Repetitive Yield Research Articles

P-stereocontrolled synthesis of oligo(nucleoside N3'→O5' phosphoramidothioate)s - opportunities and limitations.

3′-N-(2-Thio-1,3,2-oxathiaphospholane) derivatives of 5′-O-DMT-3′-amino-2′,3′-dideoxy-ribonucleosides (NOTP-N), that bear a 4,4-unsubstituted, 4,4-dimethyl, or 4,4-pentamethylene substituted oxathiaphospholane ring, were synthesized. Within these three series, NOTP-N differed by canonical nucleobases (i.e., AdeBz, CytBz, GuaiBu, or Thy). The monomers were chromatographically separated into P-diastereomers, which were further used to prepare NNPSN′ dinucleotides (3), as well as short P-stereodefined oligo(deoxyribonucleoside N3′→O5′ phosphoramidothioate)s (NPS-) and chimeric NPS/PO- and NPS/PS-oligomers. The condensation reaction for NOTP-N monomers was found to be 5–6 times slower than the analogous OTP derivatives. When the 5′-end nucleoside of a growing oligomer adopts a C3′-endo conformation, a conformational ‘clash’ with the incoming NOTP-N monomer takes place, which is a main factor decreasing the repetitive yield of chain elongation. Although both isomers of NNPSN′ were digested by the HINT1 phosphoramidase enzyme, the isomers hydrolyzed at a faster rate were tentatively assigned the RP absolute configuration. This assignment is supported by X-ray analysis of the protected dinucleotide DMTdGiBuNPSMeTOAc, which is P-stereoequivalent to the hydrolyzed faster P-diastereomer of dGNPST.

3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and α4β2 Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo alpha(2)betagammadelta and expressed human alpha4beta2 nAChRs with [(3)H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of alphaTyr(198) in agonist binding site Segment C of the principal (+) face in both alpha subunits and of gammaLeu(109) and gammaTyr(117) in Segment E of the complementary (-) face, with no labeling detected in the delta subunit. For affinity-purified alpha4beta2 nAChRs, [(3)H]epibatidine photolabeled alpha4Tyr(195) (equivalent to Torpedo alphaTyr(190)) in Segment C as well as beta2Val(111) and beta2Ser(113) in Segment E (equivalent to Torpedo gammaLeu(109) and gammaTyr(111), respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the alpha-gamma transmitter binding site, whereas it binds in two distinct orientations in the alpha4beta2 nAChR.

Identification of Binding Sites in the Nicotinic Acetylcholine Receptor for TDBzl-etomidate, a Photoreactive Positive Allosteric Effector

Etomidate, one of the most potent general anesthetics used clinically, acts at micromolar concentrations as an anesthetic and positive allosteric modulator of gamma-aminobutyric acid responses, whereas it inhibits muscle-type nicotinic acetylcholine receptors (nAChRs) at concentrations above 10 microm. We report here that TDBzl-etomidate, a photoreactive etomidate analog, acts as a positive allosteric nAChR modulator rather than an inhibitor, and we identify its binding sites by photoaffinity labeling. TDBzl-etomidate (>10 microm) increased the submaximal response to acetylcholine (10 microm) with a 2.5-fold increase at 60 microm. At higher concentrations, it inhibited the binding of the noncompetitive antagonists [(3)H]tetracaine and [(3)H]phencyclidine to Torpedo nAChR-rich membranes (IC(50) values of 0. 8 mm). nAChR-rich membranes were photolabeled with [(3)H]TDBzl-etomidate, and labeled amino acids were identified by Edman degradation. For nAChRs photolabeled in the absence of agonist (resting state), there was tetracaine-inhibitable photolabeling of amino acids in the ion channel at positions M2-9 (deltaLeu-265) and M2-13 (alphaVal-255 and deltaVal-269), whereas labeling of alphaM2-10 (alphaSer-252) was not inhibited by tetracaine but was enhanced 10-fold by proadifen or phencyclidine. In addition, there was labeling in gammaM3 (gammaMet-299), a residue that contributes to the same pocket in the nAChR structure as alphaM2-10. The pharmacological specificity of labeling of residues, together with their locations in the nAChR structure, indicate that TDBzl-etomidate binds at two distinct sites: one within the lumen of the ion channel (labeling of M2-9 and -13), an inhibitory site, and another at the interface between the alpha and gamma subunits (labeling of alphaM2-10 and gammaMet-299) likely to be a site for positive allosteric modulation.

