Amino-terminal analysis of polypeptide using dimethylaminoazobenzene isothiocyanate

A new method for qualitative and quantitative N-terminal analysis of polypeptide using dimethylaminoazobenzene-isothiocyanate is presented. The method can recover all naturally occurring N-terminal amino acids, including asparagine, glutamine, and tryptophane in a nearly quantitative yield. Less than 1 nmol of polypeptide is required for qualitative N-terminal analysis and 5 to 10 nmol of polypeptide is used for quantitative N-terminal analysis. Applications and expected limitations of this new N-terminal method are described.     

Cross-linking site in fibrinogen for alpha 2-plasmin inhibitor

A plasma proteinase inhibitor, alpha 2-plasmin inhibitor (alpha 2PI), is cross-linked with alpha chain of fibrin(ogen) by activated coagulation Factor XIII (plasma transglutaminase). alpha 2PI serves only as a glutamine substrate (amine acceptor) for activated Factor XIII in the cross-linking reaction, and the cross-linking occurs between Gln-2 of the alpha 2PI molecule and a lysine residue (amine donor) of fibrin(ogen) alpha chain, whose position was investigated. alpha 2PI and fibrinogen were reacted by activated Factor XIII. The resulting alpha 2PI fibrinogen A alpha chain complex was separated and subjected to two cycles of Edman degradation using phenyl isothiocyanate for the first cycle and dimethylaminoazobenzene-isothiocyanate for the second cycle. The aqueous phase after the cleavage stage of the second cycle, containing dimethylaminoazobenzene-thiohydantoin-Gln cross-linked with A alpha chain, was subjected to CNBr fragmentation and tryptic digestion. Only one of the peptides was found to have the peak of absorbance at 420 nm, indicating the presence of dimethylaminoazobenzene-thiohydantoin-Gln in that peptide. The peptide was identified as corresponding to residues Asn-290-Arg-348 of A alpha chain by analyses of the NH2-terminal amino acid sequence and amino acid composition. The peptide contains a single lysine at position 303, indicating that Lys-303 of fibrinogen A alpha chain is the lysine residue that forms a cross-link with Gln-2 of alpha 2PI.     

Inferring the number of evolutionary events from DNA coding sequence differences

The estimation of the amount of evolutionary divergence that has taken place between two DNA coding sequences depends strongly on the degree of constraint on amino acid replacements. If amino acid replacements are relatively unconstrained, the individual nucleotide is the appropriate unit of analysis and the method of Tajima and Nei can be used. If amino acid replacements are constrained, however, this method is shown to be inapplicable. For sequences with strong amino acid constraints, a method is outlined analogous to the Tajima and Nei method using codons as the unit of analysis. Only synonymous substitutions are used. Codon usage data can be employed to estimate the necessary parameters of the calculation, or a priori models of substitution may be employed. Sequences with significant but intermediate constraints on amino acid replacements are, in principle, unanalyzable.      

Microbiological titration of proteins and of single amino acid content in biological materials without purification and hydrolysis

A method is described for the microbiological determination of the protein content of biological materials. This method can also be adopted to titrate the concentration of a single amino acid in the protein and has the following advantages: (1) titration can be done without purification and hydrolysis of proteins; (2) the titration graph is a straight line between 25 and 800 microgram/ml; (3) protein values agree with those obtained using the Kjeldhal method; and (4) each mutant requiring one amino acid may be used to titrate the concentration of a single amino acid of the protein. The leucine content of various kinds of flour was measured with this system.     

A fast and sensitive micromethod for the manual sequencing of peptides using O-phthalaldehyde as derivatizing reagent

A general procedure for the manual sequencing of peptides using the fluorogenic reagent O-phthalaldehyde (OPA) is described. The method can be applied in two different ways. One of them involves back hydrolysis of the anilinothiazolinones resulting from the Edman degradation of the peptide and subsequent detection of the free amino acids as OPA derivatives. The other is a subtractive analysis in which the amino acid composition of the remaining peptide is determined after each degradation cycle. The direct procedure can be coupled to the subtractive one in order to assure the accuracy of the sequence analysis. The method is fast and simple, and allows determination of 10 pmol of amino acid per cycle using standard reagents and instrumentation. Sensitivity can be greatly enhanced provided that ultrapure chemicals are employed. Small peptides (8-10 residues) were sequenced from 200 pmol sample, using a high-performance liquid chromatography assembly coupled to a fluorescence detector.     

