Complete amino acid analysis of proteins from a single hydrolysate.

An analytical procedure which affords the precise amino acid composition of a protein or a peptide from a single hydrolysate is described. This method utilizes 4 N methanesulfonic acid containing 0.2% 3-(2-aminoethyl)indole, rather then 6N HCl as a catalyst for hydrolysis. The hydrolysis is carried out in vacuo (20 mu) at 115 degrees for 22 to 72 hours. Half-cystine is determined as S-sulfocysteine by treating the hydrolysate with dithiothreitol followed by an excess of tetrathionate. The values of all amino acids, including tryptophan and half-cystine, were close to the expected theoretical values for the proteins examined. The method has the advantage that the neutralized hydrolysate can be applied directly to an ion exchange column. Further, the method is capable of distinguishing between free sulfhydryl groups as S-carbosymethylcysteine and disulfides as S-sulfocysteine. A limitation of the procedure is that tryptophan remains sensitive to the presence of carbohydrate in the sample.     

The amino acid composition of proteins: a method of analysis.

Four quasiloglinear models are proposed for describing relationships between the amino acid composition of proteins and the structure of the genetic code. The models allow estimation of base frequencies in all three codon positions and can be used to investigate “interactions” between any two codon positions. The estimation procedure proposed by Ohta and Kimura (Genetics64 (1970), 387–395) is discussed and using two of the proposed quasiloglinear models an analysis of the amino acid composition of human cytochrome c is presented. The analysis suggests that of the six codons which code for leucine (CUU, CUC, CUA and CUG) do not occur in human cytochrome c.     

Single-column amino acid analysis of connective tissue proteins and related materials.

A single-column procedure is described which, in general, is useful for the amino acid analysis of any simple or complex protein, but in particular the procedure is useful for the separation and quantitation of the amino acids and amino sugars, if present, in collagen, elastin and related materials from a variety of connective tissues. In addition to the amino acids commonly found in proteins, the system resolves hydroxyproline, hydroxylysine, desmosine, isodesmosine, glucosamine, galactosamine and also a number of other ninhydrin-positive compounds ordinarily not found in acid hydrolysates of proteins. Chromatograms of the analysis of a synthetic mixture of amino acid standards and also collagen and elastin hydrolysates indicate the multiple use of the procedure and the nearbaseline separation of all the amino acids. The basic amino acids are well spread, thus providing flexibility for the isolation and identification of additional ninhydrin-positive components. The analysis, which requires approximately 2 hr and four sodium citrate buffers was performed with a Durrum (D-500) amino acid analyzer.     

A survey of amino acid side-chain interactions in 21 proteins

Based on the atomic co-ordinate data for 21 representative proteins, the frequencies of long-range interactions between side-chain groups and 15 different types of side-chain atoms have been determined. The observed frequencies are compared to the results expected for random association in order to define a scale of relative affinities. Thirty-five residue-atom pairs exhibit frequencies of interaction that differ by at least 50% from the expected values. The amino acids tend to fall into three classes: non-polar, neutral and polar amino acids. The data are regrouped in a different way to determine the average affinity of each amino acid side-chain group for all other types of side-chain groups. Fourteen side-chain pairs have at least 50% fewer interactions than expected, while 21 side-chain pairs have at least 50% more interactions than expected. Unusual patterns of association are discussed and compared with current ideas about the organization of protein structure.     

Abnormal tau proteins from Alzheimer's disease brains. Purification and amino acid analysis

Abnormal tau proteins (PHF-tau) were isolated from Alzheimer's disease brains by treatment of paired helical filament enriched-fractions with perchloric acid and boiling of the acid precipitable fraction with beta-mercaptoethanol. These proteins were purified further by a second perchloric acid treatment. The purified PHF-tau proteins were soluble in buffers devoid of sodium dodecyl sulfate. However, they were similar to the abnormal tau extracted from paired helical filaments with sodium dodecyl sulfate, also named A68, in molecular mass (68, 64, and 60 kDa), isoelectric point (pI 5.5-6.5), reactivity with anti-tau antibodies, and in requirement for alkaline phosphatase treatment to bind the Tau-1 antibody. Compared to normal tau, the soluble PHF-tau contained 100% more glycine and 35% less lysine residue. The results suggest that besides phosphorylation other types of modification may be involved in differentiating PHF-tau from normal tau.     

