A general method for highly selective cross-linking of unprotected polypeptides via pH-controlled modification of N-terminal alpha-amino groups

A method is described for the highly selective modification of the alpha-amino groups at the N-termini of unprotected peptides to form stable, modified peptide intermediates which can be covalently coupled to other molecules or to a solid support. Acylation with iodoacetic anhydride at pH 6.0 occurs with 90-98% selectivity for the alpha-amino group, depending on the N-terminal residue (as shown with a series of model hexapeptides containing a competing Lys residue). Although Cys residues must be protected (reversibly or irreversibly) before the anhydride reaction, there are no detectable side reactions of the alpha-amino moiety--of the reagent or of modified peptide--with the side chains of His, Met, or Lys. The reaction works well in denaturants, so that inhibitory effects of noncovalent structure can be minimized. In a second step the iodoacetyl-peptide can be reacted with a thiol group on a protein, on a solid chromatography matrix, on a spectroscopic probe, etc. This is illustrated by reaction of a series of N alpha-iodoacetyl-peptides with murine interferon-gamma, which contains a C-terminal Cys residue. Data are presented which suggest that this iodoacetic anhydride scheme is superior in selectivity for alpha-amino groups to conventional chemical approaches to cross-linking such as use of 2-iminothiolane or N-hydroxysuccinimide-activated carboxylic acid esters. The reaction is ideally suited for modifying peptide fragments, as pure species or as mixtures, derived from proteolytic or chemical fragmentation of proteins. Furthermore, polypeptides synthesized biosynthetically, for example via recombinant DNA techniques, can be cross-linked in this way.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relative reactivity of lysine and other peptide-bound amino acids to oxidation by hypochlorite

Antibacterial and inflammatory responses of neutrophils and macrophages produce hypochlorite as a major oxidant. Numerous side chains of amino acids found in extracellular proteins can be modified by hypochlorite, including His, Arg, Tyr, Lys, Trp, and Met. We studied the relative reactivity of each of these amino acid residues in short N-blocked peptides, where other residues in the peptide were highly resistant to hypochlorite attack. Hypochlorite treatment led to modified peptides in each case, which were detected by changes in retention on reversed-phase HPLC. A distinct single product, consuming two equivalents of hypochlorite per equivalent of peptide, was obtained from the Lys-containing peptides. UV spectroscopy, nuclear magnetic resonance (NMR), and electrospray/mass spectroscopy identified this product as the dichloramine at the epsilon-amino group of the Lys side chain. The dichloramine at Lys did not decompose to form a detectable amount of carbonyl reactive with dinitrophenylhydrazine. The dichloramine at Lys did however quantitatively revert back to Lys during HCl digestion of the tetrapeptide for amino acid analysis, with simultaneous modification of the adjacent Phe residue. The formation of the dichloramine at Lys was not blocked by peptides or acetylated amino acids that contained Tyr, His, or Arg. In contrast, the presence of equimolar Met-containing peptide, or N-Acetyl-Trp, both inhibited the formation of the dichloramine at Lys. Thus, Met and Trp side chains of proteins might be able to protect Lys from chloramine formation under some circumstances, but this interpretation must consider that Met and Trp are typically found in relatively inaccessible hydrophobic sites, whereas lysine is typically exposed on the protein surface. The hierarchy of amino acid reactivities examined here will aid in the prediction of residues in biological samples most likely to be modified by hypochlorite.     

Proteolytic excision and in situ cyclization of a bioactive loop from an REI-VL presentation scaffold.