Automated fluorometric amino acid analysis: the determination of proline and hydroxyproline.

A fluorometric method for the automated determination of the imino acids proline and hydroxyproline has been developed. The assay is based on the postcolumn reaction of the imino acids with alkaline sodium hypochlorite, which yields oxidation products amenable to detection with fluorogenic amine reagents. The method is simple and can be adapted readily to high-sensitivity amino acid analyzers which use o-phthalaldehyde for detection. As little as 10 pmol proline and 20 pmol hydroxyproline can be determined accurately. Thus the full array of natural imino and amino acids can now be determined on a high-sensitivity amino acid analyzer using o-phthalaldehyde.     

A manual subtractive end-group determination of oligopeptides using fluorescamine or o-phthalaldehyde

A new method for the end-group determination of peptides using the fluorogenic reagents fluorescamine or o-phthalaldehyde is described. The method is based on the property that the derivatives of the N-terminal amino group of peptides formed in solution after reaction with either reagent are resistant to acid hydrolysis. The N-terminal amino acid can be determined by simply comparing the amino acid analysis of the original peptide with the fluorescent derivative of the peptide. In general, the decrease of the N-terminal residue in the reacted peptides in 80–90% with fluorescamine and more than 90% with o-phthalaldehyde. Any N-terminal amino acid, with the exception of proline, can thus be determined.     

A precise method for the quantitation of proteins taking into account their amino acid composition.

A method for the quantitation of protein in biological material is described which gives the same response for all proteins irrespective of their amino acid composition. The method is based on the ninhydrin reaction of amino acids released after total acid hydrolysis of 5- to 20-μl solutions containing 1 to 100 μg of protein. The ammonia is released from the hydrolysate by diffusion and the amino acids are quantitated without fractionation using the continuous-flow system of an amino acid analyzer. Calibration is obtained with solutions of known amino acid content. The protein of a sample is calculated by multiplying the nanomoles of total amino acids found by a conversion factor F. F is the weight in micrograms of 1 nmol of the specific mixture of amino acid residues that the protein of the sample is composed of F has to be determined once for all further quantitations of the same material by quantitative amino acid analysis following standard procedures. By this method as little as 30 ng of protein per aliquot of hydrolysate analyzed can be determined.     

Chromogenic identification of oligonucleotide-directed mutants.

We describe a simple plaque color assay for identifying oligonucleotide-directed mutations in cloned DNA fragments. The basis of the method is to: fuse the sequence of interest in-frame to the E.coli lacZ gene to produce a blue plaque phage, mutate the site of interest to a stop codon to generate a white plaque phage, and revert the stop codon and surrounding nucleotides to give a blue plaque phage containing one or more desired amino acid changes. The advantages of this cyclic method are that it produces truncated as well as amino acid substituted protein molecules, it can be repeated to introduce additional mutations, and it eliminates the need for labor intensive screening. Essentially any piece of DNA can be mutated using this method if the fragment has one open reading frame. If there is an open reading frame between the site and the lacZ gene, ATG codons can be inserted at the target site. We have used this method to produce termination and amino acid substitution mutants in the yeast CUP1 gene.     

Analysis of differences in amino acid substitution patterns, using multilevel G-tests

In this paper, a new algorithm is presented, which makes possible multilevel comparison of BLOSUM protein substitution matrices based on data from different groups of organisms. As an example, a comparison between substitution matrices based on data from two groups of bacterial genomes with different GC content is presented. Our approach includes evaluating the number of amino acid pairs in BLOCKS databases created separately for the two groups of bacteria using protein sequences deposited in the COG database. Differences of distributions of amino acid pair counts are tested using the chi-squared based G-test. Different analysis levels make it possible to distinguish different patterns of amino acid substitution. Application of the algorithm reveals statistically significant differences in amino acid substitution patterns between AT-rich and GC-rich groups of bacterial organisms. The differences are particularly visible in the overall substitution pattern, amino acid conservation pattern and in comparison of substitution patterns for single amino acids. The algorithm presented in this paper can be considered a novel method for multi-level comparison of amino acid substitution patterns. The presented approach is not limited to bacterial organisms and BLOSUM substitution matrices. Statistically significant differences between substitution patterns in the two groups of bacterial organisms with respect to amino acid conservation pattern can be the evidence of different rate of evolutionary change between AT-rich and GC-rich bacterial organisms.     

