Automated carboxy-terminal sequence analysis of peptides and proteins using diphenyl phosphoroisothiocyanatidate.

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on the automation of the thiocyanate chemistry using acetic anhydride and trimethylsilylisothiocyanate (TMS-ITC) to derivatize the C-terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-terminal amino acid (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68-80). A major limitation of this approach was the need to activate the C-terminus with acetic anhydride. We now describe the use of a new reagent, diphenyl phosphoroisothiocyanatidate (DPP-ITC) and pyridine, which combines the activation and derivatization steps to produce peptidylthiohydantoins. Previous work by Kenner et al. (Kenner, G.W., Khorana, H.G., & Stedman, R.J., 1953, Chem. Soc. J., 673-678) with this reagent demonstrated slow kinetics. Several days were required for complete reaction. We show here that the inclusion of pyridine was found to promote the formation of C-terminal thiohydantoins by DPP-ITC resulting in complete conversion of the C-terminal amino acid to a thiohydantoin in less than 1 h. Reagents such as imidazole, triazine, and tetrazole were also found to promote the reaction with DPP-ITC as effectively as pyridine. General base catalysts, such as triethylamine, do not promote the reaction, but are required to convert the C-terminal carboxylic acid to a salt prior to the reaction with DPP-ITC and pyridine. By introducing the DPP-ITC reagent and pyridine in separate steps in an automated sequencer, we observed improved sequencing yields for amino acids normally found difficult to derivatize with acetic anhydride/TMS-ITC. This was particularly true for aspartic acid, which now can be sequenced in yields comparable to most of the other amino acids. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene and proteins (200 pmol to 5 nmol) noncovalently applied to Zitex (porous Teflon). The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids tested were found to sequence in good yield except for proline, which was found not to be capable of derivatization. In spite of this limitation, the methodology should be a valuable tool for the C-terminal sequence analysis of peptides and proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Improved initial yields in C-terminal sequence analysis by thiohydantoin chemistry using purified diphenylphosphoryl isothiocyanate: NMR evidence for a reaction intermediate in the coupling reaction

A thiohydantoin method for C-terminal sequence analysis of proteins on Zitex membranes involves derivatization of the free alpha-carboxyl group with diphenylphosphoryl isothiocyanate (DPPITC) plus treatment with pyridine to form a peptidylthiohydantoin derivative, cleavage of the thiohydantoin (TH) amino acid from the protein with potassium trimethylsilanolate, and identification of the released TH-amino acid by online reversed-phase HPLC. This automated chemistry, which was adapted to the Hewlett-Packard G 1009A sequencer, has been shown to identify two or three cycles on a wide variety of proteins, but suffers from low initial yields and instability of the DPPITC reagent. We report here an improved method for synthesis and purification of DPPITC. With this procedure the DPPITC reagent is a clear liquid that is stable at room temperature under vacuum for more than 9 months or for more than 24 months as a 1.0M solution in benzene at -20 degrees C. Using the purified reagent we were able to more than double the initial yield (from 30.7 to 72.4%) of TH-amino acid from a test protein and substantially decrease sequencer background. Examination of the reaction between DPPITC and the carboxylate of model N-terminally protected dipeptides with 31P NMR provides spectroscopic evidence for a postulated intermediate formed between the DPPITC and the peptide carboxylate. The reaction intermediate provides new insight into the coupling mechanism.     

Carboxylic acid-modified polyethylene: a novel support for the covalent immobilization of polypeptides for C-terminal sequencing.

