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Edman method to identify peptides with Phenylisothiocyanate (PTH)

Edman method to identify peptides with Phenylisothiocyanate (PTH)


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We all know that in this method the PTH reacts with the first amino acid (aa) from the N-terminal to the peptide and separates from it giving PTH-aa so that we can know the amino acids sequence in the peptide.

The question is what prevents the PTH from reacting with the second amino acid after it separates with the first amino acid in the same time and phase? Because if it does we can't distinguish between the first and second amino acid residue in the sequence.


The short answer is that the Edman degradation is a multi-step process. The Wikipedia page has a decent picture of the mechanism. In practice, the peptide is reacted with phenylisothiocyanate (PTH) under mildly basic conditions to give a thiourea, which is stable. The excess PTH is separated from the thiourea intermediate. The thiourea is then treated with acid to cleave the N-terminal residue from the peptide. The PTH is no longer present, so there's no concern about reacting with the second residue.


Edman Degradation diagrams

Alain Doucet , Christopher M. Overall , in Methods in Enzymology , 2011

Abstract

Edman degradation is a long-established technique for N-terminal sequencing of proteins and cleavage fragments. However, for accurate data analysis and amino acid assignments, Edman sequencing proceeds on samples of single proteins only and so lacks high-throughput capabilities. We describe a new method for the high-throughput determination of N-terminal sequences of multiple protein fragments in solution. Proteolytic processing can change the activity of bioactive proteins and also reveal cryptic binding sites and generate proteins with new functions (neoproteins) not found in the parent molecule. For example, extracellular matrix (ECM) protein processing often produces multiple proteolytic fragments with the generation of cryptic binding sites and neoproteins by ECM protein processing being well documented. The exact proteolytic cleavage sites need to be identified to fully understand the functions of the cleavage fragments and biological roles of proteases in vivo. However, the identification of cleavage sites in complex high molecular proteins such as those composing the ECM is not trivial. N-terminal microsequencing of proteolytic fragments is the usual method employed, but it suffers from poor resolution of sodium dodecylsulfate-polyacrylamide gel electrophoresis gels and is inefficient at identifying multiple cleavages, requiring preparation of numerous gels or membrane slices for analysis. We recently developed Amino-Terminal Oriented Mass spectrometry of Substrates (ATOMS) to overcome these limitations as a complement for N-terminal sequencing. ATOMS employs isotopic labeling and quantitative tandem mass spectrometry to identify cleavage sites in a fast and accurate manner. We successfully used ATOMS to identify nearly 100 cleavage sites in the ECM proteins laminin and fibronectin. Presented herein is the detailed step-by-step protocol for ATOMS.


Principle

Edman degradation allows the order of amino acids (the amino acid sequence) in a peptide to be determined by repeated end grouping. The peptide chain is gradually broken down. This reaction is used to identify the N -terminal amino acid of a peptide and consists of the reaction of a peptide with phenyl isothiocyanate , which is also known as Edman's reagent .

Since each amino acid has a different residue R , each forms a different phenylthiohydantoin derivative (PTH derivative). The N- terminal amino acid split off as PTH derivative is identified by chromatography by comparison with a standard. You can successively carry out further degradations on one and the same protein (previously shortened by an amino acid at the N -terminal end) and thus gradually determine the amino acid sequence. A sequenator is an automated device that allows the unattended execution of up to 50 degradation cycles. Since the reaction proceeds with a relative yield of> 98%, on the one hand the entrained derivatives from previous cycles and on the other hand the undesired cleavage products of proteins that have undergone one or more cleavage cycles increase with each cycle, so that after a maximum of 50 cycles the signal -Noise ratio becomes illegible. A cycle takes one to three hours, depending on the variant.