A study of the interaction of human spermatozoa membrane with ATP and cyclic-AMP

The three coat proteins of foot-and-mouth disease virus type O can be separated in milligram amounts using a SE-cellulose ionic exchanger, both from each other and viral RNA not quantitatively precipitated by acetic acid. The amino acid composition of the separated proteins has been determined and a statistical method has been used to calculate, for each, the most probable number of amino acids and molecular weight. The N-terminal amino acids of the three proteins are glycine, asparagine and threonine.     

Formation of proline thiohydantoin with ammonium thiocyanate: progress towards a viable C-terminal amino-acid-sequencing procedure.

Pure amino acid thiohydantoins are required as reference standards for development of C-terminal-sequencing procedures based on thiohydantoin formation of the C-terminal amino acids of peptides and proteins. Proline thiohydantoin was prepared using a straightforward method involving reaction of acetylproline with ammonium thiocyanate. It was characterized by UV spectrophotometry, mass spectrometry and back-hydrolysis to the free amino acid. These data establish unequivocally that the thiocyanate procedure is applicable to proline as well as to the other common amino acids. This work also validates earlier claims that proline thiohydantoin can be prepared by reaction with thiocyanic acid.     

The separation of D/L amino acid pairs by high-performance liquid chromatography after precolumn derivatization with optically active naphthylethyl isocyanate

A method for determining the optical purity of amino acids using HPLC and precolumn derivatization is described. (+)-1-(1-Naphthyl)ethyl isocyanate reacts with racemic amino acids, in high yield, to form naphthylethyl carbamoyl derivatives. The resulting diastereoisomeric pairs were separated on reversed-phase C18 columns and detected fluorometrically. Excitation maxima for naphthylethyl carbamoyl aspartic acid were 235 and 297 nm. The emission maximum was at 333 nm. Using a filter fluorometer with a zinc or cadmium lamp, less than 1 pmol of a D amino acid can be measured in the presence of 1000-fold excess of the L isomer. The column can also be monitored at lower sensitivity, using an ultraviolet detector operating at or near the absorption maximum of 222 nm. Chromatographic data are presented on the resolution of 17 amino acid pairs.     

Prediction of thermophilic proteins using feature selection technique

The thermostability of proteins is particularly relevant for enzyme engineering. Developing a computational method to identify mesophilic proteins would be helpful for protein engineering and design. In this work, we developed support vector machine based method to predict thermophilic proteins using the information of amino acid distribution and selected amino acid pairs. A reliable benchmark dataset including 915 thermophilic proteins and 793 non-thermophilic proteins was constructed for training and testing the proposed models. Results showed that 93.8% thermophilic proteins and 92.7% non-thermophilic proteins could be correctly predicted by using jackknife cross-validation. High predictive successful rate exhibits that this model can be applied for designing stable proteins.     

Support vector machines for learning to identify the critical positions of a protein

A method for identifying the positions in the amino acid sequence, which are critical for the catalytic activity of a protein using support vector machines (SVMs) is introduced and analysed. SVMs are supported by an efficient learning algorithm and can utilize some prior knowledge about the structure of the problem. The amino acid sequences of the variants of a protein, created by inducing mutations, along with their fitness are required as input data by the method to predict its critical positions. To investigate the performance of this algorithm, variants of the beta-lactamase enzyme were created in silico using simulations of both mutagenesis and recombination protocols. Results from literature on beta-lactamase were used to test the accuracy of this method. It was also compared with the results from a simple search algorithm. The algorithm was also shown to be able to predict critical positions that can tolerate two different amino acids and retain function.     