We have developed a method for the covalent immobilization of peptides, for the purpose of C-terminal sequencing, to a novel solid support, carboxylic acid-modified polyethylene (PE-COOH) film. The peptides are attached by coupling the N-terminal amino group to the activated carboxyl groups of the film. Reagents for carboxyl group activation, including 1,3-dicyclohexylcarbodiimide (DCC), 1,1'-carbonyldiimidazole (CDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and 1,3-diisopropylcarbodiimide (DICD) were compared. The best yields were obtained with DCC for a variety of tested peptides and averaged approximately 50%. The covalent attachment at pH 6.7 of peptides was shown to occur predominantly thorough the alpha-amino group for the peptide, SIGSLAK, which after attachment to the PE-COOH support permitted the C-terminal lysine residue to be sequenced in good yield, indicating that the epsilon-amino group of lysine is not covalently attached. This support offers a number of advantages over other solid supports, such as silica and polyvinylidene difluoride, for C-terminal sequencing including (1) stability to base and the high temperatures (65 degrees C) employed for C-terminal sequencing, (2) wettability with both aqueous and organic solvents, (3) a high capacity (1.6 nmol/mm2) for covalent coupling of polypeptides, and (4) easy divisibility into 1 x 5-mm pieces for use in our continuous flow reactor (CFR), which is also used for automated N-terminal sequencing (Shively, J.E., Miller, P., & Ronk, M., 1987, Anal. Biochem. 163, 517-529). Automated C-terminal sequencing on these supports is described in the companion paper (Bailey, J.M., Shenoy, N.R., Ronk, M., & Shively, J.E., 1992, Protein Sci. 1, 68-80).     

Studies in C-terminal sequencing: New reagents for the synthesis of peptidylthiohydantoins

In previous studies aimed at the sequencing of peptides and proteins from the carboxy terminus, we have derivatized the C-terminus to a thiohydantoin using acetic anhydride and trimethylsilylisothiocyanate (TMS-ITC) and subsequently hydrolyzed it to form a shortened peptide capable of further degradation and an amino acid thiohydantoin which can be identified by reverse-phase HPLC. Current limitations to this chemistry include an inability to derivatize proline and low yields with asparagine and aspartic acid residues (Baileyet al., 1992). In an attempt to solve some of these problems, we have investigated the use of reagents other than acetic anhydride for the activation of the C-terminal carboxylic acid. These include 2-fluoro-1-methylpyridinium tosylate, 2-chloro-1-methylpyridinium iodide, and acetyl chloride. Addition of TMS-ITC to peptides activated by the 2-halo-pyridinium salts formed the expected peptidylthiohydantoin, but in addition formed a peptide chemically modified at the C-terminus which was blocked to C-terminal sequence analysis. This derivative was not obtained when either acetic anhydride or acetyl chloride was used for activation. Formation of this derivative was found to require the presence of an isothiocyanate reagent in addition to the halo-pyridinium salt. Sodium thiocyanate, TMS-ITC, and a new reagent for thiohydantoin synthesis, tributyltinisothiocyanate (TBSn-ITC), were all found to be capable of forming this analogue. Structural elucidation of the C-terminally modified amino acid revealed it to be a 2-imino-pyridinium analogue. Formation of this C-terminally blocked peptide could be minimized by the use of the 2-chloro-pyridinium reagent, rather than the 2-fluoro reagent, and by performing the reaction at a temperature of 50°C or lower. The 2-halo-pyridinium reagents offer potential advantages over the use of acetic anhydride for activation of the C-terminal carboxylic acid. These include: milder reaction conditions, faster reaction times, and the ability to sequence through C-terminal aspartic acid. The TBSn-ITC reagent was found to be comparable to TMS-ITC for formation of peptidylthiohydantoins.     

Chemical carboxyl-terminal sequence analysis of peptides and proteins using tribenzylsilyl isothiocyanate

A new derivatization reagent, tribenzylsilyl isothiocyanate (TBS-ITC), is applied to C-terminal peptide and protein sequencing. It has been successfully used to sequence six C-terminal residues of house apomyoglobin and a synthetic peptide at low nanomole levels. The chemistry involves activation with acetic anhydride, derivatization with TBS-ITC, and cleavage of derivatized C-terminal amino acid thiohydantoin with sodium hydroxide. The tribenzylsilyl is a bulky, electric donor group and is a good leaving group. It facilitates the nucleophilic attack of the NCS^–1 in the coupling reaction. The efficiency for C-terminal sequencing by TBS-ITC is about the same as that of acetyl isothiocyanate (AITC), which is a derivatizing reagent for C-terminal sequencing developed by our laboratory. TBS-ITC is much more stable than AITC and trimethylsilyl isothiocyanate (TMS-ITC). TBS-ITC is a solid with relatively long shelf life, whereas AITC and TMS-ITC are liquid and not stable at room temperature.     