Mechanism

Phenylisothiocyanate is reacted with an uncharged terminal amino group, under mildly alkaline conditions, to form a cyclical phenylthiocarbamoyl derivative. Then, under acidic conditions, this derivative of the terminal amino acid is cleaved as a thiazolinone derivative. The thiazolinone amino acid is then selectively extracted into an organic solvent and treated with acid to form the more stable phenylthiohydantoin (PTH)- amino acid derivative that can be identified by using chromatography or electrophoresis. This procedure can then be repeated again to identify the next amino acid. A major drawback to this technique is that the peptides being sequenced in this manner cannot have more than 50 to 60 residues (and in practice, under 30). The peptide length is limited due to the cyclical derivatization not always going to completion. The derivatization problem can be resolved by cleaving large peptides into smaller peptides before proceeding with the reaction. It is able to accurately sequence up to 30 amino acids with modern machines capable of over 99% efficiency per amino acid. An advantage of the Edman degradation is that it only uses 10 - 100 pico-moles of peptide for the sequencing process. Edman degradation reaction is automated to speed up the process. [ 2 ]


Edman degradation

Edman degradation, developed by Pehr Edman, is a method of sequencing amino acids in a peptide. In this method, the amino-terminal residue is labeled and cleaved from the peptide without disrupting the peptide bonds between other amino acid residues.

Mechanism for Edman Degradation

Phenylisothiocyanate is reacted with an uncharged terminal amino group, under mildly alkaline conditions, to form a phenylthiocarbamoyl derivative. Then, under acidic conditions, this derivative of the terminal amino acid is cleaved as a thiazolinone derivative. The thiazolinone amino acid is then selectively extracted into an organic solvent and treated with acid to form the more stable phenylthiohydantoin (PTH)- amino acid derivative that can be identified by using chromatography or electrophoresis. This procedure can then be repeated again to identify the next amino acid. A major drawback to this technique is that the peptides being sequenced in this manner cannot have more than 50 to 60 residues. This is because the Edman degradation reaction is not 100% efficient, meaning that the cleavage step does not occur every time. However, this problem can be resolved by cleaving large peptides into smaller peptides before proceeding with the reaction. It is able to accurately sequence up to 30 amino acids with 98% efficiency per amino acid. An advantage of the Edman degradation is that it only uses 10 - 100 picomoles of peptide for the sequencing process. Edman degradation reaction is automated to speed up the process. Ώ]


What Is Edman Degradation?

Edman degradation, often referred to as N-terminal sequencing or Edman sequencing, is a well-known process for identifying the N-terminal amino acid of a protein and remains a unique approach that can yield new protein sequence data. It is also the preferred technique for quick protein expression confirmation.

The chemistry of this approach was first introduced in 1950 by P. Edman from Lund University in Sweden and further developed during his work in Melbourne, Australia to what is believed to be the first automated peptide sequencing device. Proteins produced inside individual cells act as hormones, enzymes and other important regulators. Genetic mutation resulting in an abnormal protein could have serious health consequences.

The N-terminal amino acid is cleaved by treatment with one of several reagents, then hydrolyzed to form a separate amino acid. Thin layer chromatography, HPLC or electrophoresis determines the size of the amino acid, and compares it to a known size standard. The new N-terminal end of the remaining protein can be similarly cleaved for identification.

Applied Biosystems led the development of automating Edman degradation, which binds a purified protein to a membrane and treats it with phenylisothiocyanate. The free amino acid’s automatically injected into a HPLC system for identification.

Phenylisothiocyanate is reacted with an uncharged terminal amino group, under mildly alkaline conditions, to form a cyclical phenylthiocarbamoyl derivative. Then, under acidic conditions, this derivative of the terminal amino acid is cleaved as a thiazolinone derivative. The thiazolinone amino acid is then selectively extracted into an organic solvent and treated with acid to form the more stable phenylthiohydantoin (PTH)- amino acid derivative that can be identified by using chromatography or electrophoresis. This procedure can then be repeated again to identify the next amino acid. A major drawback to this technique is that the peptides being sequenced in this manner cannot have more than 50 to 60 residues.

Applied Biosystems led the development of automating Edman degradation, which binds a purified protein to a membrane and treats it with phenylisothiocyanate. The free amino acid’s automatically injected into a HPLC system for identification. N-(9-Fluorenylmethoxycarbonyloxy)succinimide (CAS No. 82911-69-1) is used in conjunction with Isothiocyanate to conduct partial Edman degradation on biologically active peptides. Because the Edman degradation proceeds from the N-terminus of the protein, it will not work if the N-terminal amino acid has been chemically modified or if it is concealed within the body of the protein.