Covalent cyclization of peptides is an important tool in structure-function analysis of bioactive peptides, because it constrains the molecule to enrich or exclude the receptor-bound conformation. Previously we described a 2-step procedure for cyclizing purified, native peptides in aqueous solution by reacting a Met or Lys side chain with an iodoacetylated N-terminus (Wood SJ, Wetzel R, 1992a, Int J Pept Protein Res 39:533-539). We show here that the cyclization reaction scheme can be extended to peptides excised from proteins by endo-LysC proteolysis, which generates fragments terminating with Lys. To illustrate the method, we used an immunoglobulin VL domain (REI-VL) with an RGD-containing sequence engineered into its CDR3 and flanked by Lys residues. This REI-VL/RGD hybrid displayed an IC50 of 24 nM for ligand competition at the platelet fibrinogen receptor alpha IIb beta 3. The RGD-containing peptide excised by endo-LysC from the REI-VL presentation scaffold exhibited an IC50 of about 50 nM, and the corresponding cyclized peptide, and IC50 of about 10 nM. Significantly, both the N alpha-acylation and the cyclization reactions occur efficiently even in the context of the other endo-LysC fragments of REI-VL, which suggests that the reaction may prove useful in converting mixtures of endo-LysC products of many proteins into the corresponding cyclic peptides in situ.     

Synthesis and kinetics of cyclization of MHC class II-derived cyclic peptide vaccine for diabetes.

Conformationally constrained cyclic peptides are known to be better vaccines because of their ability to mimic the native structure of a protein against which an immune response is sought. To test the hypothesis of using conformationally constrained, disease-associated, MHC-derived peptides as vaccines for the prevention of type I diabetes, a 22 amino acid nonobese diabetic(NOD) mouse MHC class II-derived synthetic peptide was cyclized by the formation of end-to-end disulfide bonds and used to prevent diabetes and insulitis in NOD mice. The peptide was synthesized by Fmoc chemistry and cyclized using two methods: a commercially available cyclizing resin (Ekathiox) and air oxidation. When a 10 m excess of resin was used, the Ekathiox yielded a substantial amount of cyclic peptide with few or no side reactions. The kinetics of cyclization by air oxidation at different temperatures indicated that increasing both temperature and pH decreased the cyclization time significantly. Air oxidation at pH 10 at 37-55 degrees C yielded the desired product within 2 h.     

Determination of intrinsic hydrophilicity/hydrophobicity of amino acid side chains in peptides in the absence of nearest-neighbor or conformational effects

Understanding the hydrophilicity/hydrophobicity of amino acid side chains in peptides/proteins is one the most important aspects of biology. Though many hydrophilicity/hydrophobicity scales have been generated, an "intrinsic" scale has yet to be achieved. "Intrinsic" implies the maximum possible hydrophilicity/hydrophobicity of side chains in the absence of nearest-neighbor or conformational effects that would decrease the full expression of the side-chain hydrophilicity/hydrophobicity when the side chain is in a polypeptide chain. Such a scale is the fundamental starting point for determining the parameters that affect side-chain hydrophobicity and for quantifying such effects in peptides and proteins. A 10-residue peptide sequence, Ac-X-G-A-K-G-A-G-V-G-L-amide, was designed to enable the determination of the intrinsic values, where position X was substituted by all 20 naturally occurring amino acids and norvaline, norleucine, and ornithine. The coefficients were determined by reversed-phase high-performance liquid chromatography using six different mobile phase conditions involving different pH values (2, 5, and 7), ion-pairing reagents, and the presence and absence of different salts. The results show that the intrinsic hydrophilicity/hydrophobicity of amino acid side chains in peptides (proteins) is independent of pH, buffer conditions, or whether C(8) or C(18) reversed-phase columns were used for 17 side chains (Gly, Ala, Cys, Pro, Val, nVal, Leu, nLeu, Ile, Met, Tyr, Phe, Trp, Ser, Thr, Asn, and Gln) and dependent on pH and buffer conditions, including the type of salt or ion-pairing reagent for potentially charged side chains (Orn, Lys, His, Arg, Asp, and Glu).     

Characterization of an alkaline subtilopeptidase type Pfizer.

