Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

Some methodological aspects of analytical protein research
Methodological aspects of protein amino acid sequence analysis

To determine the Amino Acid Sequence of Proteins and Polypeptides, as with any other structural analysis, the test subjects must meet several requirements. The material to be analyzed must be homogeneous, and its key physical parameters—primarily molecular weight and exact Amino Acid Composition—must be known. Meeting these conditions, however, often presents researchers with rather difficult challenges.

Structure/149.html">The problem of protein homogeneity was once solved relatively easily using traditional Analytical Methods. A preparation was considered homogeneous if it did not separate into fractions during Electrophoresis or ultracentrifugation. Today, these criteria for homogeneity have lost their significance. More sensitive Research Methods have become available, and stricter indicators of homogeneity are now mandatory. Ion-exchange Chromatography and gel electrophoresis are currently the most sensitive methods for detecting the so-called microheterogeneity of protein preparations, whatever its nature. The term 'microheterogeneity' was proposed by Synge [82] in 1943 and Colvin et al. [6] in 1954. Among gel electrophoresis methods, vertical Disc electrophoresis has proven to be the most sensitive for analyzing protein microheterogeneity. This method requires very small amounts of starting material, allows for the simultaneous analysis of A large number of samples, and is characterized by high resolution. Disc electrophoresis can detect microheterogeneity in a preparation even when the components have identical Amino acid sequences but differ in molecular conformation. This method also allows for the detection of the most subtle chemical differences.

The high resolution of chromatography using ion-exchange celluloses, dextran, and other Materials makes this method the most suitable for isolating homogeneous protein preparations.

Recently, it has become possible to determine the Amino acid composition of proteins using automatic amino acid analyzers. When in 1948 Moore and Stein [55] introduced ion-exchange chromatography In addition to classical organic chemistry methods, as well as manometric and bacteriological analysis, a turning point occurred in The Development of amino acid chemistry. Modern automatic amino acid analyzers, developed by researchers at the Rockefeller Institute, are based on ion-exchange chromatography. The operating principle of these instruments is as follows. The protein under study is hydrolyzed, and the hydrolysate is then chromatographed on a Dowex 50 x8 type resin in the Na-form. Elution is carried out by continuous delivery of a buffer solution. The eluate emerging from the Column enters a specially shaped plastic Cell, where it is mixed with a ninhydrin solution. The ninhydrin is delivered by a special pump operating synchronously with the pump delivering the buffer solution to the column. The mixture of eluate and ninhydrin then passes through a Teflon capillary immersed in a boiling Water bath. Under these conditions, ninhydrin color development occurs in the solutions, the intensity of which is measured spectrophotometrically in a flow cell. Light absorption is recorded by a chart recorder. The Use of spherical resins [80] reduced the analysis time for a single sample by about fourfold, and the use of special Cells made very small amounts of the test substance—on the order of 0.01–0.05 µmol [38]—perfectly acceptable for analysis. The Introduction of a single-column Procedure significantly simplifies the method [9, 29, 43, 60]. Using this technique, acidic, neutral, and basic Amino Acids can be determined in the same sample, which not only saves the test material but also increases accuracy and reduces analysis time. Working with a standard amino acid analyzer and using some modifications of known methods, the analysis of a single substance can be fully completed within 3 h [9].

Amino acid Sequence Determination is carried out on THE PRINCIPLE OF 'assembling a mosaic'. Suppose we have a substance with the sequence A. B. C. D. E. F. G. H. I. J. K. L. M. N and can use two methods of Specific Cleavage of peptide bonds. One of the methods cleaves the bond

... F. G ..., and the other—the bond ... H. I... Let us apply both cleavage methods to our model. The first method will yield two Peptides: A. B. C. D. E. F. and G. H. I. J. K. L. M. N. The second method will also yield two peptides: A. B. C. D. E. F. G. H. and I. J. K- L. M. N. The sequence of the original peptide is determined based on the Analysis of the four resulting fragments. To do this, we need to determine their amino acid composition, as well as their N- and C-terminal groups. The amino acid sequence of these small peptides can be determined by stepwise degradation, further partial Hydrolysis, and Determination of the Terminal Groups of the resulting smaller fragments. This makes it possible to identify 'overlapping' sequences of peptide fragments. For example, the G... H... portion of the peptide obtained by the first cleavage method has the same sequence as the C-terminal portion of the fragment ... F. G. H.—one of the peptides obtained by the second method. Using the 'mosaic assembly' principle, we can reconstruct The structure of the original peptide in this region.