Proteins must be pure and free of contaminants to accurately determine the N-terminal amino acid. Edman degradation limits require large protein molecules fragmented into small peptide chains no longer 50 amino acids.


Abstract

The proteomes of cells, tissues, and organisms reflect active cellular processes and change continuously in response to intracellular and extracellular cues. Deep, quantitative profiling of the proteome, especially if combined with mRNA and metabolite measurements, should provide an unprecedented view of cell state, better revealing functions and interactions of cell components. Molecular diagnostics and biomarker discovery should benefit particularly from the accurate quantification of proteomes, since complex diseases like cancer change protein abundances and modifications. Currently, shotgun mass spectrometry is the primary technology for high-throughput protein identification and quantification while powerful, it lacks high sensitivity and coverage. We draw parallels with next-generation DNA sequencing and propose a strategy, termed fluorosequencing, for sequencing peptides in a complex protein sample at the level of single molecules. In the proposed approach, millions of individual fluorescently labeled peptides are visualized in parallel, monitoring changing patterns of fluorescence intensity as N-terminal amino acids are sequentially removed, and using the resulting fluorescence signatures (fluorosequences) to uniquely identify individual peptides. We introduce a theoretical foundation for fluorosequencing and, by using Monte Carlo computer simulations, we explore its feasibility, anticipate the most likely experimental errors, quantify their potential impact, and discuss the broad potential utility offered by a high-throughput peptide sequencing technology.


Conclusion

A novel lipopeptide compound was isolated from 5812-A/C Streptomyces sp. INA-5812 and found to have structural and functional characteristics similar to other commercial lipopeptide antibiotics, including its presumed structural analog daptomycin. Some principal functional differences were found in comparison with daptomycin, including a direct effect on the cellular membrane, followed by rapid permeabilization and strong bactericidal effect on target populations. Moreover, 5812-A/C was uniquely able to inhibit the metabolism of bacterial cells associated with mature biofilms, which is interesting with respect to multidrug-resistant strains and clinical isolates.


Materials and Methods

Preparation of Labeled Protein.

Uniformly labeled [ 14 C]glutathione S-transferase (GST) was prepared as follows. The DNA plasmid containing mouse GST Yc (19), provided by T. Bammler (University of Washington, Seattle), was used for transformation of BL21(DE3) cells from Escherichia coli. The bacteria harboring the GST vector were grown for 26 h at 37°C with shaking, in M9 minimal medium containing ampicillin (50 μg/ml). One microliter of this cell culture was transferred into 200 μl of M9 medium containing 85 μCi (1 Ci = 37 GBq) d -[U- 14 C]glucose (323 mCi/mmol Amersham Pharmacia) with 350 μg of nonlabeled d -glucose. The culture was incubated at 37°C with shaking until the OD600 reached 0.5–1.0. Production of recombinant GST was then induced by the addition of isopropyl β- d -thiogalactopyranoside (IPTG) to a final concentration of 1 mM. The bacteria were grown for an additional 12 h at 37°C with shaking and harvested by centrifugation at 2,000 g for 5 min at 4°C. Cells were resuspended in 100 μl of cell wall disrupting reagent BugBuster (Novagen) with the nuclease Benzonase (Novagen) to reduce viscosity. Cells were gently shaken at room temperature for 20 min, and the suspension was centrifuged at 16,000 × g for 20 min at 4°C. The supernatant was applied to glutathione Sepharose 4B beads (75 μl bed volume Sigma) in Eppendorf tubes, which were equilibrated in buffer A (50 mM Tris⋅HCl, pH 7.4/200 mM NaCl/0.5 mM DTT), and incubated for 35 min at 4°C. The suspension was centrifuged at 500 g for 5 min at 4°C, and the supernatant was discarded. The pellet was washed four times with 1 ml of buffer A. The [ 14 C]GST was eluted three times with 50 μl of buffer B (200 mM Tris⋅HCl, pH 9.0/50 mM reduced glutathione) at 4°C and eluents were pooled. The buffer was replaced with PBS containing 0.5 mM DTT by using Microcon 10 (Millipore). The concentration of uniformly labeled [ 14 C]GST was determined by SDS/PAGE analysis with silver-staining using nonlabeled GST as a standard. The concentration of the standard GST was determined by amino acid analysis. Scion Image (Scion, Frederick, MD) was used for this quantitative analysis. A Wallac 1409 LSC (Wallac, Gaithersburg, MD) was used to determine the specific activity.