Equimolar Production of Amyloid β-Protein and Amyloid Precursor Protein Intracellular Domain from β-Carboxyl-terminal Fragment by γ-Secretase

We showed previously that cells expressing wild-type (WT) beta-amyloid precursor protein (APP) or coexpressing WTAPP and WT presenilin (PS) 1/2 produced APP intracellular domains (AICD) 49-99 and 50-99, with the latter predominating. On the other hand, the cells expressing mutant (MT) APP or coexpressing WTAPP and MTPS1/2 produced a greater proportion of AICD-(49-99) than AICD-(50-99). In addition, the expression of amyloid beta-protein (Abeta) 49 in cells resulted in predominant production of Abeta40 and that of Abeta48 leads to preferential production of Abeta42. These observations suggest that epsilon-cleavage and gamma-cleavage are interrelated. To determine the stoichiometry between Abeta and AICD, we have established a 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid-solubilized gamma-secretase assay system that exhibits high specific activity. By using this assay system, we have shown that equal amounts of Abeta and AICD are produced from beta-carboxyl-terminal fragment (C99) by gamma-secretase, irrespective of WT or MTAPP and PS1/2. Although various Abeta species, including Abeta40, Abeta42, Abeta43, Abeta45, Abeta48, and Abeta49, are generated, only two species of AICD, AICD-(49-99) and AICD-(50-99), are detected. We also have found that M233T MTPS1 produced only one species of AICD, AICD-(49-99), and only one for its counterpart, Abeta48, in contrast to WT and other MTPS1s. These strongly suggest that epsilon-cleavage is the primary event, and the produced Abeta48 and Abeta49 rapidly undergo gamma-cleavage, resulting in generation of various Abeta species.

Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array.

Oligonucleotide microarrays, also called "DNA chips," are currently made by a light-directed chemistry that requires a large number of photolithographic masks for each chip. Here we describe a maskless array synthesizer (MAS) that replaces the chrome masks with virtual masks generated on a computer, which are relayed to a digital micromirror array. A 1:1 reflective imaging system forms an ultraviolet image of the virtual mask on the active surface of the glass substrate, which is mounted in a flow cell reaction chamber connected to a DNA synthesizer. Programmed chemical coupling cycles follow light exposure, and these steps are repeated with different virtual masks to grow desired oligonucleotides in a selected pattern. This instrument has been used to synthesize oligonucleotide microarrays containing more than 76,000 features measuring 16 microm 2. The oligonucleotides were synthesized at high repetitive yield and, after hybridization, could readily discriminate single-base pair mismatches. The MAS is adaptable to the fabrication of DNA chips containing probes for thousands of genes, as well as any other solid-phase combinatorial chemistry to be performed in high-density microarrays.

Comparison of four fluorescence Edman reagents with benzofurazan structure for the detection of thiazolinone amino acid derivatives

Two newly synthesized fluorescence Edman reagents with the benzofurazan structure, 7-phenylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (PSBD-NCS) and 7-methylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MSBD-NCS), were compared with 7-aminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (ABD-NCS) and 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) for peptide and protein sequence analysis by the generation of fluorescent 2,1,3-benzoxadiazolylthiazolinone (TZ)-amino acids. The effects of the substituent group at the para position to the isothiocyanate moiety of these reagents on the rate of the cyclization/cleavage reaction, the repetitive yield and the fluorescence quantum yield and stability of TZ amino acids were investigated. MSBD-TZ-amino acids were most sensitively detected and the detection limit for MSBD-TZ-Pro was 7 fmol (S/N = 3). ABD-NCS afforded the highest repetitive yield in the sequencing analysis. Fewer interfering peaks were observed in the chromatogram with DBD-NCS.

Protein sequencer maintenance and troubleshooting.