Obviously, the larger the molecule of the protein being analyzed, the more difficult the problem of finding overlapping sequences, with specific hydrolysis being one of the most crucial steps in sequence determination.

A significant difficulty in determining the amino acid sequence can arise if Cysteine or cystine residues are present in the analyzed protein molecule. Oxidation of cysteine forms S–S bridges, which not only lead to erroneous Conclusions but also hinder further analysis, as proteins and polypeptides containing them are highly resistant to enzymatic cleavage. Therefore, prior to analysis, it is recommended to eliminate S–S bridges and prevent spontaneous oxidation of free SH groups. In addition, the possibility of SH/S–S exchange should be kept in mind. If free SH groups and S–S bridges are simultaneously present in the reaction mixture, rearrangements can occur in which disulfide-bonded peptide pairs exchange partners:

Class="center">R₁—S—S—R₂ + R₃—S—S—R₄⇄ R₁-S—S—R₃ + R₂—S-S-R₄.

SH and S–S groups are eliminated by quantitative and irreversible Oxidation of proteins with performic acid. As a result of this reaction, cysteine or cystine is converted to cysteic acid [86].

During oxidation with performic acid, Tryptophan is destroyed, and a significant portion of Tyrosine is converted to chlorinated tyrosine during subsequent HCl hydrolysis [85]. At low temperatures, oxidation proceeds more mildly, while under these conditions, the quantitative nature of cysteic acid formation is preserved [35].

Cleavage of S–S bonds can also be induced by a reduction reaction. If thio compounds are used as reducing agents, free reactive SH groups arise during the reaction, which can interfere with further analysis. Therefore, they must be blocked after the reduction reaction. The best blocking method is carboxymethylation. The reaction scheme is as follows:

As a result, a carboxymethylcysteine derivative is formed. If ¹⁴C-Br- or ¹⁴C-I-acetate is used in this reaction, then, in addition to cleaving S–S bonds and blocking the resulting SH groups, peptides containing cysteine residues can be radioactively labeled.

One variant of reduction is the sulfitolysis reaction. In the presence of Cu²⁺ at alkaline pH, compounds with S–S bonds (or free SH groups) are converted to S-sulfonates under METABOLISM/18.html">The Influence of sulfurous acid salts. The reaction proceeds as follows:

In protein structural analysis, the sulfitolysis reaction has never been widely used, and today it is rarely applied [5, 11, 14, 33, 59].

One of the KEY STAGES IN determining the amino acid sequence consists in the partial Cleavage of the protein molecules under study. The methods of partial hydrolysis or specific chemical cleavage used for this purpose must, in addition to the highest possible Specificity, meet the following requirements: 1) amino acids must not be destroyed during the reaction; 2) the Specificity of the reaction, i.e., cleavage at a strictly defined site, must be known in advance; 3) no side reactions, such as acyl transfer, transpeptidation, or The formation of random peptides, should occur during cleavage.

Partial hydrolysis methods are widely used, but almost all of them lack specificity and are therefore suitable only for obtaining small fragments, for example, at the final stages of cleaving large peptides. The oldest method of non-specific partial hydrolysis is Partial Acid Hydrolysis. Sanger and Tuppy [72] applied this method to determine the STRUCTURE OF THE Insulin B-chain. Having isolated and analyzed at least 60 peptides, they were able to determine only four Regions of the chain, comprising a total of 19 amino acid residues. In partial acid hydrolysates, in addition to peptides, up to 25% free Amino acids are found [54]. Tryptophan is completely destroyed during acid hydrolysis [53], and hydroxy amino acids are significantly damaged [65].

Among non-specific enzymatic cleavage methods, hydrolysis with Pepsin, Papain, and bacterial or fungal proteases is most commonly used. All Three types of Enzymes yield hydrolysates that are complex mixtures of small peptides. Therefore, they are best used at the final stages of cleaving large peptides obtained by specific hydrolysis methods. A general review of Enzymatic hydrolysis methods was provided by Hill [34]; detailed summaries of the action of pepsin, papain, and bacterial proteases were published by Bovey and Yanari [2], Smith and Kimmel [78], and Hagihara [28], respectively.

