Chromatography Protein hydrolysates

Demmelmair, H. and Schmidt, H. L. (1993) Precise 813C determination in the range of natural abundance on amino acids from protein hydrolysates by gas chromatography isotope ratio mass spectrometry. Isotopenpraxis 29, 237 250. 

Despite the problems encountered in sample preparation, the chromatography of protein hydrolysate samples is straightforward and can give good resolution in a short time. 

U Butikofer, D Fuchs, JO Bosset, W Gmur. Automated HPLC-amino acid determination of protein hydrolysates by precolumn derivatization with OPA and FMOC and comparison with classical ion exchange chromatography. Chromatographia 31 441-447, 1991. 

Finally, we have encountered situations where carbon-14 from a labeled pesticide has been incorporated into normal natural products including amino acids. We were interested in seeing if we could separate the normal amino acids from a protein hydrolysate on a single column with sufficient resolution to identify separate amino acids. Figure 11 shows a radiochromatogram of the separation of sixteen standard C-amino acids. Were this a product of a metabolism study, we could then isolate any of these fractions, convert the residual amino acid to the N-trifluoroacetyl 0-butyl derivative for gas chromatography ar.d further confirmation of structure. 

Kukman, I.L., Zelenik-Blatnik, M., and Abram, V. 1995. Isolation of low-molecular-mass hydrophobic bitter peptides in soybean protein hydrolysates by reversed-phase high-performance liquid chromatography. J. Chromatogr. 704, 113-120. 

The amino acids in a protein hydrolysate can be conveniently separated for qualitative analysis by paper or thin-layer chromatography or by elec-... 

Chlorination products (mainly chlorotyrosine) are measured by HPLC (Fig. 8) or gas chromatography-mass spectrometry. Also, 3-nitrotyrosine can be detected in protein hydrolysates by HPLC in combination with various detection systems, including UV and electrochemical detection (Cl7, C20, L23, L24, 04, S26), gas chromatography, gas chromatography-mass spectrometry (J2), electrospray mass spectrometry, and Western blotting or ELISA using antinitrotyrosine antibodies (H20, T2, V6). 

Determination of oxidized amino acids in urine is usually performed by isotope dilution gas chromatography-mass spectrometry (L9). DOPA is estimated by HPLC separation of acid protein hydrolysates with fluorescence detection (excitation 280 nm, emission at 320 nm) (A15). Other methods are based on borate-hydrochloric acid difference spectroscopy (this method suffers interference from tyrosine and tryptophan) (W2), derivatization of DOPA with nitrite and subsequent coulometric determination (W3), and fluorometric detection after derivatization with ethylenediamine (A15). 3-Hydroxylysine is quantitated by HPLC with 9-fluorenylmethyl chloroformate precolumn derivatization (M25) of amino acids obtained by gas-phase hydrolysis of proteins (F21). Other general methods to detect amino acid damage are mass spectometry methods applied to protein hydrolysates, such as tandem mass spectrometry (F6). 

Van RUlaer W, Beernaert H., Determination of Residual 1,3-Dichloro-2-propanol in Protein Hydrolysates by Capillary Gas Chromatography, Z Lebensm Unters Forsch 188. 343-345 (1989)... 

The process of separation and quantitation of amino acids has been automated. In one automated method, a single cation exchange resin column separates all the amino acids in the protein hydrolysate. The analyzer is capable of detecting as little as 1-2 nmol of an amino acid and a complete analysis can be obtained in about 4 hours. In newer procedures, the complete analysis can be performed in about Ihour and permit detection of as little as 1-2 nmol of an amino acid. Picomole amounts of amino acids can be determined when the separated amino acids are coupled to fluorescent reagents such as o-phthalaldehyde. Amino acid separation and quantitation can also be accomplished by reverse-phase high-pressure liquid chromatography of amino acid derivatives—a rapid and sensitive procedure. 

An IND, BIA, or NDA submission should list the source and country of origin for every animal- and human-derived raw material [3, 46]. Lot numbers and supplier information should be available on site during an inspection (21 CFR 211.184). It is also recommended to contact vendors to determine if less-obvious raw materials such as amino acids used in the basal tissue culture medium, enzymes used to make protein hydrolysates, cholesterol, and some detergents (e.g., polysor-bates) are animal-derived. In some cases, protein A (isolated to make chromatography resins) has been purified over human IgG immunoaffinity columns, and the use of these resins should also be tracked and reported (21 CFR 211.184) [46]. 

Yamashita et al. (65) incorporated L-methionine into a soybean protein hydrolysate by means of the plastein reaction with papain. A 10 1 mixture of a peptic hydrolysate of soybean protein isolate and L-methionine ethyl ester was incubated in the presence of papain, the conditions being similar to those mentioned above. The methionine content of the plastein was 7.22 wt %, nearly seven times the original methionine content of the soybean protein isolate. To determine the location of the incorporated methionine residues, the plastein was treated with carboxypeptidase A. Methionine was liberated much faster than any other amino acid. A second portion of the plastein was methylated and then treated with lithium borohydride to reduce the COOH to CH2OH. Hydrolysis of the chemically treated plastein with 6N HC1 gave aminols in satisfactory yields. Subsequently, the aminols were converted to their DNP-derivatives, which were separated by thin layer chromatography. These experiments, together with some others, showed that 84.9% (molar basis) of the C-terminals of the plastein molecules were occupied with methionine, whereas only 14.4% of the N-terminals contained methionine. 

High Performance Liquid Chromatography.—This technique has been applied to a wide range of natural products, i.e., steroidal phosphates, retinyl phosphate, dansylated protein hydrolysates, thiamine phosphates, dibutyl c-AMP esters, cytosine arabinoside triphosphate, and di- and tri-nucleoside phosphates. ... 

Derivatisation of samples in HPLC is undertaken principally for two reasons. First, there is no detector for HPLC that has universally high sensitivity for all solutes hence a suitable chemical transformation of the solute can greatly extend the sensitivity and versatility of a selective detector. Second, sample derivatisation may be undertaken to enhance the detector response to sample bands relative to overlapping bands of no analytical interest. This experiment involves the pre-column derivatisation of sample with dinitro-fluorobenzene so enhancing the spectrophotometric response of the sample, that is, the amino acids from a protein hydrolysate. The conversion of the amino acids to their apolar DNP derivatives allows them to be analysed using reverse phase chromatography. Thus, polar DNP-amino acids, such as DNP-aspartate, will elute early whereas apolar DNP-amino acids, such as DNP-alanine, will elute later. The DNP-amino acids are detected by an... 

Figure 4. Chromatography of a protein hydrolysate calibration mixture containing 25 pmol of each amino acid (63 pmol of tryptophan and 50 pmol of norleucine) (top panel) and of the pH 2.2 sample diluent (bottom panel). 1, Aspartic acid 2, threonine 3, serine 4, glutamic acid 5, proline 6, glycine 7, alanine 8, cystine 9, valine 10, methionine 11, isoleucine 12, leucine, 13, norleucine (internal standard) 14, tyrosine 15, phenylalanine 16, histidine 17, lysine 18, tryptophan 19, arginine.

Moore S. and Stein W. H. (1949) Chromatography of ammo acids on starch columns. Solvent mixtures for the fractionation of protein hydrolysates / Btol Chem 178, 53-77... 

Partridge S M. (1949b) Displacement chromatography on synthetic ion-exchange resins 4 The isolation of glucosamine and histidine from a protein hydrolysate Biochem. 45, 459-463... 

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