William H. Stein
Rockefeller University
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Methods in Enzymology | 1963
Stanford Moore; William H. Stein
Publisher Summary This chapter describes the chromatographic determination of amino acids by the use of automatic recording equipments. Quantitative determination of amino acids bears a relationship to the chemistry of proteins similar to that which elementary analysis bears to the chemistry of simpler organic molecules. As the precision of analytical methods for the determination of amino acids has increased, the limiting factor in deriving the amino acid composition of a protein has become the extent to which the composition of the hydrolyzate is a true reflection of the composition of the parent protein. For the highest recovery of S-carboxymethylcysteine and tyrosine, it is desirable to remove the traces of dissolved air in the HCl before the tube is sealed. The results from a typical chromatographic analysis of a hydrolyzate of bovine pancreatic ribonuclease A are presented. Air-oxidation of cysteine in the hydrolyzate to cystine, before chromatography, is desirable in order to obtain a more accurate value for the total cystine + cysteine content of the protein and also because cysteine may interfere with the determination of proline.
Journal of Clinical Investigation | 1960
M. Prince Brigham; William H. Stein; Stanford Moore
Studies on the amino acid content of physiological fluids have usually been carried out under conditions that would permit any cysteine present to be oxidized to cystine prior to analysis. Consequently, little quantitative information has been obtained concerning the relative amounts of the -SH and the -S-S- forms of the amino acid in plasma. To know whether both forms are present is of particular importance in the study of excretion of amino acids by the kidney. Cystine is an amino acid which appears in abnormal quantities in the urine in several pathological conditions (1). Failure of tubular reabsorption has usually been invoked to explain this fact (2), an explanation which tacitly assumes that it is cystine (the -S-S- form) not cysteine (the -SH form) that is circulating in the blood and is cleared by the kidney. If cysteine were to be the predominant form normally circulating, oxidation to cystine in the kidney would be required in order to account for the excretion of the disulfide in the urine. Fujita and Numata (3) and Smith and Tuller (4) have reported the presence of cysteine in blood. Another observation which could bear on the subject is that of Brand, Cahill and Harris (5) who showed that the amount of cystine in the urine of a cystinuric subject rose after feeding cysteine and methionine, but not after the ingestion of cystine, the sulfur of which appeared almost exclusively as urinary sulfate. With these thoughts in view, a method has been
Experimental Biology and Medicine | 1951
William H. Stein
Summary The amino acid content of the urine of five cystinurics has been determined employing chromatography on columns of the ion exchange resin, Dowex-50. In every case the amount of arginine, lysine, ornithine, and cystine was found to be from 50 to 100 fold the normal level. The quantity of isoleucine was about twice the normal, whereas the taurine output was diminished to about 1/3 or less of normal. All other ninhydrin positive constituents were in the normal range. It is particularly striking that the relative molar excretion of ornithine, cystine, arginine, and lysine, which was found to be about 1:1:2:4, is quite similar from one subject to the next despite the haphazard nature of the case material and the absence of any dietary control. The results suggest the hypothesis that cystinuria involves an enzymatic defect which affects simultaneously at some site or sites (probably the kidney) some phase of the metabolism of arginine, lysine, ornithine, and the sulfur amino acids, and perhaps also isoleucine and taurine.