Modern automated sequencers, both gas-phase and pulsed-liquid forms, are normally efficient and reliable; this chapter is intended for use when they are not. The author’s own experiences are almost wholly concerned with ABI (Applied Biosystems) instruments; however, the principles described will apply to most other manufacturers’ sequencers. This chapter will address the various functions individually, in an attempt to identify typical faults and remedies as outlined in Table 1 and Notes 1–15. Table 1 A Trouble-Shooting Guide for Protein Sequencers 1. Blank chromatograms-possible failure of solvent and reagent delivery (see Section 3.1.) or of PTH a a transfer (see Sections 3.4. to 3.6., and Note 2). 2. Erratic retention times on the PTH a a. chromatogram, (see Section 3.6. and Note 4). 3. New peak appearing in every PTH a a. chromatogram, close to proline (see Note 4). 4. Compacting of the hydrophobic region of the PTH a.a. separation; most noticeable on the PTH a.a. standard; can also be accompanied by peak broadening (see Note 5). 5. Increased carry over or “lag” from cycle to cycle (see Section 3.2. and Note 6). 6. Abnormally high levels of DPTU and DPU (see Sections 3.2. and 3.3. and Note 7). 7. Excessive background peaks on PTH a∶a. chromatograms, starting within the early stages of a run (see Section 3.2. and Note 8). 8. Low repetitive yield i.e., less than 90–92% (see Section 3.2. and Note 9). 9. Low yield of PTH-ser or thr, or generally low yield-due to over- or underdrying in conversion flask (see Section 4). 10. Inability to reach the C-terminus of peptides (see Note 10). 11. Artifact peaks from the addition of DTT (see Note 11). 12. Sloping baseline at the start of the PTH separation and abysmal resolution of the early-fluting amino acids (see Note 12). 13. A very concave “smiling baseline” on PTH chromatograms (see Note 13). 14. High pressure indicated on analyzer display; usually first noticed as a high pressure shutdown during a solvent purge (see Note 14). 15. Huge, off-scale peaks on the PTH a∶a. chromatograms (see Note 15).

Gas-phase microsequencing of peptides and proteins with a fluorescent Edman-type reagent, fluorescein isothiocyanate.

A fluorescent Edman-type reagent, fluorescein isothiocyanate (FITC), was adapted to a gas-phase sequencer fitted with a miniature reaction chamber. Fluorescein thiohydantion amino acids were identified by on-line gradient HPLC with fluorescence monitoring. The optimized protocol for the coupling reaction using FITC and phenylisothiocyanate, and the degree of washing to remove the excess reagents were established. Myoglobin (5 pmol) and lysozyme (5 pmol) were analyzed by the sequencer to obtain a 35-51% initial yield (I.Y.) and 82-88% repetitive yield (R.Y.) for the former, and 51-66% I.Y. and 91-92% R.Y. for the latter. These figures permitted 20-25 amino acid residues to be identified from the N-terminus of 5 pmol samples applied to the sequencer.

Synthesis and Use of an Internal Amino Acid Sequencing Standard Peptide

A sequenceable internal standard has been made that can be added to samples destined for automated amino acid sequencing and that provides a means for continuously monitoring instrument performance. The 41-residue standard peptide is composed of two unnatural amino acids, norleucine and succinyl-lysine, and has the structure [norleucine-(succinyl-lysine) 4] 8-norleucine. The phenyithiohydantoin derivatives of the latter two amino acids are well separated via reversed-phase HPLC from the phenyithiohydantoin derivatives of the 20 naturally occurring amino acids. In addition, the standard peptide can be readily synthesized and it has excellent solubility, stability, and sequencing characteristics. The placement of norleucine at every fifth residue in this peptide permits accurate repetitive yield and carryover calculations following runs that are as short as six cycles. Use of this internal standard permits the early detection of suboptimal instrument performance and is especially helpful in differentiating between two common reasons (blocked NH 2-terminus versus an instrument problem) for the failure of an automated protein sequencer to provide an NH 2-terminal sequence.

