Buffer solution amino acids

Let us consider a simple example of how a mixture of three amino acids undergoes separation. Amino acid A could have a side-chain that is positively charged in a certain buffer solution, amino acid B could be neutral and amino acid C could be negatively charged (Figure 27.12). 

Diafiltration is a process whereby an ultrafiltration system is utilized to reduce or eliminate low molecular mass molecules from a solution and is sometimes employed as part of biopharmaceuti-cal downstream processing. In practice, this normally entails the removal of, for example, salts, ethanol and other solvents, buffer components, amino acids, peptides, added protein stabilizers or other molecules from a protein solution. Diafiltration is generally preceded by an ultrafiltration step to reduce process volumes initially. The actual diafiltration process is identical to that of ultrafiltration, except for the fact that the level of reservoir is maintained at a constant volume. This is achieved by the continual addition of solvent lacking the low molecular mass molecules that are to be removed. By recycling the concentrated material and adding sufficient fresh solvent to the system such that five times the original volume has emerged from the system as permeate, over 99... 

Electrophoresis and thin-layer chromatography are analytical separations—small amounts of amino acids are separated for analysis. Preparative separation, in which larger amounts of amino acids are separated for use in subsequent processes, can be achieved using ion-exchange chromatography. This technique employs a column packed with an insoluble resin. A solution of a mixture of amino acids is loaded onto the top of the column and eluted with a buffer. The amino acids separate because they flow through the column at different rates, as explained below. 

The following procedure has been suggested. Amino acids and primary amines are dissolved in 0.5 M phosphate buffer, pH 9.3. This solution is mixed with the same volume of a freshly prepared 0.4 M pyridoxal solution in the same buffer (pyridoxal/amino acids = 5 1). 

Weber, P. L. Buck, D. R. Capillary Electrophoresis A Past and Simple Method for the Determination of the Amino Acid Composition of Proteins, /. Chem. Educ. 1994, 71, 609-612. This experiment describes a method for determining the amino acid composition of cyctochrome c and lysozyme. The proteins are hydrolyzed in acid, and an internal standard of a-aminoadipic acid is added. Derivatization with naphthalene-2,3-dicarboxaldehyde gives derivatives that absorb at 420 nm. Separation is by MEKC using a buffer solution of 50 mM SDS in 20 mM sodium borate. 

Note The pre- and post-treatment of the chromatograms with the basic tri-ethylamine solution, which can be replaced by an alcoholic solution of sodium hydroxide [1,4] or a phosphate buffer solution pH = 8.0 (c = 0.2 mol/1) [5], serves to stabilize the fluorescence of the amino derivatives [2]. A final spraying with methanolic hydrochloric acid (chci = 5 mol/1) or 70% perchloric acid renders the detection reaction highly specific for histamine [4] and for catecholamines and indolamines [5]. 

Aminosodium salt and acylated with 1 H-tetrazole-1 -acetyl chloride. The acetoxy group is then displaced by reaction with 5-methyl-1,3-4-thi-adiazole-2-thiol in buffer solution. The product acid is converted to the sodium salt by NaHCOa. 

Reactions between aziridine-2-carboxylic acids and thiols in aqueous solution have been explored by Hata and Watanabe [112]. The reactions occurred predominantly at C-2 instead of C-3 to afford 3-amino acids, with the reaction between 148 (Scheme 3.53) and thiophenol in 0.2 m sodium phosphate buffer at room tem-... 

A substrate is a substance that is the basic component of an organism. Protein substrates are amino acids, which are essential to life Protein substrates are amino acid preparations that act to promote the production of proteins (anabolism). Amino acids are necessary to promote synthesis of structural components, reduce the rate of protein breakdown (catabolism), promote wound healing, and act as buffers in the extracellular and intracellular fluids. Crystalline amino acid preparations are hypertonic solutions of balanced essential and nonessential amino acid concentrations that provide substrates for protein synthesis or act to conserve existing body protein. 

