The amino acid analyser

The basic schematic structure of the amino acid analyser is shown in 

Buffers at varying ionic strength and pH values are pumped through the column to separate the various amino acids. 

The column eluate is mixed with ninhydrin reagent and the mixture passed through the high-temperature reaction coil where the amino acid-ninhydrin complex is formed. (The amount of the coloured ninhydrin complex formed is directly proportional to the quantity of amino acid present in the eluate.) 

The mixture thereafter is fed into the photometer unit where the amount of light absorbed at specific wavelengths (see below) indicates the amount of each amino acid-ninhydrin complex. 

The photometer output is connected to a two channel chart recorder, one channel for the 570 nm output, the other channel for the 440 

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The amino acid analyser using fluorescamine as the detecting reagent has been used to measure 250 picomoles of individual amino acids routinely [262], and dansyl derivatives have been detected fluorometrically at the 10 15 M level [260]. Where the amounts of amino acid are high enough, the fluorescamine method, with no concentration step, can be recommended for its simplicity. At lower concentrations, the dansyl method, with an extraction of the fluorescent derivatives into a non-polar solvent, should be more sensitive and less subject to interferences. For proteins and peptides, the fluorescamine method seems to be the most sensitive available method. 

A strong cation-exchange resin is used in the amino acid analyser BECAUSE... 

Following adjustment to pH 6.0, the solution is applied to a SP-Sephadex C-25 column in the sodium form. Amino acids are then eluted with 0.2 M citrate phosphate buffer, pH 8.0, and the effluent evaporated to dryness at 50 °C. The residue is dissolved in 0.1 N hydrochloric acid and applied to the amino acid analyser. Amino acids are separated by passing 0.2 M, pH 8 sodium citrate solution down the column. The S-methylmethionine content can then be obtained from the chromatogram, as illustrated in Fig. 8.1. The results obtained agree reasonably well with those obtained by thin-layer chromatography [13]. 

It had been known for some years that there is more than one form of apoA-I in plasma (El, L23, 07), when Nestruck et al. (N5) reported that four forms of apoA-I could be isolated by preparative flat bed isoelectric focusing. The two major forms in human plasma (referred to as apoA-Ij and apoA-I2 by Nestruck et al., but as isoforms or isoproteins 4 and 5 in this review (following references S9, Zl, and Z6), focus at pi 5.62 and 5.53, respectively, and contain 71 and 19%, respectively, of total apoA-I. All forms had an identical apparent Mr and common antigenicity to antisera against apoA-I. The amino acid analyses of isoforms 4, 5, and 6 resembled previously published apoA-I analyses (Bl, B43) and these isoforms activated purified lecithimcholesterol acyltransferase. 

With these newer methods of protein separation and amino acid analysis he prepared serum protein fractions by serial salting out with ammonium sulfate and by the Sober and Peterson DEAE cellulose columns (42), using the sera of reptile, fowl, and mammalian blood. Some of the amino acid analyses were carried out by the automatic amino acid methods of Hirs, Moore, and Stein (18). Fortified with this plethora of data, Block now had the opportunity to re-examine not only the ratio of the basic amino acids, but at least 12 amino acids in a variety of protein fractions prepared by at least two different procedures. With the aid of a statistician he determined the significance of the constancy of the molar ratios of pairs of amino acids and found that in spite of the marked variation of the absolute amounts of an amino acid, the molar ratios of certain pairs remain relatively constant among the numerous protein components of animal sera. 

Experimentally, the first step in amino acid analysis, whose origins go back to the work of Stein and Moore in 1948, is hydrolysis of the protein. The protein sample should not contain too much salt, detergent or other additives, and dialysis against a dilute buffer or even water is recommended as a first step. The dialysed solution is then dried in vacuo the quantity needed depends on the sensitivity of the amino acid analyser (typically 1-10 nmol, with older forms of apparatus needing 10- tolOO-fold larger size samples). The dried sample is then subjected to hydrolysis 6 M HQ at 150 °C for 6 h, or 125 °C for 24 h, or 110 °C for 48 h. To exclude oxygen, 0.02 % (v/v) 2-mercaptoethanol and 0.25 % (w/v) phenol are added to the hydrolysis. With the most sensitive amino add analysers currently available, it is possible to analyse protein samples recovered from polyacrylamide gels and blotted onto PVDF-membranes. 

The amino acid analyses of the a-, 0-, and -y-keratoses, shown in Table IV indicate that 7-keratose contains more cysteic acid, proline, serine, and threonine and less alanine, aspartic acid, glutamic acid, leucine, lysine, phenylalanine, and tyrosine than is found in oxidized wool (Corfield et al., 1958). a-Keratose in contrast contains less cysteic acid, proline, serine, and threonine, and more of the other amino acids which are present in low concentration in 7-keratose. The amino acid composition of 8-keratose is similar to that of wool. 

