Guanidine hydrochloride residues

Columns can be washed with solvents and solvent combinations suitable to remove adsorbed contaminants. When considering the adsorption of analytes, think not only of the diol functionality, but also of the adsorption to residual silanols. Often, the injection of small amounts (500 /d) of dimethyl sulfoxide removes contamination that has accumulated on the column. Aqueous solutions of sodium dodecyl sulfate, guanidine hydrochloride, or urea are compatible with Protein-Pak columns. 

Step C Preparation ofthebase-A 300 ml one-necked, round-bottomed flask, equipped with a water-cooled condenser, calcium chloride tube and magnetic stirrer is charged with anhydrous methanol (150 ml) and sodium metal (5.75 g,0.25 g atom). When the reaction is complete, the solution is treated with dry guanidine hydrochloride (26.3 g, 0.275 mol) and stirred for 10 minutes. The sodium chloride that forms is removed by filtration. The solution is concentrated in vacuo to a volume of 30 ml and the residue treated with the product of Step B, heated one minute on a steam bath and kept at 25°C for 1 hour. The product is filtered, washed well with water, dissolved In dilute hydrochloric acid and the free base precipitated by addition of sodium hydroxide to give the amllorlde product base, a solid which melts at 240.5°-241.5°C. 

In 1971, adrenodoxin, an iron-sulfur protein with a single tyrosine residue and no tryptophan was shown to fluoresce at 331 nm upon 280-nm excitation at neutral pH/20 1 On cooling from room temperature to 77 K, the emission maximum shifts to 315 nm. The redox state of the iron does not have any effect on the tyrosine emission. From these results, an exciplex between the excited singlet state of tyrosine and an unidentified group was suggested as the cause of the anomalous emission energy/2031 Later studies have shown that the excitation spectrum is a red-shifted tyrosine spectrum, that removal of the iron to form the apoprotein has no effect on the emission, and that heat, low pH, guanidine hydrochloride, urea, and LiCl all cause the emission... 

Chemical analyses of the enzyme indicate that cysteine, but not cystine, residues are present (see Section II,H,1). Therefore all physical studies in the denaturing environment of 5M guanidine hydrochloride... 

The analytical method proposed by Pajot (1976) involves (a) incubating the protein of a known concentration in 6 M guanidine at pH 6.5-7 in the presence of 30 mM 2-mercaptoethanol for 30 min (if only a small amount of the protein is available, the incubation can be carried out directly in the fluorescence cuvette) and (b) measuring the fluorescence, excited at 295 nm and observed at 354 nm, yielded by the denatured protein in 6 M guanidine hydrochloride (tryptophanyl residues concentration 3-10 /xM). A linear calibration curve is obtained, thus allowing the free tryptophan fluorescence equivalents of the protein sample to be estimated by extrapolation. 

Disulfide linkages may be broken either oxidatively or reductively. The former method involves the treatment of the protein with performic add, which converts all disulfide bonds into cysteic add residues. This procedure is usually performed before a protein is hydrolyzed for amino add analysis. Cystine and cysteine are then determined as cysteic add. The reductive cleavage of disulfide bonds involves the treatment of the protein with mercaptoethanol (SH-CH2-CH2-OH), followed by the alkylation of the newly formed -SH groups. The complete disruption of all secondary interactions (that is, complete denaturation) can be achieved in most proteins with 6 M guanidine hydrochloride and 0.1 M mercaptoethanol or 8 M urea and 0.1 M mercaptoethanol. 

Ribonuclease, the enzyme that hydrolyzes ribonucleic acids (Chap. 7), contains four disulfide bonds that help to stabilize its conformation. In the presence of 6 M guanidine hydrochloride, to weaken hydrogen bonds and hydrophobic interactions, and 1 mM mercaptoethanol, to reduce the disulfide bonds, all enzymatic activity is lost, and there is no sign of residual secondary structure. On removing the guanidine hydrochloride by dialysis or gel filtration, enzymatic activity is restored, the native conformation is regained, and correct disulfide bonds are reformed. 

The Step 1 product (4.97 mmol) dissolved in 15 ml methyl alcohol was treated with a mixture of sodium ethoxide (4.97 mmol) and guanidine hydrochloride (5.22 mmol) and the reaction was then monitored by TLC using 15% EtOAc/toluene. The mixture was then concentrated and the residue dissolved in EtOAc. The residue was purified by chromatography using 15% EtOAc/toluene and the product isolated in 95% yield. 

Cysteine residues in transferrin were reduced and alkylated in a similar manner as that done for solution samples p). Modiflcation for samples prepared with ProSorb cartridge can be performed in the same ProSorb cartridge before membrane removal, while modifications were performed in an Eppendorf tube for the electroblotted samples. The membranes were incubated 15 minutes at room temperature in a 0.25 M Tris/HQ and 6 M Guanidine hydrochloride buffer containing 1 ml of mercaptoethanol and followed by the addition of 1 ml of 4-vinyl pyridine for another 15 minutes. The membnmes were washed thoroughly with 0.1% TFA afterwards. 

For QD and fluorescence spectroscopic analyses of (o-[l-Sei46]Aga-TK and co-[D-Ser46]Aga-TK, peptide samples were prepared by freshly dissolving the lyophilized peptides at a concentration of 150 ng/ml in Dulbecco s phosphate-buffered saline, pH 7.4, in the presence or absence of guanidine hydrochloride. CD spectra were recorded with a Jasco J-720WI spectropolarimeter at room temperature using a 0.1 cm path-length cell. In aU cases, the buffer base-line spectrum was subtracted, and the results were expressed in terms of the mean residue ellipticity... 

Triphenyl Guanidine.- The residue remaining in the flask after the distillation with hydrochloric acid is treated with 100 c.c. of water, and then allowed to stand for several hours, when colourless crystals of triphenylguanidine hydrochloride separate out. These are filtered off, and warmed with some dilute caustic soda solution. The salt is decomposed, and the free base obtained, which on recrystallising from alcohol forms colourless crystals. Melting-point, 1430. 

Figure 4 Inhibition of aggregation of bovine growth hormone (BGH) by a peptide fragment comprising residues 96-133 of the hormone. A 1.75 m ml solution of the protein was initially incubated at 3.5 M guanidine hydrochloride (conditions that populate the associated intermediate) and subsequently was diluted at 0.18 m ml and 0.8 M guanidine hydrochloride (conditions that induce aggregation). Turbidity was monitored by the absorbance at 450 nm. The top curve represents the kinetics of turbidity formation in the absence of the peptide, and in descending order from the top, formation of turbidity in the presence of 1, 3, 5, 7-fold molar excess of the peptide. The peptide was present from the initial conditions that populate the intermediate. (3.5 M guanidine hydrochloride). From Brems, (26) reproduced with permission.

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