Random Peptide-Bead Synthesis.

Peptide beads were designed to release equimolar amounts of each of the natural amino acids with each Edman cycle. Fluorenylmethoxycarbonyl-amino acid mixtures (Fmoc, 5 eq) were coupled with TentaGel NH2 resin (0.26 mmol/g Rapp Polymere, Tübingen, Germany) using the 1,3-diisopropylcarbodiimide/1-hydroxybenzotriazole (5 eq) method (20). Fmoc groups were removed with a 25% piperidine/dimethylformamide (DMF) solution and a step-wise coupling reaction was carried out until 10-mer peptide-beads were obtained. After Fmoc removal, the random peptide-beads were washed with DMF, methanol, and dichloromethane, and dried under vacuum.

Protein Sequencing.

Automated protein sequencing was performed on a 477A sequencer equipped with a 120A HPLC system (PE Applied Biosystems). All reagents and solvents used for the sequencer were obtained from PE Applied Biosystems and checked for 14 C content by AMS before use. The solution of [ 14 C]GST was diluted to concentrations of 10–1,000 amol/μl with 0.1% TFA in water containing β-lactoglobulin (2.5 pmol/μl). The concentration of each diluted [ 14 C]GST solution was determined with 14 C measurement by AMS. Polybrene (3.0 mg) was applied to a glass filter on the reaction chamber and subjected to three precycles. Before the addition of [ 14 C]GST, β-lactoglobulin (25 pmol) and peptide-beads (4–7 beads) were loaded on the glass filter and dried under argon gas. The sequencer program was modified so that a known amount of standard PTH-amino acids were delivered to the conversion flask each cycle before the transfer of anilinothiazolinone amino acids to the flask in addition to the amino acids from the peptide beads and lactoglobulin. HPLC fractions were collected every 30 or 60 sec into borosilicate glass culture tubes (6 × 50 mm), which were pyrolyzed before use to remove any residual carbon.

AMS Sample Preparation and Measurement.

Each sample was collected in a small glass tube and 50 μl of carbon carrier solution (40.0 mg/ml tributyrin in methanol) was added before drying in a vacuum centrifuge to yield 1.19 mg of carrier C. Three tributyrin carrier blanks were prepared with each set of fractions. The samples were graphitized for AMS as described by Vogel (21). Typical AMS measurement times were 3 min/sample, with a counting precision of 1.4–2.0% and a SD among 3–7 measurements of 1–3%. The 14 C/ 13 C ratios of unknowns were normalized to measurements of four identically prepared standards of known isotope concentration (Australian National University Sucrose ref. 22).


Pehr Victor Edman 1916-1977

Pehr Victor Edman was born in Stockholm, Sweden, in April 1916 and died in Munich, FRG, in March 1977. He was born into a lawyer's family and received his schooling in Stockholm. In 1935 he began medical studies at the Karolinska Institute and graduated with his primary medical qualifications in 1938. He became interested in research and, following graduation, continued to work at the Karolinska Institute, largely in the laboratory of Professor Eric Jorpes. He appears to have systematically taught himself organic chemistry at this time by extensive reading. During the war years his research was interrupted by a long period of service in the medical corps of the Swedish Army. He was awarded the degree of Doctor of Medicine in 1946. The subject of his thesis was the purification and analysis of angiotensin from bovine blood. His earlier published studies concerned heparin and secretin, which were interests of his mentor Jorpes.

