Edman sequenator

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

In 1950 an alternative to the Sanger procedure for identifying N-terminal amino acids was reported by Edman—reaction with phenyl-isothiocyanate to give a phenylthiocarbamide labeled peptide. When this was heated in anhydrous HC1 in nitromethane, phenylthiohy-dantoin was split off, releasing the free a-NH2 group of the amino acid in position 2 in the sequence. While initially the FDNB method was probably the more popular, the quantitative precision which could be obtained by the Edman degradation has been successfully adapted to the automatic analysis of peptides in sequenators. 

In the following section constructural details of the Sequenator will be described to the extent required for an understanding of its operation. A detailed technical description has been published in 1967 by Edman and Begg ). Fig. 3 represents a diagrammatic view of the essential components of the instrument. The central part of the Sequenator is the reaction vessel (A), a cylindrical cup of pyrex glass mounted on the shaft of an electric motor (5). Correct functioning of this device requires that the inside cylindrical surface of the cup runs absolutely true. Variance would cause untolerable turbulence within the liquid film spread on the wall of the cup. An additional requirement is constant rotational speed of the cup. Variance of the speed would cause movement of the film up or down the wall. The cup is housed in a bell jar Q) which... 

Ingenious protein sequenators have been devised to carry out the Edman degradation automatically.242 244-246 Each released phenylthiohydantoin is then identified by HPLC or other techniques. Commercial sequenators have often required 5-20 nmol of peptide but new microsequenators can be used with amounts as low as 5-10 picomoles or less.247 248... 

GLC is an important adjunct to protein sequence determination. Automatic "sequenators" based upon the approach developed by Edman are available and have been described in detail by Niall (60). The Edman degradation, summarized in Equation 9.5, makes use of methyl or phenylisothiocyanate which reacts with the N-terminus of a peptide. Exposure of the isothiocyanate derivative of the protein to acid results in cleavage of the terminal amino acid as a thiaxolinones and exposure of the next amine group on the peptide. Thus, the process can be repetitively carried out, each amino acid removed from the peptide, in a sequential manner. Thiazolinones rearrange in acid medium to form thiohydantoin derivatives of amino acids, some of which may be directly gas chromatographed others must be derivatized typically as trimethylsilyl derivatives. 

Pure peptides (native or reduced and alkylated) are sequenced by automatic Edman degradation on a pulse liquid automatic sequenator. Reagent and solvents are purchased from the manufacturer (Applied Biosystem Division). 

Analytical research applications of mass spectrometers fitted with electro-optical ion detectors are carried out at the present time at JPL and the FOM Institute. These involve" (1) at JPL a 1" EOID on sector type mass spectrometer (CEC type 21-490) in conjunction with an API (armospheric pressure ionization) source for the development of an automated assay procedure for amino acid derivatives produced by a novel Edman type sequenator (23), and (2) at the FOM Institute a 1" EOID on a sector mass spectrometer,fitted with magnetic and electrostatic quadruple "Zoom" lenses, permitting variation of dispersion for focusing a greater number of masses on the 1" detector (24,25). 

Studying chemical modification will be described. Histones are modified in vivo by acetylation (also methylation and phosphorylation, see DeLange and Smith 1971, 1974). In some histones the a-amino groups are acetylated (in addition to lysine residues) and this precludes direct examination of the internal sites of acetylation by the Edman procedure. However, histones IIb2 and III have free a-amino groups and this enabled Candido and Dixon (1972) to examine these histones (from trout testis) which had been acetylated intracellularly with " C-labeled acetate. Each residue that was released by the Edman procedure (using a sequenator) was examined for radioactive material and it was found that lysyl Residues 14 and 23 (major sites) and 9 and 18 (minor sites) were partially acetylated in histone III and lysyl Residues 5,10,13 and 18 were partially acetylated in histone IIb2. This type of approach should be applicable to many other studies of chemical modification. 

Conversion of half-cystine residues in proteins and peptides to the S-methyl derivatives is advantageous in subsequent studies of amino acid sequence. Under the usual conditions of acid hydrolysis ( 2.1), S-methylcysteine is recovered in a 90% yield (Heinrikson 1971). The phenylthiohydantoin of S-methylcysteine is readily identified by routine thin layer chromatography procedures (Rochat et al. 1970). With the increasing use of the sequenator, PTH-S-methylcysteine offers a marked advantage over derivatives such as PTH-cysteic acid, or PTFl-carboxymethylcysteine, which have to be identified by special techniques (Edman 1960, 1970). S-methylcysteinyl residues provide a new point of cleavage for cyanogen bromide (5). 

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