The physiochemical properties, amino acid composition and profile of the the tryptic peptides for an alkaline subtilopeptidase type Pfizer have been determined. The enzyme is stable in the pH range from 5 to 10, has a pH optimum of 9.5 to 10, and is relatively stable for a period of 2 h up to a temperature of 50C. Homogeneity was demonstrated by electrophoretic techniques and the mobilities indicated on isoelectric point of 8.7. The molecular weight was found to be 25,000 by gel filtration. The amino acid composition was found to be Ala32, Arg4, Aspgamma8, Glu15, Gly29, His4, Ile9, Leu13, Lys11, Met5, Phe4, Pro14, Ser31, Thr17, Tyr9, Val22, a total of 247 amino acid residues. The enzyme does not contain either disulfide bonds or cysteine, and lacks tryptophan as well. The N-terminal end-group residue is alanine: the C-terminal amino acid is arginine. Tryptic hydrolysis of the enzyme produced 15 peptides which were separated by gradient elution on Dowex 50-X2. The amino acid composition of each appropriately purified tryptic peptide was established.     

Fluorescent, internally quenched, peptides for exploring the pH-dependent substrate specificity of cathepsin B.

Cathepsin B is a cysteine protease that in tumor tissues is localized in both acidic lysosomes and extracellular spaces. It can catalyze the cleavage of peptide bonds by two mechanisms: endoproteolytic attack with a pH optimum around 7.4, and attack from the C-terminus with a pH optimum at 4.5-5.5. In this work, seven fluorescent, internally quenched, decapeptides have been synthesized using the prototypical cathepsin B selective substrate Z-Phe-Arg-AMC as a lead, and used to identify the structural factors determining the susceptibility of peptides to hydrolysis at acidic and neutral pH values. Each peptide differs from the others in one amino acid (residue 6) and contains a highly fluorescent Nma group linked to the alpha-amino function of the N-terminal Orn residue and a Dnp group linked to the side chain of the Lys(8) residue acting as a quencher. Proteolytic cleavage was monitored by measuring the increase of fluorescence at 440 nm upon excitation at 340 nm, and the cleavage sites were determined by HPLC followed by ESI-MS analysis. Peptides containing Ala or Phe at position 6 are good substrates for the enzyme at both pH 5.0 and 7.4. By contrast, those containing Glu, Asp, Lys or Val are not cleaved at all by cathepsin B at pH 7.4, and are poorly hydrolyzed at pH 5.0. These findings provide new information for the rational design of cathepsin B-activated peptide-containing anticancer drugs.     

Studies on Nα-acylated proteins: The N-terminal sequences of two muscle enolases

A standard procedure for the identification of the N-terminal amino acid in N alpha-acylated proteins has been developed. After exhaustive proteolysis, the amino acids with blocked alpha-amino groups are separated from positively charged, free amino acids by ion exchange chromatography and subjected to digestion with acylase I. Amino acid analysis before and after the acylase treatment identifies the blocked N-terminal amino acid. A survey of acylamino acid substrates showed that acylase will liberate all the common amino acids except Asp, Cys or Pro from their N-acetyl-and N-butyryl derivatives, and will also catalyze the hydrolysis of N-formyl-Met and N-myristyl-Val. Thus, the procedure cannot identify acylated Asp, Cys or Pro, nor, because of the ion exchange step, N alpha-acyl-derivatives of Arg, Lys or His. Whenever the protease treatment releases free acylamino acids, the remaining amino acids should be detected. When applied to several proteins, the procedure confirmed known N-terminal acylamino acids and identified acyl-Ser in enolases from chum and coho salmon muscle and in pyruvate kinase from rabbit muscle, and acyl-Thr in phosphofructokinase from rabbit muscle. The protease-acylase assay has been used to identify blocked peptides from CNBr- or protease-treated proteins. When such peptides were treated with 1 N HCl at 110 degrees for 10 min, sufficient yields of deacylated, mostly intact, peptide were obtained to permit direct automatic sequencing. The N-terminal sequences of rabbit muscle and coho salmon enolase were determined in this way and are compared to each other and to the sequence of yeast enolase.     