At this juncture Edman began to take the independent research direction which he followed almost uninterruptedly for the rest of his career. He accepted a grant to work for a year in the Northrop-Kunitz laboratory at the Princeton branch of the Rockefeller Institute for Medical Research. Swedish medical research had been isolated during the war and he was anxious to learn of the progress made in the United States. Moreover, his work on angiotensin had made him realize that simple compositional analysis would not be helpful in providing a basis for understanding the biological function of peptides or proteins. The realization that proteins were not colloids but that each had a definite molecular weight and a specific structure was beginning to emerge, especially as a result of the work of the Uppsala group. Edman knew that the order of aminoacids linked by peptide bonds was an essential part of the unique makeup of any given protein. At Princeton he began experiments to try to find a way to chemically decode the aminoacid sequence of proteins.

In the early years of Edman's attempts in this area two general procedures were being used to attack the sequence problem. Various reagents had been found useful in labelling the aminoterminal (or first) aminoacid through its reactive amino group and allowing identification as a derivative. One of these reagents, fluorodinitrobenzene (FDNB), which gave the dinitrophenol (DNP) derivative of the aminoterminus, was used by Sanger in his epochal work on the structure of insulin. By using the FDNB reaction with sets of overlapping peptides derived from partial cleavage of insulin, Sanger, by 1956, was able to deduce a unique structure for the insulin molecule. This was the first primary structure of a protein to be decoded, but despite the undoubted importance of the feat it was clear that the method was too cumbersome to have wide application.

Another reagent used for aminoterminal determination was phenylisocyanate (PIC), introduced for this purpose by Abderhalden and Brockmann in 1930. As with FDNB the hydrolysis to release the aminoterminal aminoacid derivative destroyed many of the other peptide bonds, leaving the remaining protein useless for analysis. In Princeton, Edman realised that if phenylisothiocyanate (PITC) were used the nucleophilic sulphur would weaken the adjacent peptide bond, raising the possibility of finding conditions for its hydrolysis that did not cleave the remainder of the molecule. This remaining peptide could then be subjected to a second reaction with PITC and the second aminoacid determined, and so on theoretically to the carboxyterminal end of the molecule. Whether Edman thought of this solution completely independently, whether some unrelated paper uncovered in his wide reading drew PITC to his attention, or whether some colleague in Princeton or Stockholm suggested its use is not known. In view of the success which the reaction ultimately achieved, the latter seems unlikely in the absence of any claims or reminiscences to this effect. In his review of aminoacid sequencing methods written in 1969 Edman is at pains to stress that the PITC reaction was not at all derived from the earlier PIC reaction, as they had different mechanisms of action. However, in view of the superficial similarities between the reagents and the similar uses to which they were put in protein chemistry, this seems a little strained. By the time Edman returned to Sweden in 1947 he had performed enough experiments to know that the idea was practicable and could form the basis of a protein sequencing technique.

Edman took up an associate professorship at Lund and continued to work almost exclusively on protein degradation. The derivative resulting from the coupling of PITC with an aminoacid, the 3-phenyl-2-thiohydantoin (PTH) aminoacid, proved to be a stable compound in almost all cases. Edman synthesised the PTH derivatives of all the amino acids found in proteins and developed chromatographic systems to identify and quantify them conditions for the coupling of PITC to the aminoterminus and the cleavage of the PTH derivative which worked smoothly for all peptide bonds were found. After two years work Edman was able to publish the chemical details of a method capable, in theory, of solving the problem of primary structure of proteins and of providing the essential information about innumerable proteins essential for further advances in protein biochemistry. The characteristic ultraviolet absorption spectra of the PTH-aminoacids made them particularly suited to quantitative studies. It permitted useful measurements of the subunit structure and molecular weight of proteins and, in conjunction with column chromatography, it was an alternative to the ninhydrin reaction for aminoacid analysis. However, these possibilities could not be fully exploited until the recent developments in high performance liquid chromatography. The method became widely known and was given the eponym 'Edman degradation' by Kai Linderstrom-Lang of the Carlsberg Laboratories.