Structure-dependent relationships between growth temperature of prokaryotes and the amino acid frequency in their proteins

We studied the amino acid frequency and substitution patterns between homologues of prokaryotic species adapted to temperatures in the range 0–102°C, and found a significant temperature-dependent difference in frequency for many of the amino acids. This was particularly clear when we analysed the surface and core residues separately. The difference between the surface and the core is getting more pronounced in proteins adapted to warmer environments, with a more hydrophobic core, and more charged and long-chained amino acids on the surface of the proteins. We also see that mesophiles have a more similar amino acid composition to psychrophiles than to thermophiles, and that archea appears to have a slightly different pattern of substitutions than bacteria. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hydrophobicity of amino acid subgroups in proteins

Protein folding studies often utilize areas and volumes to assess the hydrophobic contribution to conformational free energy (Richards, F.M. Annu. Rev. Biophys. Bioeng. 6:151-176, 1977). We have calculated the mean area buried upon folding for every chemical group in each residue within a set of X-ray elucidated proteins. These measurements, together with a standard state cavity size for each group, are documented in a table. It is observed that, on average, each type of group buries a constant fraction of its standard state area. The mean area buried by most, though not all, groups can be closely approximated by summing contributions from three characteristic parameters corresponding to three atom types: (1) carbon or sulfur, which turn out to be 86% buried, on average; (2) neutral oxygen or nitrogen, which are 40% buried, on average; and (3) charged oxygen or nitrogen, which are 32% buried, on average.     

Analysis of gamma-carboxyglutamic acid via amino acid analysis.

Modifications of the analysis of protein-bound residues of γ-carboxyglutamic acid (Gla) via alkaline hydrolysis are presented. The method described allows easier sample manipulation than that heretofore required and insures quantitative recovery of hydrolyzed amino acids. A possible explanation of the shoulder which sometimes appears near Gla on some amino acid analyzers is presented.     

Nitric oxide can modify amino acid residues in proteins.

Nitric oxide derived from sodium nitroprusside binds to the heme moiety of hemoglobin and also modifies some functional groups in the protein. As hemoglobin concentration is increased, globin modification is decreased presumably due to formation of the NO complex with heme. The SH groups of hemoglobin are probably not involved in the formation of the stable product formed by NO. In the presence of inositol hexaphosphate, which binds preferentially in the cleft between the two beta-chains of hemoglobin, formation of one modified derivative was selectively reduced. With hemoglobin specifically blocked on its N-terminal residues, globin modification was also significantly reduced. Carbonic anhydrase, which is blocked at its N-terminus, was also refractory to modification. The results suggest that the N-terminal groups of some proteins can be modified by nitric oxide, perhaps by deamination.     

Primary structure of a chick tropoelastin peptide: Evidence for a collagen-like amino acid sequence

A 73-residue tryptic fragment from chick aortic tropoelastin has been sequenced by solid state methods. This is a new structure not previously described in any tropoelastins and may be unique for avians. It contains a G V P tripeptide repeat, giving it a primary structure like that of collagen. This may explain some of the collagen-like properties observed in elastin, particularly collagenase susceptibility and the presence of hydroxyproline.     

A procedure for amino acid sequencing in internal regions of proteins

We describe a novel procedure for determining the amino acid (aa) sequence of the internal regions of proteins. This procedure has been implemented by directly determining the sequence of aa 65-75 of the product of the trpR gene of Escherichia coli, the trp repressor. This method is based on the insertion of the cleavage site of a specific protease (factor Xa) into the protein immediately before the region to be sequenced by Edman degradation. The simplicity of the procedure makes it appealing for studies of protein structure-function relationships, and of the expression of genetic information. The method is particularly useful when there is ambiguity concerning the co-linearity of the aa and nucleotide sequences.