesis. C-terminal deletions and replacement of charged residues resulted in destabilization. The stability of ATPase activity declined in proportion to the reduction in charged residues with Ala or Gly. An EK-rich motif ( 528EKEKEKEGEK5 37) proved to be a key domain for stabilization at or near 100 °C. Mutations “tuned” the Cpn for optimal protein folding at lower optimal temperatures, and Glu substitution was more potent than Lys replacement. Pf Cpn stability was enhanced by Ca 2+, especially in the mutant Cpn lacking C-terminal Lys residues. This suggests that Glu-Glu interactions between C termini might be mediated by Ca 2+. The C-terminal of a Cpn from the psychrophilic archaeon Methanococcoides burtonii was replaced by a domain from the hyperthermophile, resulting in increased thermostability and thermoactivity. We conclude that localized evolutionary variation in the C-terminus modulates the temperature range of archaeal Cpns.

Thrombin-activable Fibrinolysis Inhibitor (TAFI) Zymogen Is an Active Carboxypeptidase

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase found in human plasma, presumably as an inactive zymogen. The current dogma is that proteolytic activation by thrombin/thrombomodulin generates the active enzyme (TAFIa), which down-regulates fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin. In this study, we have shown that the zymogen exhibits continuous and stable carboxypeptidase activity against large peptide substrates, and we suggest that the activity down-regulates fibrinolysis in vivo.

Metallocarboxypeptidases: Emerging Drug Targets in Biomedicine

Metallocarboxypeptidases (MCPs) are commonly regarded as exopeptidases that actively participate in the digestion of proteins and peptides. In the recent years, however, novel MCPs comprising a wide range of physiological roles have been found in different mammalian extra-pancreatic tissues and fluids. Among them, CPU, also known as thrombin-activatable fibrinolysis inhibitor (TAFI), has been shown to cleave C-terminal Lys residues from partially degraded fibrin, acting as inhibitor of clot fibrinolysis and therefore constituting an important drug target for thrombolytic therapies. Other MCPs such as CPE, CPN, CPM, and CPD function as pro-hormone and neuropeptide processors and display several structural differences with the pancreatic-like enzymes. In addition, important advances have been made in the discovery and characterization of new endogenous and exogenous proteinaceous inhibitors; the structural determination of their complexes with several MCPs has revealed novel binding modes. Finally, the use of MCPs in antibody-directed enzyme pro-drug therapy (ADEPT) has proved to be an efficient approach for the delivery of lethal levels of chemotherapeutic drugs specifically at tumor tissues. Taken together, these recent developments may help to understand potential biomedical implications of MCPs. Future perspectives for the regulation of these enzymes through the use of more selective and potent inhibitors are also discussed in this review and combined with earlier observations in the field.

Crystal Structure of the Human Carboxypeptidase N (Kininase I) Catalytic Domain

Human carboxypeptidase N (CPN), a member of the CPN/E subfamily of “regulatory” metallo-carboxypeptidases, is an extracellular glycoprotein synthesized in the liver and secreted into the blood, where it controls the activity of vasoactive peptide hormones, growth factors and cytokines by specifically removing C-terminal basic residues. Normally, CPN circulates in blood plasma as a hetero-tetramer consisting of two 83 kDa (CPN2) domains each flanked by a 48 to 55 kDa catalytic (CPN1) domain. We have prepared and crystallized the recombinant C-terminally truncated catalytic domain of human CPN1, and have determined and refined its 2.1 Å crystal structure. The structural analysis reveals that CPN1 has a pear-like shape, consisting of a 319 residue N-terminal catalytic domain and an abutting, cylindrically shaped 79 residue C-terminal β-sandwich transthyretin (TT) domain, more resembling CPD-2 than CPM. Like these other CPN/E members, two surface loops surrounding the active-site groove restrict access to the catalytic center, offering an explanation for why some larger protein carboxypeptidase inhibitors do not inhibit CPN. Modeling of the Pro-Phe-Arg C-terminal end of the natural substrate bradykinin into the active site shows that the S1′ pocket of CPN1 might better accommodate P1′-Lys than Arg residues, in agreement with CPN's preference for cleaving off C-terminal Lys residues. Three Thr residues at the distal TT edge of CPN1 are O-linked to N-acetyl glucosamine sugars; equivalent sites in the membrane-anchored CPM are occupied by basic residues probably involved in membrane interaction. In tetrameric CPN, each CPN1 subunit might interact with the central leucine-rich repeat tandem of the cognate CPN2 subunit via a unique hydrophobic surface patch wrapping around the catalytic domain–TT interface, exposing the two active centers.