Synthesis of novel basic head-to-side-chain cyclic dynorphin A analogs: strategies and side reactions

Novel N-terminus-to-side-chain cyclic analogs of the opioid peptide dynorphin (Dyn) A-(1-11)NH(2) were prepared that retain the basicity of the N-terminal amine and restrict the backbone conformation around the important Tyr(1) residue. Cyclic peptides were synthesized in which the N-terminal amine and the N(epsilon)-amine of a Lys at position 3 or 5 were attached to the alpha-carbon and carbonyl of an acetyl group, respectively. Several synthetic strategies were explored with detailed analysis of the side reactions in order to obtain the desired cyclic peptides. One of the side reactions observed involved premature loss of the N-terminal 9-fluorenylmethoxycarbonyl (Fmoc) group during the neutralization step following deprotection of the Mtt (4-methyltrityl) protecting group from the side chain of Lys. The successful strategy involved the synthesis of the linear peptide up through Gly(2) and functionalization through the N(epsilon)-amine of Lys. A linear N-terminal alkylated analog was prepared by alkylation of the peptide on the resin with an equimolar amount of bromoacetamide, followed by treatment of the peptide with Fmoc-OSu prior to cleavage from the resin to facilitate separation by reversed phase high performance liquid chromatography of unreacted peptide from the desired alkylated product. The novel N-terminal cyclic Dyn A analogs and the linear analog were evaluated for their opioid receptor affinities. These peptides exhibited large losses in affinity for opioid receptors; the low affinity of the linear N-terminal alkylated peptide suggested that the alpha-acetamide group on the N-terminal amine resulted in unfavorable interactions with opioid receptors.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Photooxidation of specific residues in alpha-crystallin polypeptides

Singlet oxygen is a biologically important, photochemically generated species that preferentially oxidizes His, Trp, and Met residues of protein molecules. Calf alpha-crystallin was photooxidized with use of meso-tetra(p-sulfonatophenyl)porphyrin (TPPS) and uroporphyrin (UP) as singlet oxygen generators. The effects of photooxidation were monitored by analyzing the changes in alpha-crystallin peptide maps obtained by reversed-phase HPLC using a photodiode array absorbance detector. The reaction led to the loss of six specific peptides, five of which contained photooxidizable residues. Peptides containing His-97 and His-154 from the A chain and Met-68 from the B chain are preferentially photooxidized, suggesting that those residues have access to singlet oxygen. Trp residues in the N-terminal region are converted to NFK, whereas Trp-60 in the B chain is not photooxidized strongly suggesting that the former are close to the surface of alpha-crystallin while the latter Trp residue is buried. Only one peptide that is lost from the peptide maps does not contain a photooxidizable group; however, this peptide does contain an apparently undigested Lys residue. It is suggested that it forms a cross-link with a photooxidized His residue.     

The cyclopeptides with the multi-His motif as ligands for copper(II)

The coordination properties of cyclic octapeptides with multi-His motif: c(His-Gly-His-Xaa-His-Gly-His-Xaa) where Xaa = Asp or Lys, were investigated. The binding abilities of this peptides towards Cu(II) ions were studied by using different analytic methods as: potentiometry, spectroscopy and mass spectrometry. The obtained results show that the studied peptides in physiological related pH prefer formation of the species with the {4N_Im} binding mode. The efficiency of Cu(II) binding depends on additional side chain groups Asp or Lys. Additionally the analysis of results for His containing cyclopeptides with different numbers of amino acid residues in cyclopeptide ring e.g. four, eight shows that in higher pH in both cases the binding by four amide nitrogens is not observed in the case of α-amino acid peptides.     

