Stanford Moore

[117] Chromatographic determination of amino acids by the use of automatic recording equipment

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.

THE CONCENTRATIONS OF CYSTEINE AND CYSTINE IN HUMAN BLOOD PLASMA

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

12 Pancreatic Ribonuclease

Publisher Summary Bovine Ribonuclease has been a test protein in the study of a wide variety of chemical and physical methods of protein chemistry. It is widely employed in the course of the sequencing of RNA. This chapter discusses the usefulness of the cytoplasmic inhibitor of RNase to protect RNA in the course of the synthesis of complementary DNA by reverse transcriptase. It examines the use of the inhibitor to protect mRNA during the course of in vitro translation and during the preparation of rough microsomes and detached polysomes. The introduction of affinity chromatography has simplified the isolation of pure RNases by providing a highly efficient method for separating active enzymes from molecules that do not have an affinity for the coupled substrate analog. The technique can be used as an early step in purification or as a final step after ion exchange chromatography and gel filtration to isolate an RNase fraction of given charge and size.

A study of the chromatographic determination of amino acids in the presence of large amounts of carbohydrate

Abstract Synthetic mixtures of 15 of the 18 most common amino acids (cystine, methionine and tryptophan excepted) were boiled with 6N HCl in the presence and absence of carbohydrate. The decomposition products from starch and glucose do not interfere with the chromatographic determination of the amino acids on ion exchange columns. In no instance was the observed recovery of an amino acid lowered by as much as 3 per cent by the addition of carbohydrate (2 g) to 25–50 mg of amino acids per 200 ml 6 N HCl. The dilute conditions of hydrolysis correspond to those adopted for the determination of the amino acid composition of foods of high carbohydrate content.

On the desalting of solutions of amino acids by ion exchange

Abstract The removal of sodium citrate and phosphate buffers from amino acids in the effluent from ion exchange chromatograms can be accomplished by one-step desalting on basic or acidic resins. At “salt/amino acid” ratios of 1000 to 1, the amino acid recovery is quantitative. The neutral and acidic amino acids are adsorbed on a strong base resin (Dowex 2) and eluted with acetic acid. The basic amino acids and tryptophan are adsorbed on an acid resin (Dowex 50) and eluted with HCl. The resulting salt-free solutions are suitable for examination by paper chromatography or for the isolation of amino acids for further characterization.

Spectrophotometric assay of 2′,3′-cyclic nucleotide 3′-phosphohydrolase: Application to the enzyme in bovine brain

Summary Drummond et al. have shown that brain tissue is rich in an enzyme that hydrolyzes 2′,3′-cyclic nucleotides and that the amount of ribonuclease in the extracts is undetectable. The spectrophotometric method which has been used to measure the conversion of 2′,3′-cyclic cytidylate to the 3′-monophosphate by pancreatic ribonuclease has been adapted to measurement of the brain phosphodiesterase, which yields the 2′-monophosphate (2′-CMP). The spectral change at 286 nm is measured for 2–4 min on a recording spectrophotometer; the reproducibility of the assay is about 5%. The assay has facilitated study of some of the properties of the initially insoluble enzyme. The most efficient medium tested for extraction of the enzyme from an acetone powder has been 6% Tween 20-2 M guanidinium chloride at pH 7.5. The enzyme in such extracts has been gel-filtered on Sepharose 4B to give a 3-fold increase in specific activity (to 18.6 μmoles of 2′-CMP/mg protein/min against 1 mM 2′,3′-cyclic cytidylate); the position of emergence corresponds to that of a globular protein of mol. wt. 100,000. The pH optimum of the extracted enzyme is at pH 6.0. The K m of 2′,3′-cyclic cytidylate is 0.8 mM. The enzyme preparation is inhibited about 50% by a variety of mononucleotides (2′-AMP, 3′-AMP, 5′-AMP) at mM concentrations similar to that of the substrate. The results emphasize the question as to whether 2′,3′-cyclic nucleotides represent the natural substrates for this very active catalyst which several investigators have now shown is most closely associated with the myelin fractions from brain.

Column chromatography of peptides and proteins.

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.

13 Ribonuclease T1

Publisher Summary Ribonuclease T 1 was discovered by Sato and Egami as a major ribonuclease in Takadiastase, a commercial enzyme mixture from Aspergillus oryzae . This chapter traces the history between 1970 and 1980 on the chemistry of RNase T 1 and its biochemical applications. The most well recognized use of RNase T 1 is the sequence determination of RNA. The base specificity of RNase T 1 makes it a key enzyme in researches on the sequences of RNAs. Complete digestion with RNase T 1 yields oligonucleotides ending in guanosine 3′-phosphate; partial digestion is used to obtain information on the ordering of the segments. The enzyme is also used to synthesize guanylylnucleosides, oligoguanylates, and other guanosine-containing oligonucleotides with (3′-5′)-phosphodiester bonds. Affinity chromatography is used for the preparation of RNase T 1 . Hofmann and his colleagues showed the total synthesis of RNase T 1 by a solution method based on fragment condensation. They considered the peptide chain of the enzyme in terms of seven fragments, which were synthesized by stepwise procedures.

Pancreatic Ribonuclease: Enzymic and Physiological Properties of a Cross-Linked Dimer

Monomeric ribonuclease A has very low activity toward typically double-stranded RNAs; the dimeric form of ribonuclease A obtained by cross linking the enzyme by dimethyl suberimidate has more than 78 times the activity of the monomer toward polyadenylate � polyuridylate and 440 times the activity of the monomer toward the double-stranded RNA of a virus from Penicillium chrysogenum. The half-life of the dimer in the bloodstream of the rat is 12 times that of the mononmer.

THE USE OF SPECIFIC PRECIPITANTS IN THE AMINO ACID ANALYSIS OF PROTEINS

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