Chemical Modification of the Carboxyl Terminal of Nisin A with Biotin does not Abolish Antimicrobial Activity Against the Indicator Organism, Kocuria rhizophila

Nisin is an antimicrobial peptide that is widely used for food preservation. Although it has potent activity against a number of food pathogens, suggesting potential therapeutic applications, its potential for clinical use is limited by proteolytic susceptibility and poor oral bioavailability. Derivatization of nisin could overcome these issues; however, many nisin analogues, prepared by modification at the N-terminal and C-terminal have previously been shown to be inactive. A method for the C-terminal modification was developed using biotinylation as a model derivative. Purification of the modified nisin was carried out using reverse phase chromatography. Confirmation of nisin modification was confirmed by Mass Spectroscopy. The C-terminal modification of nisin resulted in only a twofold reduction in antimicrobial activity of the conjugate against the indicator organism, Kocuria rhizophila. The C-terminal modification could be used to increase the therapeutic potential of nisin by creating more favourable physicochemical characteristics. This is the first study that showed that nisin modification can be carried out successfully without destroying its antimicrobial activity.     

Role of N- and C-terminal domains and non-homologous region in co-refolding of Thermotoga maritima β-glucosidase

Thermotoga maritima β-glucosidase consists of three structural regions with 721 amino acids: the N-terminal domain, middle non-homologous region and a C-terminal domain. To investigate the role of these domains in the co-refolding of two fragments into catalytically active form, five sites coding the amino acid residue at 244, 331 in the N-terminal domain, 403 in the non-homologous region, 476 and 521 in the C-terminal domain were selected to split the gene. All the 10 resultant individual fragments were obtained as insoluble inclusion bodies and found to be catalytically inactive. However, the catalytic activity was recovered when the two fragments derived from N-terminal and C-terminal peptides were co-refolded together. It is quite interesting to find that not only the complement polypeptides such as N476/477C but also the truncated combination (N476/522C, amino acid residues from 477 to 521 is truncated) and overlapped combination (N476/245C and N476/404C, amino acid residues from 245 to 476 and from 404 to 476 are overlapped) also gave catalytically active enzymes. Our results showed that folding motifs consisted of the complete N-terminal domain play an important role in the co-refolding of the polypeptides into the catalytically active form.     

C-Terminal Protein Characterization by Mass Spectrometry using Combined Micro Scale Liquid and Solid-Phase Derivatization

A sample preparation method for protein C-terminal peptide isolation has been developed. In this strategy, protein carboxylate glycinamidation was preceded by carboxyamidomethylation and optional α- and ϵ-amine acetylation in a one-pot reaction, followed by tryptic digestion of the modified protein. The digest was adsorbed on ZipTip_C18 pipette tips for sequential peptide α- and ϵ-amine acetylation and 1-ethyl-(3-dimethylaminopropyl) carbodiimide-mediated carboxylate condensation with ethylenediamine. Amino group-functionalized peptides were scavenged on N-hydroxysuccinimide-activated agarose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were exchanged directly on the support, eliminating sample transfer between the reaction steps. By this sequence of solid-phase reactions, the C-terminal peptide could be uniquely recognized in mass spectra of unfractionated digests of moderate complexity. The use of the sample preparation method was demonstrated with low-level amounts of a model protein. The C-terminal peptides were selectively retrieved from the affinity support and proved highly suitable for structural characterization by collisionally induced dissociation. The sample preparation method provides for robustness and simplicity of operation using standard equipment readily available in most biological laboratories and is expected to be readily expanded to gel-separated proteins.     

Sequencing of peptides and proteins from the carboxy terminus.

A new chemical method for carboxy-terminal (C-terminal) protein sequencing has been developed. This approach has been successfully used to sequence 5 residues of standard proteins and 5 to 10 residues of synthetic peptides at low nanomole levels. The sequencing procedure consists of converting the C-terminal amino acid into a thiohydantoin (TH) derivative, followed by transformation of the TH into a good leaving group by alkylation. Next, the alkylated TH is cleaved mildly and efficiently with (N = C V S)- anion, which simultaneously forms a TH on the newly truncated protein or peptide. Thus, after the initial TH derivatization, there is no return to a free carboxyl group at the C-terminus. An additional benefit of this method is that the alkylating moiety can be chosen with a variety of properties allowing for variation in the detection method. This chemistry has been adapted to automated protein sequencers with a cycle time of about 1 h.     