Making Ends Meet: Microwave-Accelerated Synthesis of Cyclic and Disulfide Rich Proteins Via In Situ Thioesterification and Native Chemical Ligation

The development of synthetic methodologies for cyclic peptides is driven by the discovery of cyclic peptide drug scaffolds such as the plant-derived cyclotides, sunflower trypsin inhibitor 1 (SFTI-1) and the development of cyclized conotoxins. Currently, the native chemical ligation reaction between an N-terminal cysteine and C-terminal thioester group remains the most robust method to obtain a head-to-tail cyclized peptide. Peptidyl thioesters are effectively generated by Boc SPPS. However, their generation is challenging using Fmoc SPPS because thioester linkers are not stable to repeated piperidine exposure during deprotection. Herein we describe a Fmoc-based protocol for synthesizing cyclic peptides adapted for microwave assisted solid phase peptide synthesis. The protocol relies on the linker Di-Fmoc-3,4-diaminobenzoic acid, and we demonstrate the use of Gly, Ser, Arg and Ile as C-terminal amino acids (using HBTU and HATU as coupling reagents). Following synthesis, an N-acylurea moiety is generated at the C-terminal of the peptide; the resin bound acylurea peptide is then deprotected and cleaved from the resin. The fully deprotected peptide undergoes thiolysis in aqueous buffer, generating the thioester in situ. Ultimately, the head-to-tail cyclized peptide is obtained via native chemical ligation. Two naturally occurring cyclic peptides, the prototypical Möbius cyclotide kalata B1 and SFTI-1 were synthesized efficiently, avoiding potential branching at the diamino linker, using the optimized protocol. In addition, we demonstrate the possibility to use the approach for the synthesis of long and synthetically challenging linear sequences, by the ligation of two truncated fragments of a 50-residue long plant defensin.     

Effect of lysine residues on the deamidation reaction of asparagine side chains

The effect of lysine residues on the deamidation reaction of the asparagine side chain has been studied on the peptide and on its lysine-acetylated derivative in a wide range of pH values. The amino acid sequence of these peptides is similar to the local sequence flanking the labile Asn-67 in RNAse A. The experimental data show that Lys influences both the deamidation rate and the relative yield of the two reaction products, i.e., the aspartic acid and beta-aspartic acid containing peptide. These effects are pH dependent and can be rationalized based on the mechanism previously proposed for the deamidation reaction via succinimide derivative.     

A consensus tetrapeptide selected by phage display adopts the conformation of a dominant discontinuous epitope of a monoclonal anti-VWF antibody that inhibits the von Willebrand factor-collagen interaction

Monoclonal antibody (mAb) 82D6A3 is an anti-von Willebrand factor (VWF) mAb directed against the A3-domain of VWF that inhibits the VWF binding to fibrillar collagens type I and III in vitro and in vivo. To identify the discontinuous epitope of this mAb, we used phage display, mutant analysis, and peptide modeling. All 82D6A3-binding phages displayed peptides containing the consensus sequence SPWR that could be aligned with P981W982 in the VWF A3-domain. Next, the binding of mAb 82D6A3 to 27 Ala mutants with mutations in the A3-domain of VWF revealed that amino acids Arg963, Pro981, Asp1009, Arg1016, Ser1020, Met1022, and His1023 are part of the epitope of mAb 82D6A3. Inspection of residues Ser1020, Arg1016, Pro981, and Trp982 in the three-dimensional structure of the A3-domain demonstrated that these residues are close together in space, pointing out that the structure of the SPWR consensus sequence might mimic this discontinuous epitope. Modeling of a cyclic 6-mer peptide containing the consensus sequence and superposition of its three-dimensional structure onto the VWF A3-domain demonstrated that the Ser and Arg in the peptide matched the Ser1020 and Arg1016 in the A3-domain. The Pro residue of the peptide served as a spacer, and the side chain of the Trp pointed in the direction of Trp982. In conclusion, to our knowledge, this is the first report where a modeled peptide containing a consensus sequence could be fitted onto the three-dimensional structure of the antigen, indicating that it might adopt the conformation of the discontinuous epitope.     