Support Vector Machine-based classification of protein folds using the structural properties of amino acid residues and amino acid residue pairs

MOTIVATION: Fold recognition is a key step in the protein structure discovery process, especially when traditional sequence comparison methods fail to yield convincing structural homologies. Although many methods have been developed for protein fold recognition, their accuracies remain low. This can be attributed to insufficient exploitation of fold discriminatory features. RESULTS: We have developed a new method for protein fold recognition using structural information of amino acid residues and amino acid residue pairs. Since protein fold recognition can be treated as a protein fold classification problem, we have developed a Support Vector Machine (SVM) based classifier approach that uses secondary structural state and solvent accessibility state frequencies of amino acids and amino acid pairs as feature vectors. Among the individual properties examined secondary structural state frequencies of amino acids gave an overall accuracy of 65.2% for fold discrimination, which is better than the accuracy by any method reported so far in the literature. Combination of secondary structural state frequencies with solvent accessibility state frequencies of amino acids and amino acid pairs further improved the fold discrimination accuracy to more than 70%, which is approximately 8% higher than the best available method. In this study we have also tested, for the first time, an all-together multi-class method known as Crammer and Singer method for protein fold classification. Our studies reveal that the three multi-class classification methods, namely one versus all, one versus one and Crammer and Singer method, yield similar predictions. AVAILABILITY: Dataset and stand-alone program are available upon request.     

An ultramicro method of amino acid analysis: application to studies of protein metabolism in cultured cells.

Analysis of the quantity and specific radioactivity of amino acids derived from intra-cellular pools, aminoacyl-transfer RNA, and protein hydrolysates of cultured cells has been achieved using a radiolabeled amino group ligand, dansyl chloride. Speeific activities of ¹⁴C- or ³H-labeled amino acids are calculated after reaction with appropriately labeled dansyl chloride of known specific activity. The quantity of amino acid is determined as a function of its diluting influence on a radioactive standard. The specific activity of as little as 2 pmol of amino acid can be measured using [¹⁴C]dansyl chloride the less sensitive of the two isotopic species available. Thus, cells from a single 60-mm culture dish provide sufficient material for analysis of both intracellular and transfer RNA amino acid pools, and one can easily analyze the amino acids in hydrolysates made from individual bands in polyacrylamide gels. The method offers significant improvement in speed, sensitivity, and economy over conventional methods of amino acid analysis and, because of its double-label design, gives accurate results with a minimum of technical expertise and no major equipment other than a scintillation counter.     

Inexpensive and easily built small scale 2D electrophoresis equipment

Octopine can be estimated fluorometrically by the reduction of NAD using octopine dehydrogenase. The method is very sensitive and specific for this unusual amino acid. Octopine can be estimated from neutralized perchloric acid extracts without further treatment.     

A graphic representation of protein sequence and predicting the subcellular locations of prokaryotic proteins

Zp curve, a three-dimensional space curve representation of protein primary sequence based on the hydrophobicity and charged properties of amino acid residues along the primary sequence is suggested. Relying on the Zp parameters extracted from the three components of the Zp curve and the Bayes discriminant algorithm, the subcellular locations of prokaryotic proteins were predicted. Consequently, an accuracy of 81.5% in the cross-validation test has been achieved using 13 parameters extracted from the curve for the database of 997 prokaryotic proteins. The result is slightly better than that of using the neural network method (80.9%) based on the amino acid composition for the same database. By jointing the amino acid composition and the Zp parameters, the overall predictive accuracy 89.6% can be achieved. It is about 3% higher than that of the Bayes discriminant algorithm based merely on the amino acid composition for the same database. The prediction is also performed with a larger dataset derived from the version 39 SWISS-PROT databank and two datasets with different sequence similarity. Even for the dataset of non-sequence similarity, the improvement can be of 4.4% in the cross-validation test. The results indicate that the Zp parameters are effective in representing the information within a protein primary sequence. The method of extracting information from the primary structure may be useful for other areas of protein studies.     

An SVM method using evolutionary information for the identification of allergenic proteins

This study presents an allergenic protein prediction system that appears to be capable of producing high sensitivity and specificity. The proposed system is based on support vector machine (SVM) using evolutionary information in the form of an amino acid position specific scoring matrix (PSSM). The performance of this system is assessed by a 10-fold cross-validation experiment using a dataset consisting of 693 allergens and 1041 non-allergens obtained from Swiss-Prot and Structural Database of Allergenic Proteins (SDAP). The PSSM method produced an accuracy of 90.1% in comparison to the methods based on SVM using amino acid, dipeptide composition, pseudo (5-tier) amino acid composition that achieved an accuracy of 86.3, 86.5 and 82.1% respectively. The results show that evolutionary information can be useful to build more effective and efficient allergen prediction systems.