Daniel J. Strydom

Amino acid analysis utilizing phenylisothiocyanate derivatives

Advances in liquid chromatography have brought about the development of new techniques in amino acid analysis which take full advantage of precolumn derivatization procedures. Using phenylisothiocyanate as the reagent, detection limits under 1 pmol can be routinely achieved, allowing the analysis of submicrogram protein samples. Analysis times as short as 10 min for samples after hydrolysis and 1 h for physiologic samples are possible. Accurate, reproducible quantitation of amino acids can be obtained from complex matrices such as plasma, urine, feed, and food samples. This level of performance and flexibility gives the analyst the first realistic alternative to ion-exchange analysis without compromising desirable features of the traditional methodology.

Chromatographic separation of 1-phenyl-3-methyl-5-pyrazolone-derivatized neutral, acidic and basic aldoses

Abstract Analysis, by HPLC, of reducing monosaccharides as their 1-phenyl-3-methyl-5-pyrazolone derivatives is attractive owing to its sensitivity of detection and the generation of single derivatives of each aldose molecule [Honda et al., Anal. Biochem. 180 (1989) 351]. The present studies establish conditions for reversed-phase chromatographic analyses of hydrolysates containing neutral, basic and acidic reducing monosaccharides. In particular, glucuronic and galacturonic acids, glucosamine and galactosamine are separated completely both from one another and from the aldoses of glycoproteins commonly found. Analyses of blank hydrolysates provide the baseline for background amounts of these carbohydrates and analyses of a variety of glycoproteins illustrate the effectiveness of these separations. Elution times were established for derivatives of all tetroses, pentoses and hexoses, as well as a variety of deoxy and phosphorylated aldoses.

Characterization of human alcohol dehydrogenase isoenzymes by high-performance liquid chromatographic peptide mapping

Abstract Human liver alcohol dehydrogenase (ADH, EC 1.1.1.1) is a large and heterogeneous family of isoenzymes and the high-performance liquid chromatographic peptide mapping technique which was developed here recognizes differences and similarities between them. Isoenzymes were S -carboxymethylated, digested with trypsin, and the mixtures of tryptic peptides fractionated by reverse-phase gradient chromatography on octadecylsilane columns, using perchlorate-phosphate buffer and acetonitrile as eluants. The resultant peptide maps were reproducible, showing great similarities between the αβγ-ADH isoenzymes (now called Class I) on the one hand and remarkable differences between these and both the π- and χ-ADH isoenzymes (now called Class II and III, respectively) on the other. This implies that these three isoenzyme groups have characteristic primary structures which correspond to their typical substrate specificities and kinetics.

Characterization and sequencing of rabbit, pig and mouse angiogenins: discernment of functionally important residues and regions☆

Rabbit, pig and mouse angiogenins have been purified from blood serum and characterized, and the rabbit and pig proteins have been sequenced fully. A partial sequence of the mouse protein is consistent with the sequence deduced from the genomic DNA (Bond, M.D. and Vallee, B.L. (1990) Biochem. Biophys. Res. Commun. 171, 988-995). All three angiogenins are homologous to the pancreatic RNases and contain the essential catalytic residues His-13, Lys-40 and His-114, and the 6 half-cystines of the human protein. Like human angiogenin they display extremely low ribonucleolytic activities toward wheat-germ RNA, yeast RNA, poly(C) and poly(U). The rabbit and pig proteins induce neovascularization in vivo and also inhibit protein synthesis in vitro. The interaction of rabbit, pig and bovine angiogenins with placental ribonuclease inhibitor, a potent inhibitor of angiogenin, was examined by fluorescence spectroscopy. Rate and equilibrium binding constants indicate that rabbit angiogenin binds to the inhibitor much like human angiogenin, whereas the pig and bovine proteins show significant differences. A comparison of the five angiogenin sequences now available points to specific residues that are highly conserved among them but differ from the corresponding residues in the RNases. These residues are clustered in particular regions of the three-dimensional structure, two of which contribute to the angiogenic, second-messenger and/or protein synthesis inhibition activities of human angiogenin.

Comparison of human and bovine brain derived heparin-binding growth factors

Two growth factors have been purified to homogeneity from human brain using heparin affinity chromatography. They have apparent molecular weights of 17 Kd and 18 Kd. Their amino acid compositions differ, but are similar to those of the two heparin-binding growth factors present in bovine neural tissue. These results suggest that the heparin-binding growth factors in neural tissue can be grouped into two distinct classes.