Specific enzymatic hydrolysis can be carried out in the presence of Trypsin and Chymotrypsin. These enzymes are currently considered to possess the highest cleavage specificity, and commercially available preparations of them are highly pure.

Trypsin hydrolyzes peptide bonds formed by basic amino acids, i.e., bonds involving Lysine and Arginine residues. The Lys-Pro and Arg-Pro peptide bonds are resistant to hydrolysis. Some other peptide bonds also exhibit partial resistance to tryptic hydrolysis, such as the Lys-Lys bond in the ... Lys-Lys-X ... structure, or bonds in Arg-Arg, Arg-Lys, and Lys-Arg peptides. A 'clustering' of basic amino acids in certain regions of a peptide renders it partially resistant to hydrolysis. The same applies to the Lys-Glu and Arg-Glu peptide bonds.

Chymotrypsin contamination in a trypsin preparation can significantly distort the specificity of tryptic hydrolysis; therefore, it is extremely important to avoid contamination of trypsin with chymotrypsin. A contaminated trypsin preparation can be purified by chromatography [84]; the chymotrypsin present in the preparation can be inactivated by acid or recently developed synthetic inhibitors.

Due to the specificity of tryptic hydrolysis, it can be 'controlled' by making certain modifications to the structure of the compounds being hydrolyzed. For example, as free SH groups are aminoethylated, the number of NH₂ groups increases, and consequently, the number of peptide bonds sensitive to trypsin increases, meaning more tryptic peptides are present in the hydrolysate [48, 63]. If, on the other hand, the NH₂ groups of lysine are reversibly or irreversibly blocked, trypsin will only be able to hydrolyze peptide bonds involving arginine. In this case, large arginyl peptides are found in the hydrolysate. With reversible blocking of lysine residues, for example by trifluoroacetylation, after the isolation of arginyl peptides, they can be unblocked and then subjected to tryptic hydrolysis; this will cleave only the lysine peptide bonds.

The newest method for the reversible blocking of ε-NH₂ groups involves maleinylation [47] and citraconylation [13] reactions. Both Reagents participating in these reactions not only block amino groups but also enhance the solubility of large fragments generated during tryptic hydrolysis. Due to the reversibility of both reactions, acylating reagents of this type can be successfully employed for primary sequence determination.

The “controlled proteolysis” method outlined above plays a vital role in analyzing the amino acid sequence of high-molecular-weight proteins.

Chymotrypsin cleaves more peptide bonds than trypsin. During short-term hydrolysis for 2–3 hours, the enzyme cleaves peptide bonds involving tyrosine, phenylalanine, and leucine residues. Proline peptides retain complete resistance to hydrolysis in this case as well. “Clusters” of aromatic amino acids, such as structures resembling ... Phe-Phe ... or ... ... Tyr-Phe ... etc., exhibit partial resistance to hydrolysis. However, as the duration of hydrolysis increases, peptide bonds of many types are broken down. A detailed review of chymotrypsin was prepared by Desnuelle [7]; information regarding the specificity of chymotryptic hydrolysis can be found in Hill's review [34].

Specific chemical cleavage of peptide bonds can be achieved using two oxidizing agents. One of them, N-Bromosuccinimide, induces the cleavage of the peptide bond involving a tryptophan residue, accompanied by the simultaneous destruction and oxidation of this residue [64]. Successful application of this method has been achieved in only a few cases. In addition to studying the structure of the polypeptide isolated from tobacco mosaic virus, N-bromosuccinimide was used in analyzing the sequence of low-molecular-weight Glucagon [58] and the N-terminal sequence of Hemoglobin [79]. A major drawback of this method is that N-bromosuccinimide does not cleave all peptide bonds formed by tryptophan residues. In reactions with the same protein preparation, from 2 to 80% of these bonds may be oxidized. Furthermore, certain proteins, such as cytochrome c, prove completely resistant to the action of N-bromosuccinimide.