The SCM-cysteine content of the SCMKB proteins ranges between that of wool itself and double this value. Compared with wool these proteins contain high concentrations of proline, serine, and threonine, but low concentrations of aspartic acid, glutamic acid, histidine, lysine, and leucine. In general, reverse trends are shown by the amino acid analyses for SCMKA fractions. SCMKB2 and SCM feather rachis are unique in that they contain virtually no histidine or lysine. In this respect they resemble... 

Table XI also compares the amino acid analyses of Merino 64 s wool with that of cuticle obtained by subjecting the wool to ultrasonic irradiation in 98 % formic acid (Bradbury and Chapman, 1964). The cuticle contained less arginine, aspartic acid, glutamic acid, leucine, and phenylalanine than whole wool, but more cystine, proline, serine, and valine. Cys-teic acid is probably formed during the ultrasonic treatment. These analyses cannot be accounted for quantitatively on the basis of a simple combination of low-sulfur and high-sulfur protein fractions, but the data suggest that the cuticle would not contain as much material in the a-helical conformation as the original fiber. The values obtained by Bradbury (1960) in an earlier examination of cuticle-rich material obtained by a mechanical descaling technique are in reasonable agreement with the values in Table XI.

Our thanks go out to Steve O Neill, Kent Yamada, and Applied Biosystems for their efforts in developing and supporting the 494HS. We also thank Scott Lauren for the amino acid analyses and Hsieng Lu for his on-going support. 

The tables indicate that there is still considerable divergence of opinion regarding both the properties and chemical composition of the gonadotropic hormones. Indeed, in some reports the amino acid analyses of two LH preparations have been found to be more dissimilar than those for FSH and LH. It should especially be noted that Butt (Bll) and Stoekell Hartree (S27) analyzed the same preparation of LH and arrived at very different results with respect to amino acid content this finding sug-... 

The amino acid analyses of food products report cystine instead of cysteine. Cystine is an amino acid that is formed from the oxidation of two molecules of cysteine. 

In order to examine whether the tyrosine content, indicated by the residual band at 1513 cm , agreed with the amino acid analyses of the various proteins tested, the intensity of the 1513cm band was plotted against known tyrosine content. The best linear correlation between residual intensity at 1513 cm (Dj, for the tyrosine band in deuterated specimens) and tyrosine content was obtained with the peak intensity of the amide I band (Dj in undeuterated specimens), i.e., when Dj.jDi was plotted vs percentage of tyrosine content (Fig. 10.15). When this ratio... 

Table 2. The Amino Acid Analyses and Carbohydrate Analyses of the Cuticle Collagen of the Oligochaete Lumbricus and the Polychaete Nereis (adapted from Spiro and Bhoyroo, 1980). 

From 1,53 g of starting material, 106 mg of adrenocorticotropic hormone, fraction 8 B, was obtained. This material was almost homogeneous on electrophoresis. The yield of almost 7 per cent was half that obtained with adrenocorticotropic hormone from pigs. On the other hand, several fractions, viz., 4 A, 5 B, 6 B and 7 A, also had relatively high corticotropin activity. The amino acid analyses of these samples were almost identical. The combined weight of Fractions 4 A, 5 B,... 

Conversion of the proenzyme into its active form. We concluded that only the proenzyme form of acid protease can exist in human seminal plasma at the physiological pH of around 7.5 therefore, we investigated activation of proenzyme in acid medium. The purified proenzyme was incubated in 1 mM HCl, pH 3, for 1 hr, and then chromatographed on Sephadex G-50 column. The proenzyme was converted into an active form and some peptide of small molecular weight was released (Fig.2). As shown in Table II, when the amino acid analyses of the proenzyme, the active form, and activation peptide were carried out, the number of each amino acid residue of the proenzyme agreed well with the additive value between the number of that amino acid in the active form and activation peptide. This supported the conversion of the proenzyme to an active form. The amino acid composition of the active protease and of the proenzyme were comparable to those of bovine pepsin (10) and pepsinogen (11). However, definite differences are present. About forty residues which carried most of the basic amino acids, were released from pepsin, while sixty-nine residues, which carried about 30% of basic amino acid of the precursor were liberated from the proenzyme of seminal plasma acid protease. 

Thus 0.34% cytochrome is definitely not homogeneous on electrophoresis, and we have no proofs of its homogeneity the 0.43% cytochrome, however, is homogeneous on electrophoresis, and the amino acid analyses have so far not shown any definite deviation from integers in the ratio of moles amino acid to atoms of iron. Further criteria of purity would clearly be required before one could definitely assert that even this cytochrome c is chemically pure. Cytochrome c with 0.34% iron, on the other hand, is in our opinion definitely impure. 

The amino acid analyses in the purest preparations with 0.43% iron gave the results shown in Table II. Cystine, lysine, tyrosine, glutamic and aspartic acids, and leucine were isolated in a pure state from cytochrome c. 

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