Over and above the usual requirements such as the elimination of chemical and mechanical impurities by recrystallization or reprecipitation [1] peptides have to be dried with exceptional care. Free peptides, often lyophilized materials, retain moisture tenaciously. In addition to water they might contain acetic acid as well. In order to obtain reproducible values the samples have to be dried in vacuo, at elevated temperature, for prolonged periods of time. The temperature of drying should be based on the melting point or decomposition point of the material. While it is desirable to choose a temperature as high as 11 0 °C or even 130 °C, drying should not be accompanied by melting or decomposition.
Protected peptides are usually insoluble in water. Nevertheless, they can be hygroscopic in a sense: they might absorb water from moist air. Hence, for the sake of valid analyses such samples must be dried at 110 to 130 °C and allowed to cool in a desiccator in the presence of phosphorus pent oxide. In particularly difficult cases the sample should be weighed and dried, by the microanalyst, in a "piggy", which is closed while still hot and reweighed after cooling.
Probably because of the presence of weak basic centers, such as nitroguanidines, the imidazole of histidine or the amide groups themselves, protected pep tides can contain acetic acid or trifluoroacetic acid, even after drying. This possibility should be considered at the evaluation of the value of elemental analysis. For best information all the elements present in the compound should be determined and the sum should amount to 100%.
Lengthy considerations and experience taught the authors to value the results of combustion analysis. Spectral methods should complement but not replace the determination of elemental composition.
2.2 Amino Acid Analysis
If the synthetic material is available in sufficient amounts, a 3-5 mg sample should be used and weighed with an accuracy of 0.1 mg. This allows the calculation of "recovery" from the values of amino acid analysis and thus the
determination of the peptide content of the analyzed substance. The peptide content of a sample can be far below 100% since peptides, both free and protected, can retain significant amounts of moisture and/or non-volatile inorganic impurities.
Most commonly, constant boiling hydrochloric acid is used for hydrolysis. This is prepared by diluting concentrated hydrochloric acid with an equal volume of water and distilling the mixture from an all-glass apparatus. The first part of the distillate about 10% is discarded and about 10% of the acid is left undistilled. The distillate, about 5.7 N HCI, is stored in several small glassstoppered bottles or in sealed ampoules to prevent the absorption of ammonia from the air during frequent opening.
The sample is weighed into a soft glass ampoule with a narrow neck and dissolved in a small volume (e.g. 0.5 ml) of constantly boiling hydrochloric acid. The solution is cooled in an ice-water bath, evacuated with the help of a water aspirator and sealed under vacuum by melting the narrowed part of the tube with a small Bunsen burner. The sealed ampoule is placed into a cavity of an electrically heated metal block and kept at 110°C for 16 hours.
Peptides containing valine and/or isoleucine residues [2J as next neighbors in their sequence require longer periods for complete hydrolysis, sometimes as long as 3-4 days. On long hydrolysis, however, significant decomposition of serine and threonine takes place. Therefore, for a precise determination of the amino acid ratios both short and long hydrolyses are necessary. From several analyses, e.g. after hydrolysis for 16,32 and 64 hours, the true values for valine and isoleucine can be found by graphical extrapolation to infinite time while extrapolation to zero hour gives the hypothetical amount of serine and threonine in the hydrolysate in the absence of decomposition [3].
Tryptophan is destroyed in hot hydrochloric acid, particularly in the presence of air and heavy metal impurities. With highly purified hydrochloric acid, tryptophan-containing peptides can be hydrolyzed in vacuo and the tryptophan content determined [4]. Hydrolysis with 3 molar f3-mercaptoethanesulfonic acid or 4 molar methanesulfonic acid causes no significant decomposition of the indole. Methionine sulfoxide is converted in part to methionine during hydrolysis with hydrochloric acid. The conversion is complete if f3-mercaptoethanol (1 mg per ml) is added. The sulfoxide remains intact during hydrolysis with 3 N p-toluenesulfonic acid.
On completion of the hydrolysis the sealed ampoules are cooled to room temperature and carefully [5J opened. The hydrolysate is transferred with a pipet into a small beaker, the ampoule rinsed with distilled water and the acid is removed on a steam bath with the help of a stream of nitrogen. A small volume of distilled water is added and similarly evaporated. The residue is dissolved in a buffer of pH 2.2 and, after appropriate dilution, applied to the column of the amino acid analyzer. The applied volume depends upon the kind of instrument used. The buffer, glass vessels, pipet, etc. must be clean, especially if a sensitive method of analysis involving samples of 10 nanomoles or less is
210 Preparation of Analytical Samples
applied. Several amino acids, aspartic acid, serine and glycine particularly, are present in fingerprints in amounts which can distort the results of amino acid analyses.
2.3 NMR Spectra
A tendency for solvent retention often complicates the recording of NMR spectra. It is advisable, therefore, to dissolve the (dried) sample in the de ute rated solvent, to evaporate the solvent in vacuo and to redissolve the residue.
The most commonly used solvent in NMR spectroscopy, CDCI3 , has limited applicability in peptide chemistry. Only relatively small molecules such as protected and activated amino acids and small peptides are soluble in chloroform. Deuterated dimethylformamide, (CD3)2NCDO, although expensive, is more generally useful, as is deuterio-dimethylsulfoxide. Very good results were obtained in the authors' laboratory with fully deuterated acetic acid, CD3COOD. The latter, unlike triftuoroacetic acid, which is also used in NMR spectroscopy of peptides, leaves most protecting groups intact. Also, the chemical shifts in CD3COOD are quite close to those recorded in CDCl 3 and thus allow a better comparison with chemical shifts reported in the literature. Triftuoroacetic acid has a major effect on the chemical shifts and renders the use of published values rather difficult. Last, but not least, in CD3COOD exchangable protons on oxygen and nitrogen atoms are displaced by deuterium. This results in a welcome simplification of the spectra. To bring the exchange to completion the samples are dissolved in CD3COOD, the solvent removed by evaporation [6J, the residue redissolved, and the evaporation repeated. Finally, the solution of the residue in CD3COOD is applied for the recording of the spectra. The small amount of CHD2 COOD present in the solvent can serve as internal reference.
1. Amino acids and their derivatives can generally be sublimed in vacuo. In the authors' laboratory a cyclo-pentapeptide was sublimed without any decomposition at about 270 "C and 4 Pa.
2. Some residues with blocked functional side chains, e.g. S-benzyl-cysteine, have similar influence on the rate of the hydrolysis of the peptide bonds surrounding them.
3. Some protected peptides remain insoluble in hot constant boiling hydrochloric acid. A mixture of equal volumes of concentrated hydrochloric and acetic acid can be helpful in such instances. It should be noted, however, that hydrolysis with this mixture is conducive to racemization.
4. The most practical method for the determination of the tryptophan content of a peptide is the recording of the nv. spectrum. Tryptophan has a molar absorption coefficient of 5500 at 280 nm. Only tyrosine absorbes in the same order of magnitude (1370 at 278 nm). Phenylalanine (about 200 at 260 nm) interferes less with this determination, but nitroarginine has too high molar absorption to allow the exact determination of tryptophan content.
5. The eyes must be protected during breaking of the sealed part of the ampoule. 6. The solvent can be removed by lyophilization (sublimation from the frozen solution) as well.