Polypeptide synthesis using an expressed peptide as a building block for condensation with a peptide thioester: application to the synthesis of phosphorylated p21Max protein(1-101).

An expressed peptide proved to be useful as a building block for the synthesis of a polypeptide via the thioester method. A partially protected peptide segment, for use as a C-terminal building block, could be prepared from a recombinant protein; its N-terminal amino acid residue was transaminated to an alpha-oxoacyl group, the side-chain amino groups were then protected with t-butoxycarbonyl (Boc) groups, and. finally, the alpha-oxoacyl group was removed. On the other hand, an O-phosphoserine-containing peptide thioester was synthesized via a solid-phase method using Boc chemistry. These building blocks were then condensed in the presence of silver ions and an active ester component. During the condensation, epimerization at the condensation site could be suppressed by the use of N,N-dimthylformamide (DMF) as a solvent. Using this strategy, a phosphorylated partial peptide of the p21Max protein, [Ser(PO3H2)2.11]-p21Max(1-101), was successfully synthesized.     

Dichain structure of botulinum neurotoxin: identification of cleavage sites in types C, D, and F neurotoxin molecules

Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum as about a 150-kDa single-chain polypeptide. Posttranslational modification by bacterial or exogenous proteases yielded dichain structure which formed a disulfide loop connecting a 50-kDa light chain (Lc) and 100-kDa heavy chain (Hc). We determined amino acid sequences around cleavage sites in the loop region of botulinum NTs produced by type C strain Stockholm, type D strain CB16, and type F strain Oslo by analysis of the C-terminal sequence of Lc and the N-terminal sequence of Hc. Cleavage was found at one or two sites at Arg444/Ser445 and Lys449/Thr450 for type C, and Lys442/Asn443 and Arg445/Asp446 for type D, respectively. In culture fluid of mildly proteolytic strains of type C and D, therefore, NT exists as a mixture of at least three forms of nicked dichain molecules. The NT of type F proteolytic strain Oslo showed the Arg435 as a C-terminal residue of Lc and Ala440 as an N-terminal residue of Hc, indicating that the bacterial protease cuts twice (Arg435/Lys436 and Lys439/Ala440), with excision of four amino acid residues. The location of cleavage and number of amino acid residue excisions in the loop region could be explained by the degree of exposure of amino acid residues on the surface of the molecule, which was predicted as surface probability from the amino acid sequence. In addition, the observed correlation may also be adapted to the cleavage sites of the other botulinum toxin types, A, B, E, and G.     

An improved chemical approach toward the C-terminal sequence analysis of proteins containing all natural amino acids.

An improved chemical method, capable of derivatizing all natural amino acids to their corresponding thiohydantoins, is described. This involves activation by acetyl chloride in TFA followed by derivatization with ammonium thiocyanate. Possible interference of reactive side chains was investigated by reacting N-acetylamino acids as well as several peptides with propionyl chloride instead of acetyl chloride. The products were characterized by PDMS mass spectrometry and 1H-NMR. This chemical method allows, for the first time, complete derivatization of N-acetylproline to proline thiohydantoin. Applying this chemistry to peptides with a C-terminal proline, the yields for formation of proline thiohydantoin were found to be up to 60%, depending on the peptide sequence. The previous inability to derivatize C-terminal proline to thiohydantoin was thought to stem from the fact that proline cannot form the oxazolonium ion required for efficient reaction with the thiocyanate ion. However, we have found mass spectrometric evidence for the existence of a proline oxazolonium ion, under basic as well as under acidic conditions. This improvement in derivatization of C-terminal amino acids including proline is a major step forward in the development of a general chemical C-terminal sequencing method that permits the C-terminal sequence analysis of proteins of any amino acid composition.     