In the early 1950s Mr 'Jack' Holt, a well-known Victorian racehorse trainer, died and his will provided that the income from his estate, to be held in trust, was to be used for medical research at St. Vincent's Hospital in Melbourne. By 1956 the hospital authorities had decided to establish a separate research institution in the hospital rather than disburse the funds as research grants to existing hospital units. The decision had also been taken to develop a non-clinical basic science area, preferably biochemistry. The School of Medical Research, as it was originally known, had its own governing board and for practical purposes functioned independently of the hospital administration.

Edman applied for the position of Director of Research. He was clearly the outstanding candidate and moreover his interests coincided with the preference for biochemistry as the focus of the School. In 1957 Edman accepted the offer of the position of first Director of Research at St Vincent's School of Medical Research in Melbourne. His reasons for this move, which were said to have been a mixture of general dissatisfaction with scientific resources in Lund and the impending breakdown of his marriage, must have been strong and not primarily directed to career improvement. He had just accomplished an outstanding piece of individual biochemical research which would have made him welcome in many leading centres in the northern hemisphere. In Australia, in Melbourne, he would be largely isolated from these centres the School was a new institution, with no traditions, no established workers, nor any support staff and although situated in a teaching hospital of the University of Melbourne, it had itself no academic or university affiliation. Moreover, Australia at that time was not known for generous government research funding. Despite this formidable array of disincentives Edman decided to move alone to Melbourne to continue his work, initially without trained help and without close colleagues.

In Australia Edman completed a few small projects on other aspects of protein structure that he had begun in Sweden, but otherwise he worked almost entirely on the phenylisothiocyanate (PITC) degradation. This work fell into three phases: improvements in the conditions for the degradation, largely focussed on the elimination of side reactions 'automation' of the reaction sequence and application of the degradation to various sequence problems. The latter phase overlapped the other two and usually involved the interests of visiting scientists who had come to Edman's laboratory to learn or use his technique.

By 1960-61 the three-stage degradation reaction had been essentially perfected. Its universal application and repetitive nature suggested to G.S. Begg, Edman's Australian technical assistant, that it would be suitable for automation. Edman realized that the number of existing proteins (about ten million) made manual sequencing an impossible task and was quickly converted to the idea of automation. The close control of reaction conditions possible with automation also gave promise of higher and more constant repetitive yield than were possible manually. High repetitive yields are crucial to repetitive processes, whether synthetic or degradative. Geoffrey Begg had been one of the early technical staff employed by Edman after his arrival in Melbourne. He had no formal qualifications but from a combination of courses at technical college and self-instruction had achieved a remarkable expertise in practical chemistry and glassblowing, mechanical engineering, and electronics. The sequenator project provided a perfect opportunity to use these multiple talents which complemented Edman's academic and theoretical knowledge. Edman and Begg worked as a team on the sequence automation project, with no sustained input from other workers. It was typical of Edman's thorough approach to all tasks that he became a sufficiently adept toolmaker in this period to do much of the fitting and turning himself.

The basis of what was to become the protein sequenator was developed to a prototype stage in a period of a few weeks in the autumn of 1961 – the glass cup spinning on its cylindrical axis, addition of reagents via a catheter, reactions in a thin liquid film on the wall of the spinning cup, and extractions by solvent moving upwards over the film into a groove. Within two years Edman and Begg had built, in their own workshop, a machine capable of reliably carrying out the reactions of the degradation. They had found new conditions and reagents suitable for the physical conditions of the spinning cup for example, the open cup in general required less volatile chemicals and the narrow delivery and effluent tubes demanded special attention to the surface tension and rheological properties of the solvents. Edman's wide knowledge of classical organic chemistry enabled quick progress in converting the manual reaction to its automated form. In 1964 Edman reported his preliminary findings to a meeting in Scotland. In 1967 in the first issue of the European Journal of Biochemistry with Begg as co-author he published his definitive paper demonstrating an unbroken automated determination of the aminoterminal sixty aminoacids of humpback whale myoglobin at the rate of one residue per hour. The extent of this advance can be gauged from the knowledge that at that time the most extensive manual degradation encompassed about fifteen residues at a rate of one per day. Many laboratories could not establish the manual degradation at all, owing to a failure to appreciate the importance of pure reagents in eliminating side reactions. During the next few years Edman's aim was to improve the repetitive yield obtained from the machine an increase from the 98% of the 1967 paper to 99% was calculated to double the length of determinable sequence. The protein sequenator in Melbourne remained unique until late in 1969 when the Beckman Instrument Company in the United States put on the market a commercial version based on Edman's design. Edman played no part in the commercialisation of his machine. The Board of the School discussed the possibilities of patenting the sequenator but soon accepted Edman's strong view that he should publish fully without patent protection. Edman was elected a Fellow of the Australian Academy of Science in 1968 and a Fellow of the Royal Society of London in 1974. Edman became an Australian citizen in the mid sixties.

