Lysine, reversed-phase

A dansyl-containing lysine analogue, monodansylcadaverine (MDC), was used in initial TGase linking, because the dansyl UV absorption peak allowed quantification by reverse-phase HPLC using the absorbance at 280 nm. The reacted Qll was dissolved in TFA along with a known dansyl standard. Peak areas of the standard were then compared with product to establish the amormt of MDC present into Qll. Six Qll peaks were measured and ascribed to Qll with zero to five MDCs attached (Fig. 33). 

In this case, unilamellar vesicles with a large capture volume were prepared by the reverse phase evaporation technique and alginate was used to microencapsulate the liposome s. The alginate spheres were double coated, first with poly-L-lysine and then with polyvinyl amine (Wheatley and Langer in press). 

A problem with the chromatographic determination of cysteic acid is that there is almost no retention of cysteic acid. For both reversed-phase HPLC and ion-exchange amino acid analyzers (usually employing cation-exchange resins), cysteic acid is essentially eluted within or near the void volume of the column. This makes it more susceptible to unknown chromatographic interferences from various matrices. When cysteine is alkylated by 3-bromopropylamine, the product (S-3-aminopropylcysteine) looks very similar to lysine in structure. Hale et al. (90) show that this alkylated species affords excellent chromatographic separation on four different commercially available amino acid analysis systems and that, indeed, it does elute very near lysine in each case (see Fig. 4). 

Lysine is an essential amino acid. Since lysine is a fairly acid-stable amino acid, its analysis as total lysine by the traditional hydrochloric acid hydrolysis is straightforward. Fairly recent examples for the successful determination of total lysine employing either ion-exchange (82) or reversed-phase (101) HPLC are available. 

Similar experiments were undertaken joining AMP to a dipeptide hippuryl-lysine. This particular dipeptide was used because a comparison of the retention times of lysine and hippuryllysine revealed that the addition of the hip-puric acid to the lysine reduced the polarity of the latter. However, a determination of the retention times of the dipeptide and AMP on a reversed-phase column (Fig. 2.16B) reveals both to be polar. Nevertheless, their conjugate has a longer retention time than either of the starting materials. Note, however, that the decrease in polarity of this conjugate is very much less than what was observed following the summation of the AMP and lysine (Fig. 2.16,4). 

The DNP derivatives are analyzed either in normal or reversed phase. Disadvantages of this method are the lower detection sensitivity (60 times less sensitive compared to dabsyl detection) and the lower separation resolution. However, this approach has proven useful for the determination of lysine in food materials. 

Another strategy for stable isotope labeling of N-terminus and lysine side-chain amines is reductive animation by nondeuterated and deuterated formaldehyde. Dimethylation of peptides occurs by the formation of a Schiff base, followed by reduction with cyanoborohydride. Peptides thus labeled differ by a mass of 4 Da for each labeled pair. The labeled peak pairs show minimum isotope effects on reverse-phase separation and hence coelute (93). Dimethylation using formaldehyde with the natural isotopic abundance (hydrogen and 12C) versus a heavy form with two deuteriums and one atom of 13C... 

A simple assay based on potent and specific inhibition of jack bean a-mannosidase has been devised for determining low concentrations of 162 (up to 0.5 cm ) in M anisopliae cultures (110). The new assay was used to demonstrate that the addition of L-lysine (163) to the culture medium stimulated production of the alkaloid by approximately fourfold. Other early metabolic precursors of 162 in this fungus, including a-aminoadipic acid, saccharopine (164), L-pipecolic acid (165), and L-lysine itself, were quantified by reverse-phase HPLC analysis of mycelial extracts derivatised with 9-fluorenylmethyl chloroformate (FMOC) (111). 

The primary structure was assessed by peptide mapping and N- and C-terminal sequencing. N-terminal sequence analysis showed that a single sequence was detected, MKAIFVLNAA, which corresponds exactly to the first 10 amino acids at the N terminus of P40 as predicted from the DNA sequence. Reverse phase HPLC analysis of a digestion of P40 with a lysine-specific endopeptidase, i.e. endoproteinase Lys-C, was used for identification and primary structure confirmation (Fig. 9). Endoproteinase Lys-C hydolyzes peptide bonds at the carboxylic side of lysine residues. The seventeen peaks resolved were characterized by mass spectrometry, allowing the confirmation of 99 % of the primary sequence. 

The X-ray structure of spinach Rubisco identifies 10 loop regions in the active-site (8). The major photolabeled peptide isolated by ion-exclusion and ion-exchange chromatography, and reverse phase HPLC corresponded to the Rubisco LSu tryptic peptide Val-42 to Arg-79 (Table 1), an active-site peptide (9). Two portions of the tryptic peptide Asp-95 to Lys-128, the peptide which comprises loop B in the crystal structure (8) were also recovered. Also recovered were two peptides (Table 1) which include residues in two other loop regions of the Rubisco active-site (8). The peptide Asp-202 to Arg-213 (loop 2 in ref. 8) is adjacent to the activator lysine (10). The peptide Gln-259 to His-267 is the N-terminal fragment of Gln-259 to Arg-285 the tryptic peptide which encompasses loop 4. Thus, all four of the photoaffinity labeled LSu peptides have been previously shown to be active-site peptides based on X-ray crystallography or chemical modification or by both techniques. 

To confirm that the incorporated radioactive moiety was mono(ADP-ribose), the acid-insoluble fractions of SR vesicles incubated with labeled NAD in the presence and absence of poly L-lysine were treated with alkali and the respective supernatant was analyzed by reverse phase HPLC. In both cases, radioactive peaks co-eluted with authentic ADP-ribose and 5 AMP (data not shown). 

Reverse-phase chromatography - in which the amino acids react with the reagent to form fluorescent or ultraviolet-absorbing derivatives, which are then separated using a more polar mobile phase (e.g. acetate buffer with a gradient of acetonitrile) and a less polar stationary phase (e.g. octadecyl-bonded silica). The availability of amino adds to the animal can be estimated by chemical methods. For example, for lysine there are colorimetric methods that depend on the formation of compounds between lysine and dyes (see Chapter 13). 

Having determined the structure of the SH-peptide we started investigating the fraction of peptides soluble in 0.1% TFA. The peptides were separated by reversed phase HPLC (Figure 4). Save for one exception all the peptides were obtained in pure state and their sequences were established in a solid-phase protein sequencer in the case of peptides with C-terminal lysine, or in the spinning cup sequenator in the case of peptides C-terminated with arginine (Figure 1,3). 

The major drawback to the FMOC derivatization is that unlike OPA, FMOC is itself fluorescent and reacts with water to give a fluorescent product. If excess FMOC is not removed, it elutes in a broad band in the middle of the profile interfering with amino acid quantification. The excess reagent can be dealt with in two ways. A very hydrophobic amine such as 1-amino-adamantane can be added. This will then elute after lysine, the last amino acid to elute with reversed-phase chromatography. This, of course, lengthens the chromatography run time. The FMOC can also be extracted from the reaction by liquid-Uquid extraction using pentane. This is usually done twice. The liquid-liquid extraction complicates automation of the reaction. 

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