Another oxidizing agent, Cyanogen bromide, is a more reliable reagent [26]. It breaks down peptide bonds involving a Methionine residue. The Mechanism of this reaction is similar to the cleavage by iodoacetamide [45]. Cyanogen bromide oxidation is widely used as a specific method for obtaining large peptide fragments. It has been very successfully employed alongside other methods in elucidating the amino acid sequence of Ribonuclease [27], Myoglobin [17], trypsinogen [36], and aldolase [42, 68, 69].

Even with specific enzymatic or chemical cleavage of a protein or medium-molecular-weight polypeptide, a rather complex mixture of peptides is obtained. There is a set of diverse methodological techniques for fractionating this mixture and purifying individual components. It is hardly possible to propose a single fractionation scheme equally applicable to all protein hydrolyzates. The first step typically involves preliminary fractionation according to peptide charge or size, followed by the final purification of the peptides using electrophoresis, Ion Exchange, or other chromatographic Procedures.

Preliminary Separation of a peptide mixture according to their electrostatic charge can be achieved using free ionophoresis (or multicompartment electrophoresis), which was successfully employed by Sanger and Tuppy [72]. However, this method has now been entirely superseded by Gel filtration, i.e., separation according to molecular size. Following the pioneering work in this direction [44, 83], the extremely useful method of gel filtration was developed by Porath and Flodin [61]; Flodin later provided its theoretical foundation [20].

In gel filtration, Two Types of gels are most commonly used: gels obtained by cross-linking dextran (Sephadices) and synthetic acrylamide gels (Bio-Gels). Acrylamide gels exhibit low adsorption capacity; therefore, for an equivalent resolving power, they are characterized by a higher yield of the fractionated substance compared to dextran gels.

Following preliminary fractionation, further purification of the peptide mixture is conveniently performed using paper electrophoresis. There are four variants of this method: 1) vertical paper electrophoresis without cooling [51]; 2) vertical paper electrophoresis with toluene cooling [50]; 3) horizontal paper electrophoresis with “sandwich”-type cooling [25]; 4) horizontal paper electrophoresis with cooled-plate cooling [10].

Horizontal paper electrophoresis allows for the analysis of peptide mixtures via the “fingerprint” method and the isolation of micro-amounts of various fractions. Paper electrophoresis in two buffer systems is convenient for these tasks, as it enables the clear separation of acidic and basic Components of the mixture in a single-step experiment [8]. After medium-duration hydrolysis, a mixture of acidic and basic peptides is typically present in the reaction mixture. The simultaneous application of two different Buffer solutions facilitates a more complete separation of such peptides.

The use of volatile buffers in electrophoresis makes it possible to elute the separated peptides immediately after drying the paper and to carry out their further fractionation.

Among volatile buffers, the pyridine–acetic acid and acetic acid–formic acid systems with pH values of 6.5, 3.5, and 1.9 are most frequently used.

During purification, paper chromatography is applied to separate peptides following electrophoresis at different pH values.

It is standard practice to identify peptides using the ninhydrin reaction. The addition of Cd²⁺ to the ninhydrin solution not only increases its sensitivity but also improves color stability. N-acyl peptides can be detected via the chlorine reaction [67]. Certain amino acids are identified by specific color reactions, for example, Histidine by the Pauli reaction and arginine by the Sakaguchi reaction. These same reactions can also be used to detect peptides containing histidine and arginine.

While paper electrophoresis and chromatography can be classified as micro-preparative methods, the separation of peptide mixtures via ion-exchange chromatography should arguably be considered macro-preparative. Its main advantage lies in the fact that it allows for the Processing of large quantities of material and yields unquestionably higher recoveries of fractions. The use of volatile buffers in ion-exchange chromatography makes it possible to avoid the time-consuming Desalting procedure that complicated previously proposed methodologies [49, 92]. A major breakthrough in ion-exchange chromatography is the introduction of spherical resins. Their use helps increase the flow rate of fractionated substances through the column and significantly shortens the duration of preparative separation. Spherical resins in automated amino acid analyzers ensure reproducible, high-resolution comparative chromatography of peptides, i.e., they automate the “fingerprint” analysis method.