Identification of Precise Electrostatic Recognition Sites between Cytochrome c6 and the Photosystem I Subunit PsaF Using Mass Spectrometry

The reduction of the photo-oxidized special chlorophyll pair P700 of photosystem I (PSI) in the photosynthetic electron transport chain of eukaryotic organisms is facilitated by the soluble copper-containing protein plastocyanin (pc). In the absence of copper, pc is functionally replaced by the heme-containing protein cytochrome c6 (cyt c6) in the green alga Chlamydomonas reinhardtii. Binding and electron transfer between both donors and PSI follows a two-step mechanism that depends on electrostatic and hydrophobic recognition between the partners. Although the electrostatic and hydrophobic recognition sites on pc and PSI are well known, the precise electrostatic recognition site on cyt c6 is unknown. To specify the interaction sites on a molecular level, we cross-linked cyt c6 and PSI using a zero-length cross-linker and obtained a cross-linked complex competent in fast and efficient electron transfer. As shown previously, cyt c6 cross-links specifically with the PsaF subunit of PSI. Mass spectrometric analysis of tryptic peptides from the cross-linked product revealed specific interaction sites between residues Lys27 of PsaF and Glu69 of cyt c6 and between Lys23 of PsaF and Glu69/Glu70 of cyt c6. Using these new data, we present a molecular model of the intermolecular electron transfer complex between eukaryotic cyt c6 and PSI.

Structural/Functional Characterization of the α2-Plasmin Inhibitor C-Terminal Peptide

The alpha(2)-plasmin inhibitor (A2PI) is a main physiological regulator of the trypsin-like serine proteinase plasmin. It is composed of an N-terminal 15 amino acid fibrin cross-linking polypeptide, a 382-residue serpin domain, and a flexible C-terminal segment. The latter, peptide Asn(398)-Lys(452), and its Lys452Ala mutant were expressed as recombinant proteins in Escherichia coli (r-A2PIC and r-A2PICmut, respectively). CD and NMR analyses indicate that r-A2PIC is flexible, loosely folded, and with low content of regular secondary structure. Functional characterization via intrinsic fluorescence ligand titrations shows that r-A2PIC interacts with the isolated plasminogen kringle 1 (r-K1) (K(a) approximately 69.9 mM(-)(1)), K4 (K(a) approximately 45.7 mM(-)(1)), K5 (K(a) approximately 4.3 mM(-)(1)), and r-K2 (K(a) approximately 3.2 mM(-)(1)), all of which are known to exhibit lysine-binding capability. The affinities of these kringles for r-A2PIC are consistently larger than those reported for the ligand N(alpha)-acetyllysine, a mimic of a C-terminal Lys residue. The r-A2PICmut, with a C-terminal Ala residue, also interacts with r-K1 and K4, although with approximately 5-fold lesser affinities relative to r-A2PIC, demonstrating that while Lys(452) plays a major role in the binding, internal residues in r-A2PIC tether the kringles. (1)H NMR spectroscopy shows that key aromatic residues within the K4 lysine-binding site (LBS), namely, Trp(25), Trp(62), Phe(64), Trp(72), and Tyr(74), selectively respond to the addition of r-A2PIC and r-A2PICmut, indicating that these interactions proceed via the kringles' canonical LBS. We conclude that r-A2PIC docks to kringles primarily through lysine side chains and that Lys(452) most definitely enhances the binding. This suggests that multiple Lys residues within A2PI could contribute, perhaps in a zipper-like fashion, to its binding to the in-tandem, multikringle array that configures the plasmin heavy chain.