Microsequence analysis of electroblotted proteins: II. Comparison of sequence performance on different types of PVDF membranes

The influence of different types of polyvinylidene difluoride (PVDF) membranes on gas phase sequence performance has been evaluated. These PVDF membranes have been classified as either high retention (Trans-Blot and ProBlott) or low retention membranes (Immobilon-P) based on their ability to bind proteins during electroblotting from gels. Initial yields, repetitive yields, and extraction efficiency of the anilinothiazolinone amino acid derivatives have been compared for several standard proteins that have been either electroblotted or loaded onto PVDF membranes by direct adsorption. These results show that the major differences in initial sequence yields between membranes arise from differences in the amount of protein actually transferred to the membrane rather than sequencer-related factors. In contrast to several previous observations from other laboratories, more tightly bound proteins do not sequence with lower initial yields and initial yields are not affected by the ratio of surface area to protein. The stronger binding on high retention PVDF membranes does not adversely affect recoveries of difficult to extract, or very hydrophobic, amino acid derivatives. Several amino acids, especially tryptophan, are actually recovered in dramatically higher yield on high retention membranes compared with either Immobilon or glass filters. At the same time, the protein and peptide binding properties of high retention membranes will frequently improve the repetitive yield by minimizing sample extraction during the sequencer cycle. Stronger protein binding together with improved electroblotting yields offer substantially improved sequence performance when high retention PVDF membranes are used.

Evaluation of aminolysis of anilinothiazolinones to phenylthiocarbamoyl amino acid methyl amides as an alternative conversion method in protein sequencing

The aminolysis of products of sequential degradation of proteins and peptides by methylamine is an alternative method of conversion of the unstable 5-alkyl-2-anilino-4-thiazolinones into the stable methyl amides of N α -phenylthiocarbamoyl amino acids. The volatility of methylamine permits use in the gas phase during both manual and automatic sequential degradation. Two procedures were studied: (mode A) aminolysis by methylamine in the sequencer reaction chamber after liberation of the thiazolinones by trifluoroacetic acid and (mode B) aminolysis by methylamine vapors passed through a 1-chlorobutane solution of thiazolinones in the conversion flask of the sequencer. The sequencing program was modified for both procedures by making use of the standard sequencer functions. The yields of aminolysis in the conversion flask (mode B) are comparable to those obtained by standard conversion in 25% trifluoroacetic acid and the procedure does not affect the repetitive yield. Aminolysis on the glass filter (mode A) requires a major modification of the degradation process, yet gives higher yields of the degraded amino acid derivatives. A disadvantage of both procedures, especially of mode A, is the presence of N-methyl- N′-phenylthiourea in the methyl amide samples. We have not been able to achieve the expected improvement of the yields of degraded hydroxy amino acids. Therefore the replacement of acid conversion of anilinothiazolinones to phenylthiohydantiones by aminolysis for routine degradation cannot be recommended. High yields of methyl amides make aminolysis a promising candidate for the incorporation of fluorescent or other labels in the products of sequencing degradation.

Single-step electroelution of proteins from SDS-polyacrylamide gels and immobilization on diisothiocyanate-glass beads in prepacked capillary columns for solid-phase microsequencing

A new method for immobilization of proteins purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) prior to sequencing is described. It utilizes a simple apparatus that permits the simultaneous electroelution of proteins from gel slices and attachment to diisothiocyanate-activated glass beads prepacked in capillary tubes [S-P. Liang and R. A. Laursen, Anal. Biochem. 188, 366–373 (1990)]. Transfer/attachment yields of >80% within 90 min were observed for several 125I-labeled proteins with a range of molecular weights using 0.2 m sodium phosphate (pH 8.9) buffer containing 0.1% SDS. The method has the advantage of high capacity, relative simplicity, and insensivity to the presence of SDS and Coomassie blue stain. The highest transfer yields were obtained when proteins were run on gels which had been aged for at least 12 h. For 100- to 1000-pmol samples, the sequenceable amount of protein, including transfer, was generally 30–60%, with an average repetitive yield of 95%. Factors which influence sample recovery and sequencing yield are discussed.