A key role in sequence determination belongs to the identification of terminal amino acids, which possess either a free NH₂ group at the N-terminus or a free COOH group at the C-terminus of the peptide. For this reason, Sanger's classical method represented a major breakthrough in Protein Chemistry; its principle is as follows. The analyzed protein or peptide is treated under very mild conditions with 2,4-dinitrofluorobenzene (DNFB). This results in the dinitrophenylation of N-terminal amino groups as well as certain reactive side chains containing the following groups: ε-NH₂, phenolic –OH, imidazole –NH, and –SH. Subsequently, the dinitrophenyl (DNP) derivatives of proteins or peptides are subjected to acid hydrolysis, and ether extraction is used to isolate the α-DNP-amino acids that occupied the N-terminal position in the protein or peptide. Following extraction, only water-soluble DNP-arginine and DNP-lysine remain in the hydrolyzate. Chromatographic analysis of the ether extract is then used to precisely identify the terminal amino acid using column [1, 62], paper [46, 66], or Thin-Layer Chromatography [3].

The dansyl method, developed by Gray and Hartley in 1963 [24], is in many respects similar to the dinitrofluorobenzene method. The primary reagent in the dansyl method is dimethylaminonaphthalenesulfonyl chloride. The dansyl group attaches to the same peptide groups as DNFB, but due to intense fluorescence, dansyl derivatives can be detected in very small amounts (down to 10^-4 μmol).

Another METHOD FOR DETERMINING the N-terminal amino acid is Edman Degradation using phenylisothiocyanate [15, 16]. Phenylisothiocyanate converts N-terminal amino acids into phenylthiocarbamyl derivatives, which are cleaved off as phenylthiohydantoin derivatives under conditions that do not damage other peptide bonds. This method enables the stepwise degradation of polypeptides and peptides. The cleaved amino acid residue can be identified by chromatography of the phenylthiohydantoin derivatives [76] or by comparing the amino acid composition of shortened peptides with that of the starting material. Recently, the Edman reaction has commonly been combined with the dansyl method, since this approach requires a very small amount of the degraded peptide to determine the terminal amino acid.

The N-terminal sequence can also be determined using the enzyme aminopeptidase, which specifically hydrolyzes N-terminal peptide bonds (i.e., bonds formed by an amino acid with a free α-NH₂ group) [77, 81]. The amino acids liberated after hydrolysis are identified chromatographically.

Similarly, C-terminal Amino Acids and C-terminal sequences can be determined using Carboxypeptidases A and B. If a basic amino acid occupies the C-terminal position, it can be cleaved off exclusively by carboxypeptidase B [21].

Following Sanger's pioneering work, the Primary Structure of numerous polypeptides and proteins has been elucidated in recent years. After the amino acid sequence of insulin was determined, the sequences of ribonuclease, tobacco mosaic virus polypeptide, several cytochrome c preparations, various hemoglobin chains, trypsin inhibitor, chymotrypsinogen and chymotrypsin, trypsin, glyceraldehyde-3-phosphate dehydrogenase, etc., were decoded. Along with the sequences of certain Peptide Hormones, these data were included in the “Atlas of Protein Sequence and Structure,” which was first published in 1966 and subsequently reissued multiple times¹.

The second edition of this atlas, published in 1967–1968, is twice the size of the first and constitutes an exceptionally valuable summary of sequence determination results. It presents the sequences of cytochrome c, ferredoxin, hemoglobin chains, myoglobin, various IMMUNOGLOBULINS and their L-chains, trypsinogen, trypsin, chymotrypsinogen, chymotrypsin, subtilisin, α-lactalbumin, tryptophan synthase, lysozymes, ribonuclease, trypsin inhibitor, peptide hormones, venoms, toxins, viral proteins, etc. Including species variations, the number of proteins with elucidated structures reaches nearly two hundred.

Elucidating the entire sequence of the studied protein is not at all necessary for solving certain biological problems. For instance, when investigating structure-function relationships, analyzing species specificity, or addressing A number of other problems, it is required to determine the sequence of a specific part of the structure of that “marked” peptide which directly participates in the process under consideration. Depending on the function being studied, THE CONCEPT OF a “marked” peptide can be quite broad. It may be an Active Site, a binding region, or, finally, a specificity locus.