TAFI Meets the Sticky Ends

SummaryThe coagulation and fibrinolytic system are often considered as separate entities, despite the obvious connection that one produces fibrin, the substrate for the other. A potent example of a more subtle and yet important connection lies in thrombin’s ability to generate an inhibitor of fibrinolysis. This exemplifies the pivotal role of thrombin in haemostasis, a consequence of its broad range of activities. Thrombin activates the zymogen form of a basic carboxypeptidase, generating an active enzyme called carboxypeptidase U, carboxypeptidase R, plasma carboxypeptidase B or TAFIa, thrombin activable fibrinolysis inhibitor (reviewed in 1-4; references from the reviews are not cited here). The activated carboxypeptidase removes C-terminal basic residues from fibrin. C-terminal Lys residues are key to binding t-PA and plasminogen, and thus to generating plasmin activity, a mechanism by which partially-lysed fibrin amplifies plasmin formation. The removal of these C-terminal Lys residues therefore decreases local fibrinolytic activity (Fig. 1). Several recent studies, two of which appear in this issue of Thrombosis and Haemostasis (5, 6), add to our knowledge of this activity and its potential role in thromboembolic disease.

Characterization of Heterodimeric Alkaline Phosphatases from Escherichia coli: An Investigation of Intragenic Complementation

Escherichia coli alkaline phosphatase (EC 3.1.3.1) belongs to a rare group of enzymes that exhibit intragenic complementation. When certain mutant versions of alkaline phosphatase are combined, the resulting heterodimeric enzymes exhibit a higher level of activity than would be expected based upon the relative activities of the parental enzymes. Nine previously identified alkaline phosphatase complementation mutants were re-examined in this work in order to determine a molecular explanation of intragenic complementation in this experimental system. The locations of these mutations were determined by DNA sequence analysis after PCR amplification of the phosphatase-negative phoA gene. Most of the mutations involved ligands to metal-binding sites. Each of the mutant enzymes was re-created by site-specific mutagenesis, expressed, purified, and kinetically characterized. To investigate cooperativity between the two subunits, we analyzed heterodimeric forms of some of the site-specific mutant enzymes. To enable the isolation of the heterodimeric alkaline phosphatase in pure form, the overall charge of one subunit was altered by replacing the C-terminal Lys residue with three Asp residues. This modification had no effect on the kinetic properties of the enzyme. Heterodimeric alkaline phosphatases were created using two methods: (1) in vitro formation by dissociation at acid pH followed by reassociation at slightly alkaline pH conditions in the presence of zinc and magnesium ions; and (2) in vivo expression from a plasmid carrying two different phoA genes. Increases in k cat, as well as a large reduction in the p-nitrophenyl phosphate K m were observed for certain combinations of mutant enzymes. These results suggest that the structural assembly of E. coli alkaline phosphatase into the dimer induces cooperative interactions between the monomers necessary for the formation of the functional form of the holoenzyme.

Genomic organization of three novel toxins from the scorpion Buthus martensi Karsch that are active on potassium channels