Effect of lysine side chain length on intra-helical glutamate--lysine ion pairing interactions

Ion-pairing interactions are important for protein stabilization. Despite the apparent electrostatic nature of these interactions, natural positively charged amino acids Lys and Arg have multiple methylenes linking the charged functionality to the backbone. Interestingly, the amino acids Lys and Orn have positively charged side chains that differ by only one methylene. However, only Lys is encoded and incorporated into proteins. To investigate the effect of side chain length of Lys on ion-pairing interactions, a series of 12 monomeric alpha-helical peptides containing potential Glu-Xaa (i, i+3), (i, i+4) and (i, i+5) (Xaa = Lys, Orn, Dab, Dap) interactions were studied by circular dichroism (CD) spectroscopy at pH 7 and 2. At pH 7, no Glu-Xaa (i, i+5) interaction was observed, regardless of the Xaa side chain length. Furthermore, only Lys was capable of supporting Glu-Xaa (i, i+3) interactions, whereas any Xaa side chain length supported Glu-Xaa (i, i+4) interactions. Side chain conformational analysis by molecular mechanics calculations showed that the side chain length of Lys enables the Glu-Xaa (i, i+3) interaction with lower energy conformations compared to residues with side chain lengths shorter than that of Lys. Furthermore, these calculated low energy conformers were consistent with conformations of intra-helical Glu-Lys salt bridges in a non-redundant protein structure database. Importantly, the CD spectra for peptides with Glu-Lys interactions did not alter significantly upon changing the pH because of a greater contribution to these interactions by forces other than electrostatics. Incorporating side chains just one methylene shorter (Orn) resulted in significant pH dependence or lack of interaction, suggesting that nature has chosen Lys to form durable interactions with negatively charged functional groups.     

Helicity of short E-R/K peptides

Understanding the secondary structure of peptides is important in protein folding, enzyme function, and peptide‐based drug design. Previous studies of synthetic Ala‐based peptides (>12 a.a.) have demonstrated the role for charged side chain interactions involving Glu/Lys or Glu/Arg spaced three (i, i + 3) or four (i, i + 4) residues apart. The secondary structure of short peptides (<9 a.a.), however, has not been investigated. In this study, the effect of repetitive Glu/Lys or Glu/Arg side chain interactions, giving rise to E‐R/K helices, on the helicity of short peptides was examined using circular dichroism. Short E‐R/K–based peptides show significant helix content. Peptides containing one or more E‐R interactions display greater helicity than those with similar E‐K interactions. Significant helicity is achieved in Arg‐based E‐R/K peptides eight, six, and five amino acids long. In these short peptides, each additional i + 3 and i + 4 salt bridge has substantial contribution to fractional helix content. The E‐R/K peptides exhibit a strongly linear melt curve indicative of noncooperative folding. The significant helicity of these short peptides with predictable dependence on number, position, and type of side chain interactions makes them an important consideration in peptide design.     

Investigating the cationic side chains of the antimicrobial peptide tritrpticin: hydrogen bonding properties govern its membrane-disruptive activities

The positively charged side chains of cationic antimicrobial peptides are generally thought to provide the initial long-range electrostatic attractive forces that guide them towards the negatively charged bacterial membranes. Peptide analogs were designed to examine the role of the four Arg side chains in the cathelicidin peptide tritrpticin (VRRFPWWWPFLRR). The analogs include several noncoded Arg and Lys derivatives that offer small variations in side chain length and methylation state. The peptides were tested for bactericidal and hemolytic activities, and their membrane insertion and permeabilization properties were characterized by leakage assays and fluorescence spectroscopy. A net charge of +5 for most of the analogs maintains their high antimicrobial activity and directs them towards preferential insertion into model bacterial membrane systems with a similar extent of burial of the Trp side chains. However the peptides exhibit significant functional differences. Analogs with methylated cationic side chains cause lower levels of membrane leakage and are associated with lower hemolytic activities, making them potentially attractive pharmaceutical candidates. Analogs containing the Arg guanidinium groups cause more membrane disruption than those containing the Lys amino groups. Peptides in the latter group with shorter side chains have increased membrane activity and conversely, elongating the Arg residue causes slightly higher membrane activity. Altogether, the potential for strong hydrogen bonding between the four positive Arg side chains with the phospholipid head groups seems to be a determinant for the membrane disruptive properties of tritrpticin and many related cationic antimicrobial peptides.     