Advances in Protein Chemistry | 1956
Stanford Moore; William H. Stein
Publisher Summary The chapter presents a summary of recent experiments on the column chromatography of peptides and a description of the current status of chromatography as a method for purifying proteins. Columns of ion exchange resins have received particular attention because they were shown to yield quantitative results with amino acids, and hence would be expected to separate peptides quantitatively as well. In addition, such columns are not sensitive to the presence of neutral salts, and can thus be used directly for the analysis of physiological fluids or tissue extracts. When amino acids are chromatographed on sulfonated polystyrene resins, separations of peptides depend upon the affinity of the resin for both the ionic and the nonionic portions of the molecules. The great usefulness of paper chromatography for the separation of peptides has led to several attempts to extend the principles of liquid-liquid or liquid-gel system to columns packed with cellulose or starch. In the study of protein chromatography, many of the laboratory procedures employed are similar to those devised for experiments with amino acids and peptides. The actual protein concentrations can be calculated only if the ninhydrin color values per milligram or the extinction coefficients of the individual proteins are known. Successful chromatography requires that the proteins move down a column as discrete zones and give rise to relatively sharp and symmetrical peaks on an effluent curve. The chapter explores that sulfonic acid resins are employed in two quite different ways in the course of protein purifications.
Annals of the New York Academy of Sciences | 1946
William H. Stein; Stanford Moore
The immediate goal which protein chemists hope to attain with the aid of amino acid analysis is the ability to express the empirical formulae of numerous proteins in terms of their constituent amino acids, in much the same manner as the empirical formulae of simpler compounds are expressed in terms of their constituent elements. The protein chemist, as a consequence, should be able to analyze a protein for its constituent amino acids with the accuracy and dispatch attending the elementary analysis of simple compounds. Accuracy primarily, and secondarily dispatch, therefore, will be the criteria by which the methods to be discussed in this communication will be judged. In this discussion, a “specific precipitant” will be defined as any reagent which, when added to a protein hydrolysate, will unite with, and cause to precipitate, one or a small group of amino acids by the formation of insoluble salts or molecular complexes. Reagents which combine chemically with amino acids through covalent linkages to form insoluble derivatives will not be considered. The use of precipitants for the exploration of the amino acid composition of protein hydrolysates has a long history. This history may be divided roughly into its qualitative and quantitative aspects. Our extensive knowledge of the distribution of amino acids in nature is a result largely of the qualitative or semi-quantitative use of specific precipitants. This phase of the subject has been dealt with elsewhere,1~2~8 and hence will not be discussed here. We shall be concerned, rather, with the quantitative aspects of the subject. In the past, many specific precipitants have been employed as quantitative tools for the determination of specific amino acids, or small groups of amino acids, in protein hydrolysates. The information thus obtained has been of the greatest importance, and is the foundation upon which the newer quantitative work must build. The question may be raised, however, whether cjuantitative isolation procedures offer promise of future rapid advances in the amino acid analysis of proteins. In the course of this discussion, an attempt will be made to answer this question. In order to do this, an evaluation of methods is necessary. The methods selected are
Structure–Function Relationships of Proteolytic Enzymes#R##N#Proceedings of the International Symposium, Copenhagen, June 16–18, 1969, No. 37 in the Series of the International Union of Biochemistry Sponsored Symposia | 1970
William H. Stein
Publisher Summary This chapter focuses on chemical studies on purified pepsin. There are at least three carboxyl groups at or near the active site of pepsin. Two required for catalysis, and one more for reaction with a diazo reagent. There are probably even more than that, because p-bromophenacyl bromide does not cause complete inactivation of the enzyme and, hence, cannot react with either of the two catalytically essential groups. After reaction with p-bromophenacyl bromide, the substituted enzyme can still combine with and be inactivated by a diazo reagent in the presence of copper. Hence, p-bromophenacyl bromide does not react with the same carboxyl group with which a diazo reagent reacts. The pepsin can act over a broad pH range and the optimum pH for hydrolysis depends upon the structure of the substrate. If a series of carboxyl groups with overlapping pKs are present at the active site, the identity of those that may participate in binding will depend upon the nature of the substrate.
Analytical Chemistry | 1958
Darrel H. Spackman; William H. Stein; Stanford. Moore
Journal of Biological Chemistry | 1954
Stanford Moore; William H. Stein
Journal of Biological Chemistry | 1948
Stanford Moore; William H. Stein
Journal of Biological Chemistry | 1963
Arthur M. Crestfield; Stanford Moore; William H. Stein