Another approach for the synthesis of enantiopure amino acids or amino alcohols is the enantioselective enzyme-catalyzed hydrolysis of hydantoins. As discussed above, hydantoins are very easily racemized in weak alkaline solutions via keto enol tautomerism. Sugai et al. have reported the DKR of the hydantoin prepared from DL-phenylalanine. DKR took place smoothly by the use of D-hydantoinase at a pH of 9 employing a borate buffer (Figure 4.17) [42]. 

Amino acids containing nucleobases like uracil and adenine, as well as imidazole, such as / -(uraciM-yl)-a-alanine, j5-(adenin-9-yl)-a-alanine, and j8-(imidazol-l-yl)-a-alanine, can also be polycondensed by CDI in aqueous imidazole buffer solution at pH 6.8 at 0 °C. The polycondensation leads to low conversion (yields of polymer 1% after four days), but pure polypeptides resulted from the reaction. Thus, compared to other alternative procedures for polycondensation, that using CDI proved to be the most effective 503... 

Jorgenson reported the use of glass capillaries for free solution electrophoresis 25 years ago (Jorgenson andLukacs, 1981,1983). Aplug of analyte was introduced into a buffer-filled capillary and separated at high electric fields. Capillaries of 75 im inner diameter were employed, and detection of labeled amino acids and peptides was based on fluorescence. 

E. L. Shock (1990) provides a different interpretation of these results he criticizes that the redox state of the reaction mixture was not checked in the Miller/Bada experiments. Shock also states that simple thermodynamic calculations show that the Miller/Bada theory does not stand up. To use terms like instability and decomposition is not correct when chemical compounds (here amino acids) are present in aqueous solution under extreme conditions and are aiming at a metastable equilibrium. Shock considers that oxidized and metastable carbon and nitrogen compounds are of greater importance in hydrothermal systems than are reduced compounds. In the interior of the Earth, CO2 and N2 are in stable redox equilibrium with substances such as amino acids and carboxylic acids, while reduced compounds such as CH4 and NH3 are not. The explanation lies in the oxidation state of the lithosphere. Shock considers the two mineral systems FMQ and PPM discussed above as particularly important for the system seawater/basalt rock. The FMQ system acts as a buffer in the oceanic crust. At depths of around 1.3 km, the PPM system probably becomes active, i.e., N2 and CO2 are the dominant species in stable equilibrium conditions at temperatures above 548 K. When the temperature of hydrothermal solutions falls (below about 548 K), they probably pass through a stability field in which CH4 and NII3 predominate. If kinetic factors block the achievement of equilibrium, metastable compounds such as alkanes, carboxylic acids, alkyl benzenes and amino acids are formed between 423 and 293 K. 

Measure the absorbance of the solutions at 335 nm. Determination of the number of amines present in a particular sample may be done by comparison to a standard curve generated by use of an amine-containing compound (i.e., an amino acid) dissolved at a series of known concentrations in the bicarbonate sample buffer and assayed under identical conditions. 

Table 6.3 displays the solution pKa values of the promoters capable of facilitating the electron transfer of Cu, Zn-SOD listed in Table 6.1. The —COOH-termi-nated SAMs are mostly negatively charged in phosphate buffer (pH 7.0). Although the bovine erythrocyte Cu, Zn-SOD has a net negative charge at pH 7.0 (p/ = 4.9), an electrostatic interaction is still expected to occur between the SAMs and the positively charged amino acid moieties (typically —NI13). Besides, the hydrogen bonding between —COOH groups and the amino acid residues is believed to comprehensively...

A fluorescence method that would measure either free or combined amino acids, depending upon the pH of the solution, was originally proposed by Udenfriend et al. [282] and adapted for seawater by North [283] and Packard and Dortch [284], In this Fluran method, peptides normally yield maximum fluorescence by pH 7, while amino acids fluoresce best at pH 9. With the proper choice of buffers, the fluorescence of peptides and proteins can be differentiated from that due to free amino acids. 

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