Amino acids purified

New methods use combined HPLC/Mass spectrometry to identify modified amino acids. Purified recombinant human insulin-like growth factor separated into two peaks on reverse phase HPLC (Cl 8 column/acidified water) even though other methods indicated it was completely pure. Plasma desorption mass spectrometry of the individual peptides detected a single methionine sulfoxide molecule that was sufficient to decrease the hydrophobicity of the whole protein significantly. Most of the oxidation occurred when the secreted fusion protein was cleaved with hydroxylamine under not strictly anaerobic conditions, but about 5% occurred during die . coli fermentation. 

The synthesis of phosphonic analogues of aspartic acid and asparagine is accomplished by addition of diethyl phosphite to diethyl acctamidomcthylcncmalonate The sttongly activated double bond readily adds diethyl phosphite in an exothermic reaction catalyzed by EtONa (Scheme 8.84). Hydrolysis of the crude addition product with concentrated HCl at reflux delivers the amino acid purified using acidic ion-exchange resin (Dowex SOW). 

W. C. Rose and coworkers (67) were able to maintain N-balance in young men with high caloric diets constructed from racemate containing mixtures of nine crystalline amino acids, purified fats, carbohydrates and vitamin mixtures. Since the nine essential amino acids of those diets had to be converted in part to the ten unessential amino acids, the requirement of each of-the nine essential amino acids appears to be about twice that shown in Table V. 

When the sample is a solid, a separation of the analyte and interferent by sublimation may be possible. The sample is heated at a temperature and pressure below its triple point where the solid vaporizes without passing through the liquid state. The vapor is then condensed to recover the purified solid. A good example of the use of sublimation is in the isolation of amino acids from fossil mohusk shells and deep-sea sediments. ... 

The potentiometric titration curve shown here was recorded on a 0.4300-g sample of a purified amino acid that was dissolved in 50.00 ml of water and titrated with 0.1036 M NaOH. Identify the amino acid from the possibilities listed in the following table. 

Miscellaneous Reactions. Sodium bisulfite adds to acetaldehyde to form a white crystalline addition compound, insoluble in ethyl alcohol and ether. This bisulfite addition compound is frequendy used to isolate and purify acetaldehyde, which may be regenerated with dilute acid. Hydrocyanic acid adds to acetaldehyde in the presence of an alkaU catalyst to form cyanohydrin the cyanohydrin may also be prepared from sodium cyanide and the bisulfite addition compound. Acrylonittile [107-13-1] (qv) can be made from acetaldehyde and hydrocyanic acid by heating the cyanohydrin that is formed to 600—700°C (77). Alanine [302-72-7] can be prepared by the reaction of an ammonium salt and an alkaU metal cyanide with acetaldehyde this is a general method for the preparation of a-amino acids called the Strecker amino acids synthesis. Grignard reagents add readily to acetaldehyde, the final product being a secondary alcohol. Thioacetaldehyde [2765-04-0] is formed by reaction of acetaldehyde with hydrogen sulfide thioacetaldehyde polymerizes readily to the trimer. 

A potentially general method of identifying a probe is, first, to purify a protein of interest by chromatography (qv) or electrophoresis. Then a partial amino acid sequence of the protein is deterrnined chemically (see Amino acids). The amino acid sequence is used to predict likely short DNA sequences which direct the synthesis of the protein sequence. Because the genetic code uses redundant codons to direct the synthesis of some amino acids, the predicted probe is unlikely to be unique. The least redundant sequence of 25—30 nucleotides is synthesized chemically as a mixture. The mixed probe is used to screen the Hbrary and the identified clones further screened, either with another probe reverse-translated from the known amino acid sequence or by directly sequencing the clones. Whereas not all recombinant clones encode the protein of interest, reiterative screening allows identification of the correct DNA recombinant. 

These methodologies have been reviewed (22). In both methods, synthesis involves assembly of protected peptide chains, deprotection, purification, and characterization. However, the soHd-phase method, pioneered by Merrifield, dominates the field of peptide chemistry (23). In SPPS, the C-terminal amino acid of the desired peptide is attached to a polymeric soHd support. The addition of amino acids (qv) requires a number of relatively simple steps that are easily automated. Therefore, SPPS contains a number of advantages compared to the solution approach, including fewer solubiUty problems, use of less specialized chemistry, potential for automation, and requirement of relatively less skilled operators (22). Additionally, intermediates are not isolated and purified, and therefore the steps can be carried out more rapidly. Moreover, the SPPS method has been shown to proceed without racemization, whereas in fragment synthesis there is always a potential for racemization. Solution synthesis provides peptides of relatively higher purity however, the addition of hplc methodologies allows for pure peptide products from SPPS as well. 

Evidence soon emerged that the endogenous opioids were peptides rather than simple morphine-like molecules (9). The first direct evidence for endogenous opioids in brain extracts was provided in 1975 when two pentapeptides were purified that differed only in the carboxyl terminal amino acids (10) (Table 1). These peptides were called methionine- (Met-) and leucine- (Leu-) enkephalin, from the Greek term meaning "in the head."... 

The key enzyme in this sequence, isopenicillin N synthase (IPNS), has been purified from E. coli (59) and the recombinant enzyme shown to be a single polypeptide of 336 amino acids containing two cysteines, numbers 106 and 255 from the /V-teiminus, and probably a ferrous ion in a nonheme environment. The enzyme has been crystallized and studies undertaken to obtain suitably sized crystals for diffraction studies. 

Isoxanthopterin (2-amino-4,7-dihydroxypteridine) [529-69-1] M 179.4, m>300°, pKj -0.5 (basic), pKj 7.34 (acidic), pKj 10.06 (acidic). Purified by repeated pptn from alkaline solutions by acid (preferably AcOH), filter, wash well with H2O then EtOH and dried at 100°. Purity is checked by paper chromatography [Rp 0.15 (n-BuOH, AcOH, H2O, 4 1 1) 0.33 (3% aq NH4OH). [Goto et al. Arch Biochem... 

Polypeptides. These are a string of a-amino acids usually with the natural 5(L) [L-cysteine is an exception and has the R absolute configuration] or sometimes "unnatural" 7f(D) configuration at the a-carbon atom. They generally have less than -100 amino acid residues. They can be naturally occurring or, because of their small size, can be synthesised chemically from the desired amino acids. Their properties can be very similar to those of small proteins. Many are commercially available, can be custom made commercially or locally with a peptide synthesiser. They are purified by HPLC and can be used without further purification. Their purity can be checked as described under proteins. 

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