Preparation of an N-acetyl-octapeptide of cholecystokinin: The role of N-acetylation in protecting the octapeptide from degradation by smooth muscle tissues

The C-terminal octapeptide of cholecystokinin (CCK-8) was acetylated on its lone N-terminal amino group using acetic anhydride in N,N-dimethylformamide. The acetylated derivative (Ac-CCK-8) and unreacted CCK-8 were separated from acetic anhydride and other reaction products by fractionation on Sephadex LH-20. Final purification was by thin-layer isoelectric focusing in a pH 2.5–4.0 gradient. The immunochemical properties of the octapeptide were unaffected by acetylation as measured by radioimmunoassay. The N-acetylated-octapeptide was equally as effective as unmodified CCK-8 in producing concentratiion-dependen isometric tension development in isolated cat gallbladder strips. Acetylation did, however, protect CCK-8 from N-terminal degradation by soluble peptidases that eluted from gallbladder and other smooth muscle tissues of the cat. Unmodified CCK-8 was degraded rapidly in the presence of these tissues and in buffers previously exposed to the same tissues. In contrast, the Ac-CCK-8 was resistant to N-terminal degradation under the same conditions. Degradation of CCK-8 from its N-terminus produces biologically inactive derivatives and could adversely affect in vitro studies. Since the acetylated-CCK-8 retained full biological and immunological activity, its use would eliminate the effect of extracellular proteolysis on CCK-8 action.     

Isolation and characterization of porcine beta-casein.

Porcine beta-casein was isolated by chromatography on DEAE-cellulose. The protein had a molecular weight of 24 900 as determined by gel filtration on Sephadex G-100 in guanidine-HCl. Its amino acid composition differed from bovine beta-casein especia-ly in respect to serine, alanine and leucine. In common with bovine beta-casein the N-terminal amino acid was arginine; the C-terminal was either alanine or valine, while the C-terminal of bovine beta-casein is valine. At any temperature porcine beta-casein was more sensitive to Ca2+ than bovine beta-casein, while at a fixed Ca2+ concentration porcine beta-casein aggregated at a lower temperature than bovine beta-casein. Porcine beta-casein was susceptible to hydrolysis by calf chymosin but the proteolytic specificity differed from that of calf chymosin on bovine beta-casein.     

Primary structure of mannuronate lyases SP1 and SP2 fromTurbo cornutus and involvement of the hydrophobic C-terminal residues in the protein stability

The complete amino acid sequences of two isoforms, SP1 and SP2, of mannuronate lyase from a wreath shell,Turbo cornutus, were determined to elucidate amino acid residues responsible for causing the more stable protein conformation of SP2. The sequences of the two isoforms were identical except for two hydrophobic C-terminal amino acid residues of SP2, Ile and Leu, which were additionally attached to Thr of the C-terminal residue of SP1 (253 residues in total). The molecular weight of SP2 was calculated to be 28,912 from the amino acid sequence data. Two disulfide bond cross-linkages were found to be between 106 and 115 and between 145 and 150, and a partially buried single SH group was located at 236. A carbohydrate chain that consisted of 3 GlcNAc, 3 Fuc, and 1 Man was anchored on Asn-105 in a typical carbohydrate-binding motif of Asn-X-Ser. This is the first evidence of the primary structure of mannuronate lyase, and no significant homology of the amino acid sequence among other proteins was found. The C-terminal truncated SP2, which was produced by digestion with carboxypeptidase Y and corresponded structurally to SP1, showed a thermal stability identical to that of SP1. These results indicate that the higher stability of SP2 than SP1 arises from the presence of the C-terminal two hydrophobic amino acid residues.     

Synthesis of different types of dipeptide building units containing N- or C-terminal arginine for the assembly of backbone cyclic peptides.

Different types of dipeptide building units containing N- or C-terminal arginine were prepared for synthesis of the backbone cyclic analogues of the peptide hormone bradykinin (BK: Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). For cyclization in the N-terminal sequence N-carboxyalkyl and N-aminoalkyl functionalized dipeptide building units were synthesized. In order to avoid lactam formation during the condensation of the N-terminal arginine to the N-alkylated amino acids at position 2, the guanidino function has to be deprotected. The best results were obtained by coupling Z-Arg(Z)2-OH with TFFH/collidine in DCM. Another dipeptide building unit with an acylated reduced peptide bond containing C-terminal arginine was prepared to synthesize BK-analogues with backbone cyclization in the C-terminus. To achieve complete condensation to the resin and to avoid side reactions during activation of the arginine residue, this dipeptide unit was formed on a hydroxycrotonic acid linker. HYCRAM technology was applied using the Boc-Arg(Alloc)2-OH derivative and the Fmoc group to protect the aminoalkyl function. The reduced peptide bond was prepared by reductive alkylation of the arginine derivative with the Boc-protected amino aldehyde, derived from Boc-Phe-OH. The best results for condensation of the branching chain to the reduced peptide bond were obtained using mixed anhydrides. Both types of dipeptide building units can be used in solid-phase synthesis in the same manner as amino acid derivatives.     