The cDNA and genomic DNA of three novel toxins from the scorpion Buthus martensi Karsch that are active on K+ channels, designated BmKTX (where KTX is kaliotoxin), BmTX1 and BmTX2, were cloned and sequenced. On the basis of their known amino acid sequences, gene-specific primers for 3ʹ and 5ʹ rapid amplification of cDNA ends (RACE) were designed and synthesized. By overlapping the two partial cDNA sequences obtained by 3ʹ and 5ʹ RACE, their full-length cDNA sequences were completed. BmKTX encodes a signal peptide of 22 amino acid residues and a mature toxin of 38 residues, whereas BmTX1 and BmTX2 encode signal peptides of 20 and 21 residues respectively and a mature toxin of 38 residues for each. Their cDNA-deduced amino acid sequences were totally consistent with those determined except that the C-terminus of BmKTX had an additional Gly residue, which was removed during post-translational processing and was indispensable for the amidation of its C-terminal Lys residue. In addition, the first deduced amino acid for both BmTX1 and BmTX2 is Gln instead of pyro-Glu in the reported toxins, which obviously also undergoes post-translational processing. The genomic DNA species of these three toxins were also amplified by PCR, then cloned and sequenced. They all consisted of two exons disrupted by a small single intron. All of these introns were inserted within the signal peptides at position -6 for BmKTX and at position -5 for both BmTX1 and BmTX2 upstream of the mature toxins, and consisted of 87, 87 and 80 bp respectively.

Genomic organization of three novel toxins from the scorpion Buthus martensi Karsch that are active on potassium channels

The cDNA and genomic DNA of three novel toxins from the scorpion Buthus martensi Karsch that are active on K(+) channels, designated BmKTX (where KTX is kaliotoxin), BmTX1 and BmTX2, were cloned and sequenced. On the basis of their known amino acid sequences, gene-specific primers for 3' and 5' rapid amplification of cDNA ends (RACE) were designed and synthesized. By overlapping the two partial cDNA sequences obtained by 3' and 5' RACE, their full-length cDNA sequences were completed. BmKTX encodes a signal peptide of 22 amino acid residues and a mature toxin of 38 residues, whereas BmTX1 and BmTX2 encode signal peptides of 20 and 21 residues respectively and a mature toxin of 38 residues for each. Their cDNA-deduced amino acid sequences were totally consistent with those determined except that the C-terminus of BmKTX had an additional Gly residue, which was removed during post-translational processing and was indispensable for the amidation of its C-terminal Lys residue. In addition, the first deduced amino acid for both BmTX1 and BmTX2 is Gln instead of pyro-Glu in the reported toxins, which obviously also undergoes post-translational processing. The genomic DNA species of these three toxins were also amplified by PCR, then cloned and sequenced. They all consisted of two exons disrupted by a small single intron. All of these introns were inserted within the signal peptides at position -6 for BmKTX and at position -5 for both BmTX1 and BmTX2 upstream of the mature toxins, and consisted of 87, 87 and 80 bp respectively.

Regulatory mechanism of fibrinolysis system by thrombin activatable fibrinolysis inhibitor (TAFI)

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a 60-kDa plasma protein that has been shown to be identical to plasma carboxypeptidase B (CPB) and carboxypeptidase U (CPU). TAFI is activated by thrombomodulin (TM)-bound thrombin and specifically removes the C-terminal Lys and Arg by its CPB activity. One of its target substrates is the C-terminal Lys residue in the alpha-chain of plasmin-digested fibrin, which is critical for plasminogen binding to fibrin. Thus, its removal seems to be the main mechanism through which TAFI inhibits fibrinolysis. In this article, relevance of C-terminal Lys of plasmin-digested fibrin in fibrinolysis is described, and then possible roles of TAFI and TM-bound thrombin in a cross-talk between coagulation and fibrinolysis are discussed.