Isolation of proteins for peptide mapping, amino acid analyses, and sequencing using disulfide crosslinked polyacrylamide gels

Conditions for recovery of small amounts of proteins (1–50 μg) from disulfide crosslinked polyacrylamide gels have been examined. Procedures were developed for solubilization and precipitation of Coomassie blue-stained protein bands excised from gels after electrophoretic separations. The precipitated protein was then resolubilized for use in peptide mapping, amino acid analyses, or microsequencing. The amino acid compositions of standard proteins (bovine albumin, ovalbumin, phosphorylase b, and β-galactosidase) isolated by this method were in good agreement with the values for the corresponding conventionally purified proteins. Sequencing was done with high repetitive yield on samples of 100 pmol or below. The method has been successfully applied to several proteins and protein fragments.

Solid-phase sequencing on the gas-phase sequencer

Automated Edman degradation has been successfully used for determining the primary structure of numerous peptides and proteins. Quantitative solid-phase Edman degradation has great potential use for amino acid sequence analysis of synthetic peptides assembled on resin support by the Merrifield procedure. We report here the combined use of a modified gas-phase sequencer program and our improved reversed-phase HPLC analysis for PTH-amino acids to carry out the sequence analysis on synthesized peptide resins. This approach is far more sensitive than using glass beads on the conventional solid-phase sequencer. The peptide was assembled on copoly (styrene-1% divinylbenzene) resin beads at an initial substitution of 0.54 mmol/g. On a routine basis, 10–15 resin beads are used, and a repetitive yield of 94% is obtained: as few as 4 beads can be successfully sequenced. The HPLC PTH-amino acid analysis is sensitive down to subpicomole quantities. This procedure offers a sensitive and rapid analytical tool for checking the purity of peptides as they are being assembled on solid support.

Improved procedures for automated liquid phase sequence analyses of protein and peptide.

For the sequence analysis of histones rich in lysine, we modified the subprograms for two reagents of a JEOL JAS-47KS protein sequence analyzer. Together with this modification, the use of a synthetic carrier, Polybrene, the minimization of aldehyde contamination in Quadrol buffer, and the introduction of hydrophilic groups into epsilon-N-amino groups of lysine residues, markedly increased the repetitive yield of PTH-amino acids. Tetrahymena histones H3 and H4 were thus sequenced up to residues 104 and 92, respectively, in each consecutive analysis (Hayashi, T., Hayashi, H., Fusauchi, Y., & Iwai, K. (1984) J. Biochem. 95, 1741-1749; Hayashi, H., Nomoto, M., & Iwai, K. (1984) J. Biochem. 96, 1449-1456). The details for these improved procedures and results are described here.

On the synthesis of a 32P-labelled Edman reagent for the sensitive identification of amino-acid derivatives.

A phosphorus-32 containing derivative of phenylisothiocyanate was prepared to increase the sensitivity of amino-acid sequence determination. The respective compound 2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinane 2-oxide showed about the same reactivity, stability, and polarity as the Edman reagent itself. A repetitive yield of 94% was obtained in the stepwise degradation of insulin B chain using a solid phase sequencer. The synthesis of this radioactive reagent was achieved within 5 h but with a specific activity of 1 Ci/mol. Eight amino acids were reacted with the 32P-labelled reagent and identified by autoradiography after two dimensional thin-layer chromatography.

Rapid synthesis of oligodeoxyribonucleotides. VII. Solid phase synthesis of oligodeoxyribonucleotides by a continuous flow phosphotriester method on a kieselguhr-polyamide support.

A new kieselguhr-polydimethylacrylamide support has been used in a continuous flow, column system for solid phase synthesis of oligodeoxyribonucleotides by a phosphotriester procedure. Using only protected mononucleotides a 14-mer, 20-mer and 27-mer were assembled in high repetitive yield using a simple manually operated, bench top apparatus.