Locating the “marked” peptide is far from straightforward. Nature rarely endows a structure of interest to the experimenter with any kind of label. However, such a label does exist, for example, in the heme-binding peptide of cytochrome c. The part of the peptide directly involved in carrying out the biological function is covalently linked via a thioether bond to the prosthetic group of the cytochrome. This stable bond facilitates the isolation and analysis of this peptide. Tuppy et al. [87–91] successfully compared the sequences of heme-binding peptides isolated from Cytochromes c of various species origins. Some of these data are presented in Table 6.

¹ One of the latest editions was published in 1972 (vol. 5) edited by Dayhoff; a Supplement to it was released in 1973 (see References). — Ed. note.

Table 6 Heme-binding peptides from cytochromes of various species

As can be seen from the data in Table 6, cytochromes from even very phylogenetically distant species exhibit a striking similarity in their heme-binding peptides, particularly within the region containing the ... Cys-His-Thr ... sequence. Taken together with the results of later studies, these findings strongly support the principle of "similar structure — similar function" widely accepted in protein chemistry.

Another remarkable example of The Study of "functional" peptides can be found in The history of esterase research. Jansen et al. [39] discovered that equimolar concentrations of diisopropyl fluorophosphate (DFP) are capable of irreversibly inhibiting The activity of certain proteases and esterases. Hydrolysates of these enzymes yielded specific peptides bound to the radioactive inhibitor, allowing their structures to be determined. The results of these analyses are presented in Table 7. In all the compared enzymes, the amino acid residues located adjacent to the DFP-blocked, Serine-containing side chain were found to be identical or closely related. This comparison further validates the principle of "similar structure — similar function." In protein chemistry, however, this principle must be applied with a certain caveat: an analogous structure does not always correspond to an identical function, but rather to a similar or genetically related one. Similar structures may be genetically related and arise, for instance, from a common ancestral protein through Amino Acid Substitutions resulting from a single-base change in their codons.

Treatment of dehydrogenases with radioactive iodoacetate, followed by the fractionation of the hydrolysate, makes it possible to determine the amino acid sequence surrounding the reactive SH-groups in these enzyme molecules. This approach has been successfully used to decode the active-site sequences of glyceraldehyde-3-phosphate dehydrogenase and Alcohol dehydrogenases [30, 31, 47]. The sequence of the corresponding region in Lactate dehydrogenase was determined in a similar manner [23, 37]. The results of these investigations are summarized in Table 8.

Table 7 Serine-containing peptides of esterases

The structural analogies shown in Table 8 allow dehydrogenases to be divided into two groups. The first group comprises lactate and alcohol dehydrogenases, while the second group consists of the two types of triose dehydrogenases. Despite some apparent contradictions, our current understanding of the Functions of these enzymes largely Supports this Classification. For example, it has been demonstrated quite conclusively that the reactive SH-groups of the two triose dehydrogenases listed in the table participate in a similar manner in the formation of an intermediate acyl-enzyme complex. Both triose dehydrogenases are analogous to each other in their corresponding structural regions, yet show no similarity to the Enzymes of the first group. Thus, structural analogy is indeed accompanied by functional similarity.

