Analysis of protein-bound and free amino acids

The analysis of free amino acids – thosse amino acids (AAs) not bound in protein, is relatively simple; we first extract the samples in weak acid and then separate them by high pressure liquid chromatography (HPLC). By contrast, analyzing the concentrations of protein-bound amino acids requires an acid hydrolysis step that destroys some of the AAs. Depending on the AAs of interest it may be necessary to treat the sample in different ways, e.g. with a performic acid oxidation preceding acid hydrolysis. The HPLC step is the same as for free AAs. There are many systems for measuring AAs, including ion-exchange chromatography. We determine the concentration of AAs by first hydrolysing the samples according to Llames and Fontaine (1994) and then separating the AAs by HPLC using the method of Cohen et al. (1989) and Cohen and Michaud (1993), essentially the AccQ.Tag{SUP()}TM{SUP} method (Waters, Milford, MA,USA). This method attaches a fluorescent tag to AAs that are separated by HPLC and detected by a fluorescence detector. An advantage of this method is that many chemistry laboratories already have an HPLC fitted with a fluorescence detector..

It is important in many branches of biology to know the concentrations of AAs in a sample and the relative amounts of free- and protein-bound AAs. For example, many animals have a dietary requirement for AAs, rather than nitrogen (crude protein) and some have an intolerance of free AAs (DeGabriel et al. 2002). The analysis we describe here is typical of many amino acid analyses, whereby an acid hydrolysis liberates protein-bound AAs that then react with a label that makes them detectable during chromatography, in this case a fluorescing compound. We extract the free AAs with diluted hydrochloric acid at room temperature while stirring. Protein hydrolysis does not occur under these conditions, which means that all of the AAs in solution were free AAs in the sample.

__Before proceeding read the Material Safety Data Sheet (MSDS) for each substance.__ These are widely available on the www.
#Acid hydrolysis of protein-bound amino acids
**__Oven__ – an oven that can be set to 112C. Ideally, this will be acid-resistant. If not, it needs frequent cleaning and regular inspection for both corrosion and for electrical safety.
**__6N HCl – (For 2L)__ Note that the HCl sold as “36%” is w/w and not w/v. Thus there is 360 g HCl per kg. The specific gravity of this solution is 1.18. Therefore it contains 360g in 848mL or 425g per L. Thus, 36% w/w HCl is, in fact, 11.8N and not 10N as many believe. For the purposes of this assay it can be considered 12N. Prepare 2 L of 6N HCl reagent by placing 2 g of phenol ===(EXTREME CARE REQUIRED)=== in a glass flask containing exactly 1000mL of MilliQ water. Carefully add exactly 1000mL of HCl 36% and mix.
**__Internal standard__ – -amino-butyric acid (2.498 mM). (MW = 103.1). __(For 2L)__. Dissolve 0.5151 g of dry -amino-butyric acid in 200 mL of 1N HCl. Transfer to a 2L volumetric flask and make to volume with MilliQ water. Store at 5C. Use at room temperature.
**__Sodium hydroxide__ (7.5M) (MW = 40) (hydrolysate pH correction). ===(EXTREME CARE REQUIRED)===. __(For 1L).__ Dissolve, with stirring, 300g of AR sodium hydroxide in about 700 mL of MilliQ water in a 4 L flask. Make up to approximately 1L when cool.
**__Citrate buffer__ (0.067M trisodium citrate at pH 2.2 – sample dilution buffer for diluting the hydrolysate). __(For 4L).__ In a 5L flask dissolve with stirring 78.2 g of dry trisodium citrate (MW = 294.10) and 4g of phenol ===(EXTREME CARE REQUIRED)=== in 2000 mL of MilliQ water. Add 20 mL thiodiglycol and 64mL of 36% HCl. Dilute the solution with 1800mL of MilliQ water. Adjust the pH to 2.2 using 1N HCL or NaOH. Transfer evenly to two 2L volumetric flask and make up to volume. Transfer to a storage container and store at 5C.
**Millipore (or similar quality) water
**plastic vial with O-ring seal (e.g., Sarstedt 72.694.100)
**drying oven or freeze drier
**50 mL bottle fitted with an acid proof (PBTP) screw cap with Teflon seal (Schott AG, Mainz, Germany)
**conical flasks (ca 100 mL) – one per sample
**volumetric flasks (250 mL) – one per sample
**0.2 μm syringe filter %%% %%%
#Materials for chromatographic analysis%%%__Before proceeding read the Material Safety Data Sheet (MSDS) for each substance.__
**__Eluent A – aqueous acetate-phosphate buffer__ – This is mixed as a concentrate and then diluted 1 in 8 (e.g. 250mL in 2L using volumetric flasks) as needed. Store both the concentrate and the running eluent at 5C. The compositions are shown in table 1.
;:__Table 1. Eluent A__
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**__Acetonitrile__ – mixed with water (60 MeCN:40 H{SUB()}2{SUB}O) in a two eluent system and denoted as eluent B. Foams when filtered. Therefore, filter the MeCN and H{SUB()}2{SUB}O separately before mixing. Degas by sonication.
**__AccQ.Fluor reagent__ – This is the fluorescent derivatising reagent, 6-aminoquinolyl-N-hydroxysuccinimydyl carbamate (supplied in the Waters kit, although we synthesise it ourselves according to the method of Cohen and Michaud, 1993)
**__Borate buffer – 0.2-0.5M__ (supplied in the Waters kit).
**__Amino acid standards kit__
**Nova-Pak C18, 4 μm column
**vortex or sonicator
**Fluorescence detector (in our case a Waters 474 scanning fluorescence detector)

To determine the amino acid composition of diets, feed ingredients and eucalypt leaf, dry the samples in either an oven at 60C or in a freeze-drier. Grind to a particle size of less than 1 mm and then use the following method – a modification of that of Llames and Fontaine (1994), to prepare the samples for chromatography.
#Hydrolyse about 500 ∓ 20 mg of sample with 25 mL of 6N HCl (containing phenol) in a 50 mL bottle (Schott) at 112 C for 22 h. To prevent bottles breaking during the hydrolysis leave the lids loose for the first hour and then tighten them securely. #After cooling, add a weighed amount (two decimal places is fine) of internal standard (ca 10 mL of 2.498mM

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