Protein solid-aqueous buffer solution

The purification and separation first requires removal of the enzyme. This is, of course, soluble ir. the aqueous buffer solution at pH 8, but acidification and heating denatures the protein (rather like heating egg white) and destroys its structure. The solid material filtered off is the denatured enzyme The separation in ethanol works because the very polar amino acid is soluble only in water but the more organic amide is soluble in ethanol. The amide has an extra organic group and only one ver polar group (CO2H). In addition, it does not form a zwitterion. 

Reaction of AMCA-NHS with proteins proceeds efficiendy in the pH range 7—9. Avoid buffers containing amines that can compete in the coupling reaction, such as Tris or glycine, and avoid imidazole buffers since they promote hydrolysis of the NHS ester. AMCA-NHS is relatively insoluble in aqueous buffers. The compound must be first dissolved in organic solvent prior to addition of a small aliquot to the reaction mixture. A concentrated stock solution may be prepared in DMSO and stored up to 2 weeks refrigerated or frozen without loss of activity. The solid and all solutions of AMCA-NHS should be protected from light to avoid photodecomposition of the fluorophore. 

KLH also should not be frozen. Freeze-thaw effects cause extensive denaturation and result in considerable amounts of insolubles. Commercial preparations of KLH are typically freeze-dried solids that no longer fully dissolve in aqueous buffers and do not display the protein s typical blue color due to loss of chelated copper. The partial denatured state of these products often makes conjugation reactions difficult. Pierce Chemical is the only commercial source of KLH that includes special (proprietary) stabilizers to provide the protein in a lyophilized form that is almost completely soluble upon reconstitution and with its blue copper-binding characteristics still intact. Reconstitution of the Pierce product with water yields a buffered solution ready for conjugation reactions. 

The solid-state and solution chemistry of triethanolamine complexes has been investigated. While the solid-state structure was maintained in organic solvent (38), a different structure was observed in aqueous solution.262 170 NMR spectroscopy was used to demonstrate that the two oxo groups were different and in combination with H and 13C NMR data, defined the structure as (39).262 Speciation studies and a detailed characterization of this class of compounds were important because the ligand is a commonly used buffer in biology and the complexes are model systems for interactions with proteins.61,263 The thermodynamic parameters were determined for several derivatized diethanolamine ligand-vanadium(V) complexes, and represent some of the few vanadium complexes for which such parameters are known.62 The structure of (nitrilotriacetato)dioxovanadate was reinvestigated.2 4... 

After collection and dechorionation at room temperature, washed, dechorio-nated embryos should be quick-frozen in liquid N2 and stored at -70 C. Storage for periods of up to 1 year or longer seems to be without adverse effect. Immediately before fractionation, chilled (4°C) extraction buffer (Buffer E) should be prepared Buffer E contains 5 m Af MgC, 50 mM NaCl, 50 m M Tris-HCl, pH 7.5, 250 mM sucrose, 2.5 mM N-ethylmaleimide (NEM), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM L-tosylamide 2-phenyIethyl chloromethyl ketone (TPCK). NEM, PMSF, and TPCK should be included as protease inhibitors. About 20-40 min before fractionation begins, all protease inhibitors are added in solid form to otherwise complete Buffer E in amounts greater or equal to those specified. After addition of protease inhibitors. Buffer E should be stirred continuously at 4°C. Both PMSF and TPCK are incompletely soluble in aqueous solution at the concentrations specified. However, residual undissolved reagents constitute a solid reservoir that becomes completely depleted during embryo fractionation, due to either hydrolysis, protein modification, or both. 

Crude Solid. The simplest way to use enzymes in organic solvents is to suspend a precipitate or a lyophilisate. The enzyme does not need to be of high purity, but some care should be taken during the preparation. In aqueous solution, the enzyme has an optimal pH, dictated by the ionization state under which the amino acids involved in the catalysis must be to allow activity. The solid enzyme must be in the same ionization state when used in organic solvents (15). For this purpose, it is important to precipitate the enzyme or lyophilize it from a solution buffered at this pH. This applies to the other forms of solid enzyme preparations. The other important point is the drying of the preparation. It has been observed that the secondary structure of proteins can be affected by lyophilization (16). This can be avoided by the use of lyoprotectants such as sorbitol (17) or salts such as KCl (18). 

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