In 1972 Edman resigned from St Vincent's School of Medical Research and became Director of Protein Chemistry I at the Max Planck Institute for Biochemistry at Martinsreid near Munich. In 1968 he had remarried his second wife Agnes Henschen had come from Stockholm and this had given him a reason to think of a return to Europe. In the ten years since his arrival in 1957 the School had remained small, and attempts to raise support for expansion on the basis of the success of the sequenator project proved not very successful. Now, as many years before in Lund, he believed that the importance of his work was not properly recognised and that he would continue to have inadequate resources in Melbourne. A move to the new laboratories of the Max Planck Institute seemed to provide an answer to his needs. Edman set up his laboratory in Munich along the lines of that in Melbourne and with the same aim of increasing the efficiency of the degradation. In addition, with his aid, Agnes Henschen began to make substantial progress in her studies of fibrinogen structure. Sadly, Edman developed a cerebral tumour and died after a short period of coma in 1977.

Edman played little if any role in broader scientific administration or politics in Australia. Although his School had no formal academic affiliation, there is no evidence that he would not have been accepted in these arenas. Some efforts to arrange a personal appointment at the University of Melbourne came to nothing. Thus he remained something of an enigma in the scientific community. He was slow to publish, with approximately one and a half papers per year during his Australian period, which made difficulties for those wishing to implement the method. If his impact on the Australian scene was limited, it was paradoxically the result of his single-minded pursuit of the sequence degradation. Such work, despite Edman's reputation, was not very attractive to students and he never built up a tradition of a flow of graduate students. Once the initial work on the manual or especially the automated reaction was complete, the details would easily have been completed by others in his or other laboratories. One cannot help thinking that his impact would have been so much greater had he seen himself able to move strongly into new areas of protein structure and function. Biological research often requires the appreciation of the importance of an approximate result for advancement.

Nevertheless the Fellowships of the Australian Academy of Science and the Royal Society of London indicate in how much esteem his work was held internationally, and this judgement has been supported by later events. Technical advances in related fields, especially in liquid chromatography and sensitive ultra violet detectors, have led to the development of low-level or microsequencing techniques which still employ the reactions described by Edman forty years ago and which play an indispensible role in gene isolation and molecular cloning.

Edman's reputation as a reclusive person, often difficult to deal with, did not arise from those who worked with him in the laboratory. He was reserved by Australian standards, but courteous and always helpful, often humorous, and took pleasure in organising social occasions both at his home and outdoors in the country. To those who came to know him in the laboratory two aspects probably had a lasting influence. In those days he was a rare example in the hospitals and the world of Australian medical research of someone who devoted himself full-time to nonclinical research. This served as an example, to those who came across him, of the possibility of such a career. At a time when biochemistry in Australia was largely concerned with the intricacies of metabolic pathways, an area where the great discoveries had already been made, Edman understood and stressed the importance of the information-containing macromolecules. The double helical structure of DNA had been proposed by Watson and Crick only a few years before Edman's arrival in Australia. The possibility of obtaining a corresponding understanding of the more complex structures of proteins which Edman's work opened up inspired his colleagues with the belief that they were in a position to participate directly in a new era of biological investigation. fic basis for consciousness. Cognitive Studies 5, 95-109.