The NH 2-terminal amino acid sequence of cellular retinoic-acid binding protein from rat testis

Volume 135, number 1 FEBS LETTERS November 1981 THE NH2-TERMINAL AMINO ACID SEQUENCE OF CELLULAR RETINOIC-ACID BINDING PROTEIN FROM RAT TESTIS Ulf ERIKSSON, Johan SUNDELIN, Lars RASK and Per A. PETERSON Department of Cell Research, The Wallenberg Laboratory, University of Uppsala, Box 562, S-751 22 Uppsala, Sweden Received 5 October 1981 1. Introduction Several intracellular water-soluble proteins with high affinity for vitamin A compounds have been identified [1-5]. Two of these proteins, the cellular retinol-binding protein (CRBP) and the cellular retinoic acid-binding protein (CRABP) have been proposed to mediate the physiological action of vitamin A in some tissues [6,7]. CRBP has a wide- spread tissue distribution and occurs in epithelial tissues as well as in the liver [8]. CRABP is present in the adult only in a few tissues, among them the gonads [8]. Both proteins have been purified to homogeneity from several sources [9-13]. The tentative amino acid sequence of rat liver CRBP has been determined [14] and it displays sig- nificant homology with the myelin protein P2 [ 15 ]. Work in progress is aimed at elucidating the three- dimensional structure of rat liver CRBP by X-ray crystallography [16]. Rat CRABP has not yet been investigated in terms of its primary structure. However, we report here the NH2-terminal amino acid sequence of rat testis CRABP. The data demonstrate that the 2 retinoid-binding pro- teins are related to each other as well as to the myelin protein P2. 2. Materials and methods 2.1. Isolation of CRABP CRABP was isolated from rat testis essentially as in [10]. Upon high-resolution SDS-polyacrylamide gel electrophoresis the purified protein separated into 2 very closely migrating bands. However, this hetero- geneity, whether of genetic origin or due to post- translational modification is not derived from the NH2-terminal region. This can be inferred from the observation that the highly purified CRABP gave rise to a single NH2-terminal amino acid sequence with the expected yield of phenylthiohydantoin-amino acids in the degradation steps. 2.2. Protein sequence determination CRABP, extensively reduced and alkylated, was exhaustively dialysed against water, and 36 nmol were subjected to automatic NH2-terminal amino acid sequencing using a Beckman 890C sequencer. The details of the procedure have been given [17]. The phenylthiohydantoin-amino acids were analysed by reversed-phase, high-pressure liquid chromatog- raphy [18]. The repetitive yield, calculated from the yields of the leucine residues in positions 18,19,22 and 28 was found to be ~98%. The same calculation based on the alanine residues in positions 4,21,26 and 32 gave a repetitive yield of 96%. 3. Results and discussion Highly purified rat testis CRABP was subjected to NH2-terminal amino acid sequence analysis. The amino acid sequence obtained allowed unambiguous identification of 32 residues (fig. 1 ). The NH2-terminal amino acid could not be identified due to the high background in this step. The comparison of the CRABP sequence with the amino acid sequences of rat liver CRBP in [14] and the bovine myelin protein P2 in [15], reveals that to maximize homologies the CRABP sequence has to start with a gap, i.e., CRABP appears to be one amino acid shorter than CRBP and P2 in the NH2-terminal Published by Elsevier/North-Holland Biomedical Press 70 00145793/81/0000-0000/$02.75 © 1981 Federation of European Biochemical Societies

A gas-liquid solid phase peptide and protein sequenator.

A new miniaturized protein and peptide sequenator has been constructed which uses gas phase reagents at the coupling and cleavage steps of the Edman degradation. The sample is embedded in a matrix of Polybrene dried onto a porous glass fiber disc located in a small cartridge-style reaction cell. The protein or peptide, though not covalently attached to the support, is essentially immobile throughout the degradative cycle, since only relatively apolar, liquid phase solvents pass through the cell. This instrument can give useful sequence data on as little as 5 pmol or protein, can perform extended sequence runs (greater than 30 residues) on subnanomole quantities of proteins purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and can sequence hydrophobic peptides to completion. The sequenator is characterized by a high repetitive yield during the degradation, low reagent consumption, low maintenance requirements, and a degradative cycle time of only 50 min using a complete double cleavage program.

The protein sequenator: a new extraction principle and a metal reaction cup.

A simpler and more efficient extraction procedure is proposed for solvent extraction of the Edman degradation procedure in the protein sequenator. The method is less likely than the conventional one to cause wash-out of peptides or proteins from the reaction cup while the very small solvent volumes required substantially decrease the running costs of the instrument. It has been used successfully (98% repetitive yield) with a metal reaction cup in the sequenator.