Table 8 Cysteine-containing "active" peptides in various dehydrogenases

References

1. Bailey K., Biochem. J., 49, 23 (1951).

2. Bovey F. A., Yanari S. S., in: Boyer P., Lardy H., Myrbäck K., The Enzymes, Academic Press, New York (1960).

3. Brenner A., Niederwieser A., Pataky G., Experientia, 17, 145 (1961).

4. Butler P. J. E., Harris J. I., Hartley B. S., Leberman R., Biochem. J., 112, 679 (1961).

5. Cecil R., Loening U. E., Biochem. J., 76, 146 (1960).

6. Colvin J., Smith D. B., Cook W. H., Chem. Rev., 54, 687 (1954).

7. Desnuelle P., in: Boyer P., Lardy H., Myrbäck K., The Enzymes, Academic Press, New York (1966).

8. Devenyi T., Magyar Kem. Folyóirat, 69, 538 (1963).

9. Devenyi T., Acta Biochim. Biophys. Acad. Sci. Hung., 3, 429; 4, 297 (1968, 1969).

10. Devenyi T., Sajgo M., Horvath E., Szeneyi B., Magyar Kem. Folyóirat, 70, 123 (1964).

11. Dinh van Hoang, Rovery M., Desnuelle P., Biochim. Biophys. Acta, 69, 188 (1963).

12. Dixon G. H., Kauffmann D. L., Neurath H., J. Biol. Chem., 233, 1373 (1958).

13. Dixon H. B. F., Perham R. N., Biochem. J., 109, 312 (1968).

14. Dlouha V., Keil B., Sorm F., Coll. Czechoslov. Chem. Commun., 28, 2969 (1963).

15. Edman P., Acta Chem. Scand., 4, 283 (1950).

16. Edman P., Acta Chem. Scand., 7, 700 (1953).

17. Edmundson A. B., Nature, 198, 354 (1963).

18. Engström L., Biochim, Boiphys. Acta, 56, 606 (1962).

19. Fisher E. H., Graves D. J., Crittenden E. R. S., Krebs E. G., J. Biol. Chem. 234, 1698 (1959).

20. Flodin P., J. Chromatogr., 5, 103 (1961).

21. Gladner J. A., Folk J. E., J. Biol. Chem., 231, 393 (1957).

22. Glander J. A., Laki K., J. Am., Chem., Soc., 80, 1263 (1958).

23. Gold A. H., Segal H., Biochemistry, 4, 1506 (1965).

24. Gray W. R., Hartley R. S., Biochem., J, 89, 379 (1963).

25. Gross D., Nature, 176, 72 (1955).

26. Gross E., Witkop B., J. Am. Chem. Soc., 83, 1510 (1961).

27. Gross E., Witkop B., J. Biol. Chem., 237, 1856 (1962).

28. Hagihara B., in: Boyer P., Lardy H., Myrbäck K., The Enzymes, Academic Press, New York, 4, 193 (1960).

29. Hamilton P. B., Anal. Chem., 35, 2055 (1963).

30. Harris J. I., Nature, 203, 30 (1964).

31. Harris J. I., Meriwether P. B., Park J. H., Nature, 198, 154 (1963).

32. Hartley B. S., J. Cell and Compar Physiol., 54, 203 (1959).

33. Hartley B. S., VI Intern. Biochem. Congr., IV Sympos. Publishing House of the USSR Academy of Sciences, Moscow (1962).