Characterization of the Interaction between the Wilson and Menkes Disease Proteins and the Cytoplasmic Copper Chaperone, HAH1p

Wilson disease (WD) and Menkes disease (MNK) are inherited disorders of copper metabolism. The genes that mutate to give rise to these disorders encode highly homologous copper transporting ATPases. We use yeast and mammalian two-hybrid systems, along with an in vitro assay to demonstrate a specific, copper-dependent interaction between the six metal-binding domains of the WD and MNK ATPases and the cytoplasmic copper chaperone HAH1. We demonstrate that several metal-binding domains interact independently or in combination with HAH1p, although notably domains five and six of WDp do not. Alteration of either the Met or Thr residue of the HAH1p MTCXXC motif has no observable effect on the copper-dependent interaction, whereas alteration of either of the two Cys residues abolishes the interaction. Mutation of any one of the HAH1p C-terminal Lys residues (Lys(56), Lys(57), or Lys(60)) to Gly does not affect the interaction, although deletion of the 15 C-terminal residues abolishes the interaction. We show that apo-HAH1p can bind in vitro to copper-loaded WDp, suggesting reversibility of copper transfer from HAH1p to WD/MNKp. The in vitro HAH1/WDp interaction is metalospecific; HAH1 preincubated with Cu(2+) or Hg(+) but not with Zn(2+), Cd(2+), Co(2+), Ni(3+), Fe(3+), or Cr(3+) interacted with WDp. Finally, we model the protein-protein interaction and present a theoretical representation of the HAH1p.Cu.WD/MNKp complex.

Plasminogen kringle 4 binds the heptapeptide fragment 44-50 of the plasminogen N-terminal peptide.

The interaction between the plasminogen kringle 4 module and a synthetic peptide corresponding to the tryptic heptapeptide fragment Ala-Phe-Gln-Tyr-His-Ser-Lys (AFQYHSK), segment 44-50 of the plasminogen N-terminal peptide (Wiman and Wallén, Eur J Biochem 1975; 50:489-494), has been investigated by 1H-NMR spectroscopy. AFQYHSK, as well as the shorter fragments thereof, FQYHSK, QYHSK and YHSK, all bound to kringle 4 with equilibrium association constant (Ka) values ranging between 2.5 and 8.5 mM-1. The NMR evidence also indicates that binding is mediated by the canonical kringle lysine binding site and involves the C-terminal Lys residue of the ligand peptide. The results (a) support a potential interaction between plasminogen Lys-binding kringles and the N-terminal activation peptide, and (b) unambiguously demonstrate the capability of such kringles to bind polypeptides ending with C-terminal lysine.

Epitope characterization by modifications of antigens and by mapping on resin-bound peptides Discriminating epitopes near the C-terminus and N-terminus of Escherichia coli ribosomal protein S13

The feasibility of using antigen modifications and synthetic resin-bound peptides to distinguish closely related epitopes has been demonstrated in this report. Epitopes recognized by five monoclonal antibodies (MAbs) specific to Escherichia coli ribosomal protein S13 have been located near the C-terminus and N-terminus of the S13 molecule; these epitopes were characterized by modifications of antigens and by utilization of peptides of increasing length synthesized on aminomethyl crosslinked polystyrene resin. Three of these MAbs react with the C-terminal peptide S13 84–117 which has five Lys residues clustered within the last 16 amino acids. Phthalylation of Lys residues almost eliminated the binding of two MAbs and reduced binding of the third by 50%. Removal of the C-terminal Lys residue(s) at the S13 C-terminus with carboxypeptidase B has no effect on the binding of these three MAbs. A 23-residue peptide corresponding to S13 95–117 was synthesized by a modified Merrifield solid phase method. Samples of resin with peptides of increasing length were obtained after each cycle of amino acid coupling. The peptides were deprotected without hydrolysis of the peptide-resin linkage and used in an enzyme immunoassay to detect the extent of MAb interaction with the lengthening peptides. The epitopes recognized by the two MAbs more sensitive to Lys modification have been localized in S13 97–117. The third MAb binds to S13 98–117 but binds more strongly when the sequence is lengthened. Two MAbs directed toward the N-terminal 22 residues of S13 were similarly characterized. Binding of one MAb, little affected by phthalylation, required the N-terminal residue of S13 to be present in the synthetic peptide. The other MAb, whose binding was inhibited by phthalylation, bound to the synthetic S13 2–22 and bound more strongly to the synthetic S13 1–22.