Fibril formation by primate, rodent, and Dutch-hemorrhagic analogues of Alzheimer amyloid beta-protein.

Deposition of extraneuronal fibrils that assemble from the 39-43 residue beta/A4 amyloid protein is one of the earliest histopathological features of Alzheimer's disease. We have used negative-stain electron microscopy, Fourier-transform infrared (FT-IR) spectroscopy, and fiber X-ray diffraction to examine the structure and properties of synthetic peptides corresponding to residues 1-40 of the beta/A4 protein of primate [Pm(1-40); human and monkey], rodent [Ro(1-40); with Arg5-->Gly, Tyr10-->Phe, and His13-->Arg], and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D) [Du(1-40); with Glu22-->Gln]. As controls, we examined a reverse primate sequence [Pm*(40-1)] and an extensively substituted primate peptide [C(1-40); with Glu3-->Arg, Arg5-->Glu, Asp7-->Val, His13-->Lys, Lys16-->His, Val18-->Asp, Phe19-->Ser, Phe20-->Tyr, Ser26-->Pro, Ala30-->Val, Ile31-->Ala, Met35-->norLeu, Gly38-->Ile, Val39-->Ala, and Val40-->Gly]. The assembly of these peptides was studied to understand the relationship between species-dependent amyloid formation and beta/A4 sequence and the effect of a naturally occurring point mutation of fibrillogenesis. The three N-terminal amino acid differences between Pm(1-40) and Ro(1-40) had virtually no effect on the morphology or organization of the fibrils formed by these peptides, indicating that the lack of amyloid deposits in rodent brain is not due directly to specific changes in its beta/A4 sequence. beta-Sheet and fibril formation, judged by FT-IR, was maximal within the pH range 5-8 for Pm(1-40), pH 5-10.5 for Du(1-40), and pH 2.5-8 for Ro(1-40).(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation and determination of alpha-amino acids by boroxazolidone formation

Reaction of an alpha-amino acid (alpha-AA) with 1,1-diphenylborinic acid (DPBA) leads to the formation of a kinetically stable adduct at pH 2-5 in which both the alpha-amino and the alpha-carboxyl groups are bound to boron forming a cyclic mixed anhydride termed a boroxazolidone. In this adduct, the greater than N:B bond is coordinate, involving the free electron pair of nitrogen, thereby satisfying the octet rule for the second electron shell of boron (Group IIIA). Consequently, the alpha-amino function of the boroxazolidone can be primary, secondary, or tertiary, as demonstrated by boroxazolidone formation with glycine, N-methylglycine, and N,N-dimethylglycine. On reaction with DPBA, the alpha-AA moiety of N-terminal gamma-glutamyl peptides is also derivatized as demonstrated by the formation of a glutathione boroxazolidone. The 1,1-diphenylboroxazolidone adducts of alpha-AA may be separated by reversed-phase (RP)-HPLC (AA-DPBA/RP-HPLC) enabling the derivatization procedure to be used as a precolumn reaction for alpha-AA analysis. Under the conditions we describe here, DPBA is not stably reactive with the epsilon-amino group of lysine. Furthermore, it does not complex with amide bonds of the peptide backbone or to any side chains of the common amino acids. Reaction of an alpha-AA mixture with DPBA, followed by RP-HPLC (AA-DPBA/RP-HPLC) is then a simple method by which to analyze alpha-AA in a mixture with peptides and amines. Precolumn reaction with DPBA may be used to separate peptides from alpha-AA and from those peptides which contain an alpha-AA moiety. Unreacted peptides are bound only weakly to the HPLC column and thus are separated from reacted alpha-amino acids which are retained as 1,1-diphenylboroxazolidones until their selective elution. This method is particularly suited for the analysis of alpha-amino acids that are derived from post-translational modification of protein side chains.