34. Hill R. L., Adv. Protein Chem., 37,107 (1965).

35. Hirs C. H. W., J. Biol. Chem., 219, 611 (1956).

36. Hoffmann T., Walsh K- A., Rauffmann D., Neurath H., Fed. Proc., 22, 528 (1963).

37. Holbroock J. J., Pfleiderer G., Schnetger J., Diemair S., Biochem. Z., 344, 1 (1966).

38. Hubbard R. W., Rremer D. M., Anal. Biochem., 12, 593 (1965).

39. Jansen E. F., Curl A. L., Laurence A., Batts A. R., J. Biol. Chem., 179, 189 (1949).

40. Jansz H. S., Brons D., Biochim. Biophys. Acta, 33, 396 (1959).

41. Jansz H. S., Brons D., Warringa M. G., Biochim, Biophys. Acta, 34, 573 (1959).

42. Lai C. Y., Arch. Biochim. Biophys., 128, 22 (1968).

43. Larsen I., Scientific Tools, 12, 24 (1965).

44. Lathe G. H., Ruthaven G. R. J., Biochim. J., 62, 665 (1965).

45. Lawson W. B., Gross E., Folitz C. H., Witkop B., J. Am. Chem. Soc., 85, 1509 (1961).

46. Levy A., Nature, 174, 126 (1954).

47. Li T. R., Vatlee B. L., Biochemistry, 3, 869 (1964).

48. Lindley H., J. Am. Chem. Soc., 77, 4027 (1956).

49. Margoliash E., J, Biol. Chem., 237, 2161 (1961).

50. Michl M., Monatsh. Chemie, 82, 489 (1951).

51. Mikes O., Coll. Czechoslov. Chem. Commun., 22, 831 (1957).

52. Milstein C., Sanger F., Biochim. J., 79, 456 (1961).

53. Monier R., Jutisz M., Bull Soc. Chim. Biol., 32, 228 (1950).

54. Moore S., Bergmann M., J. Biol. Chem., 154, 191 (1944).

55. Moore S., Stein W. H., J. Biol. Chem., 176, 367 (1948).

56. Naughton M. A., Sanger F., Hartley B. S., Shaw D. C., Biochim. J., 77, 149 (1960).

57. Oesterbaan R. A., wan Andrichen M. E., Biochim. Biophys. Acta, 27, 423 (1958).

58. Patchornik A., Lawson W. B., Cross E., Witkop B., J. Am. Chem. Soc., 82, 5923 (1960).

59. Pechere J. F., Dixon G. H., Mayburry R. H., Neurath H., J. Biol. Chem., 233, 1364 (1968).

60. Piez K. A., Morris L., Anal. Biochem., I, 187 (1960).

61. Porath J., Flodin. O., Nature. 183, 1657 (1959).

62. Porter R. R., Sanger F-, Biochem. J., 42, 287 (1948).

63. Raftery M. A., Cole R. D., Biochem. Biophys. Res. Commun., 10, 464 (1963).

64. Ramachandran L. R., Witkop B., J. Am. Chem. Soc., 81, 4028 (1959).

65. Rees M. W., Biochem., J., 40, 632 (1946).

66. Rovery M., Fahre S., Bull. Soc. Chim. Biol., 35, 541 (1953).

67. Rydon H. N., Smith P. W. G., Nature, 169, 922 (1952).

68. Sajgó M., Acta Biochim. Biophys. Acad. Sci. Hung., 4, 385 (1969).

69. Sajgó M., FEBS Lett., 12, 349 (1971).

70. Sanger F., Proc. Chem. Soc., p. 76 (1963).

71. Sanger F., Shaw D. C., Nature, 187, 872 (1960).

72. Sanger F., Tuppy H., Biochem. J., 49, 463; 481 (1951).

73. Schaffer N. K-, Harmann S., Engle R. J., J. Biol. Chem., 214, 799 (1955).

74. Schaffer N. R-, May C. S., Summerson W. H., J. Biol. Chem., 206, 201 (1954).

75. Schwarts J. H., Crestfield A. M., Lipmann T., Proc. Nat. Acad. Sci. (Wash) 49, 722 (1963).

76. Sjöquist J., Biochim. Biophys. Acta, 41, 20 (1960).

77. Smith E. L., Fed. Proc., 16, 801 (1957).

78. Smith E. L., Kimmel J. R., in: Boyer P., Lardy H., Myrbäck K., The Enzymes, Academic Press, New York, 133 (1960).

79. Sosakowa S., J. Biochem. (Tokyo), 53, 188 (1963).

80. Spackman D. H., Fed. Proc. 22, 244 (1963).

81. Spackman D. H., Smith E. L., Brown D. M., J. Biol. Chem., 212, 255 (1955)

82. Synge R. L. M., Chem. Rev., 32, 135 (1943).

83. Synge R. L. M., Tiselius A., Biochem., J., 46, P41 (1950).

84. Tatian H. H., Biochim. Biophys. Acta, 27, 407 (1958).

85. Thompson E. O. P., Biol. Chem., 207, 563 (1954).

86. Toennies G., Homiller R. P., J. Am. Chem. Soc., 64 , 3054 (1942).

87 Tuppy H., Z. Naturforsch., 12b, 784 (1957).

88. Tuppy H., Naturwissenschaften, 46, 35 (1959).

89. Tuppy H., Bodo G., Monatsh. Chemie, 85 , 807, 1024, 1182 (1954).

90. Tuppy H., Dus K., Monatsh. Chemie. 89, 407 (1958).

91. Tuppy H., Peteus S., Acta Chem. Scand., 9, 363 (1955).

92. Vanecek J., Meloun B., Kostka V., Keil B., Sorm F., Biochim. Biophys. Acta, 37, 169 (1960).

Recommended reading

Colowick S. P., Kaplan N. O. eds., Methods in Enzymology, vol. XXV, Academic Press, New York, London, 1972.

Dayhoff M. O., Atlas of Protein Sequence and Structure, vol. 5, 1972, Suppl. 1, Natl. Biomed. Res. Foundation Georgetown Univ. Med. Centr. Washington D. C., 1973.

Needleman S. B. ed., Protein Sequence Determination, Springer Verlag, Berlin, Heidelberg, New York, 1970.