Automated carboxy-terminal sequence analysis of peptides.

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on solution phase conditions for formation of the peptidylthiohydantoins with trimethylsilylisothiocyanate (TMS-ITC) and for hydrolysis of these peptidylthiohydantoins into an amino acid thiohydantoin derivative and a new shortened peptide capable of continued degradation (Bailey, J. M. & Shively, J. E., 1990, Biochemistry 29, 3145-3156). The current study is a continuation of this work and describes the construction of an instrument for automated C-terminal sequencing, the application of the thiocyanate chemistry to peptides covalently coupled to a novel polyethylene solid support (Shenoy, N. R., Bailey, J. M., & Shively, J. E., 1992, Protein Sci. I, 58-67), the use of sodium trimethylsilanolate as a novel reagent for the specific cleavage of the derivatized C-terminal amino acid, and the development of methodology to sequence through the difficult amino acid, aspartate. Automated programs are described for the C-terminal sequencing of peptides covalently attached to carboxylic acid-modified polyethylene. The chemistry involves activation with acetic anhydride, derivatization with TMS-ITC, and cleavage of the derivatized C-terminal amino acid with sodium trimethylsilanolate. The thiohydantoin amino acid is identified by on-line high performance liquid chromatography using a Phenomenex Ultracarb 5 ODS(30) column and a triethylamine/phosphoric acid buffer system containing pentanesulfonic acid. The generality of our automated C-terminal sequencing methodology was examined by sequencing model peptides containing all 20 of the common amino acids. All of the amino acids were found to sequence in high yield (90% or greater) except for asparagine and aspartate, which could be only partially removed, and proline, which was found not be capable of derivatization. In spite of these current limitations, the methodology should be a valuable new tool for the C-terminal sequence analysis of peptides.     

Structure-function relationships in diphtheria toxin channels: I. Determining a minimal channel-forming domain

Diphtheria Toxin (DT) is a 535 amino acid exotoxin, whose active form consists of two polypeptide chains linked by an interchain disulphide bond. DT's N-terminal A fragment kills cells by enzymatically inactivating their protein synthetic machinery; its C terminal B chain is required for the binding of toxin to sensitive cells and for the translocation of the A fragment into the cytosol. This B fragment, consisting of its N-terminal T domain (amino acids 191–386) and its C-terminal R domain (amino acids 387–535) is responsible for the ion-conducting channels formed by DT in lipid bilayers and cellular plasma membranes. To further delineate the channel-forming region of DT, we studied channels formed by deletion mutants of DT in lipid bilayer membranes under several pH conditions. Channels formed by mutants containing only the T domain (i.e., lacking the A fragment and/or the R domain), as well as those formed by mutants replacing the R domain with Interleukin-2 (Il–2), have single channel conductances and selectivities essentially identical to those of channels formed by wild-type DT. Furthermore, deleting the N-terminal 118 amino acids of the T domain also has minimal effect on the single channel conductance and selectivity of the mutant channels. Together, these data identify a 61 amino acid stretch of the T domain, corresponding to the region which includes -helices TH8 and TH9 in the crystal structure of DT, as the channel-forming region of the toxin.This work was supported by NIH grants AI22021, AI22848 (R.J.C.), T32 GM07288 (J.A.M.) and GM29210 (A.F.).     

A manual subtractive end-group determination of oligopeptides using fluorescamine or o-phthalaldehyde

A new method for the end-group determination of peptides using the fluorogenic reagents fluorescamine or o-phthalaldehyde is described. The method is based on the property that the derivatives of the N-terminal amino group of peptides formed in solution after reaction with either reagent are resistant to acid hydrolysis. The N-terminal amino acid can be determined by simply comparing the amino acid analysis of the original peptide with the fluorescent derivative of the peptide. In general, the decrease of the N-terminal residue in the reacted peptides in 80–90% with fluorescamine and more than 90% with o-phthalaldehyde. Any N-terminal amino acid, with the exception of proline, can thus be determined.