Sites of modification of human angiogenin by bromoacetate at pH 5.5

Human angiogenin is inactivated by treatment with bromoacetate at pH 5.5. Use of [14C]bromoacetate and tryptic peptide mapping have identified the sites of carboxymethylation as His-13 and His-114, with His-114 reacting approximately 1.5-fold more rapidly than His-13. At later stages in the reaction, both His-13 and -114 become modified with His-114 in part forming a bis derivative. Comparison with carboxymethylhistidine derivatives of known structure obtained from bovine pancreatic ribonuclease A indicates that the reaction order is N-1 of His-114 greater than N-3 of His-13 greater than N-3 of His-114.

Sensitive Analysis of Cystine/Cysteine using 6-Aminoquinolyl-N-Hydroxysuccinimidyl Carbamate (AQC) Derivatives

Publisher Summary This chapter focuses on sensitive analysis of cystine/cysteine using 6-aminoquinolyl-N-hydroxysuccimniidyl carbamate (AQC) derivatives. A need for high-sensitivity amino acid analyses exists as protein recognition, and isolation and sequencing techniques are becoming more sensitive. Difficulties in cystine analysis are compounded by this general need to analyze subnanomole quantities of amino acids and the more specialized need to analyze low pmol and sub-pmol amounts of protein. The recent development of the highly sensitive and stable AQC derivatives for fmol analyses of amino acids provides the framework for the generation of workable analyses of cysteine at such low levels. The chapter describes the combined use of three approaches to cystine analysis with derivatization to AQC derivatives, and focuses on the success of disulfide interchange in providing analyses of cysteine to fmol sensitivity. The cleanliness of the chromatographic environment surrounding the elution position of one such derivative and the stability of the derivatives combine to provide an opportunity to quantify low nanogram quantities of proteins in the presence of the ubiquitous background of amino acids.

On-line separation of phenylthiohydantoin derivatives of hydrophilic modified amino acids during sequencing

Abstract All 20 commonly occurring phenylthiohydantoin (PTH) amino acids can be separated by a new reversed-phase chromatography method without interference from breakthrough peaks that prevail in other methods, and with good resolution from the most common reagent-related background peaks. Separation is compatible with automated sequencing in a cartridge sequencer. A large chromatographic area exists between the breakthrough peaks and the first- and second-eluting common PTH-amino acids, PTH-His and PTH-Asn, and is consequently available for detection and separation of hydrophilic amino acid derivatives. Derivatives such as phosphotyrosine, β-hydroxyaspartic acid and N-glycosylated asparagine elute as readily identifiable peaks. The method uses chromatography on octadecylsilane with isocratic elution followed by a simple linear gradient from acetonitrile—methanol to isopropanol—methanol to provide complete separation in 23 min. The turnaround time of 34 min includes a period of column washing to eliminate sequencer byproducts. PTH-Amino acids are generally detected with single pmol (1 to 4 pmol) sensitivity. PTH-His and PTH-Arg are eluted earlier than conventionally with a consequent sharpening of peaks and higher practical sensitivity.

The hydrolysis process and the quality of amino acid analysis: ABRF-94AAA collaborative trial

Publisher Summary Amino acid analysis remains an indispensable tool in a variety of biological research and development fields, for example, the biochemical study of proteins, quality control in biotechnology and nutrition, and in clinical analyses. This chapter discusses primarily on discriminating between hydrolysis and all other aspects of amino acid analysis as potential sources of error. Two samples are distributed, one pre-hydrolyzed and one to be hydrolyzed, and their absolute yields are determined with very similar accuracy and standard deviations. The average errors in determining individual amino acids, however, are significantly different for the two samples, with smaller errors in the pre-hydrolyzed sample. However, direct cystine analysis of the pre-hydrolysate is as successful as that of the protein sample, suggesting that chromatography and derivatization, and not hydrolysis are the most important factors in successful analysis of cystine by this simple methodology. Thus, it is clear that excellent analyses can be and are done by any of the available technologies, with proper concern for the many pitfalls peculiar to each method.

A micromethod for the determination of carboxyl-terminal amino acids of peptides and proteins: Phenylisothiocyanate reaction with hydrazinolysates

Proteins and peptides are digested with anhydrous hydrazine and the free amino acids, released from their carboxyl termini, are analyzed by reverse-phase chromatography as phenylthiocarbamyl (PTC) derivatives. Most of the potentially interfering hydrazides are removed due to the insolubility of their derivatives in the solution used for dissolving the phenylthiocarbamyl-amino acids. This methodology can be used to analyze peptides and proteins in quantities smaller than 100 pmol. The method is here employed for the screening of carboxyl termini in peptide fractions originating from an HPLC chromatogram.