Francis E. Dwulet

Purification and characterization of undegraded human ceruloplasmin

Abstract Human ceruloplasmin, which is usually cleaved by limited proteolysis into three major fragments during preparation ( M r ⋍ 18,650 , 50,000, and 70,000) was isolated in good yield as an undegraded single-chain protein ( M r ⋍ 135,00 ). The cryosupernatant from fresh frozen plasma (100 liters) was fractionated with polyethylene glycol (PEG 4000) at + 5°C yielding a ceruloplasmin-enriched fraction in the 20% PEG supernatant. Three steps of chromatography on DEAE-Sephacel, hydroxyapatite, and Sephadex G-200 produced a homogeneous protein with maximal enzymatic activity and the A 610 A 280 ratio of 0.046 corresponding to 98–100% purity. Two forms of ceruloplasmin having this absorbance ratio were obtained; Form I was predominant and was studied further. The procedure separated both forms from apoceruloplasmin and degraded ceruloplasmin. The single-chain ceruloplasmin (Form I) had an NH2-terminal sequence of Lys-Glu-Lys-His-Tyr-Tyr-Ile-, the same as for the 70,000 fragment, and is suitable for structural study by sequence analysis and physicochemical methods.

Evolution of the amino acid substitution in the mammalian myoglobin gene

SummaryMultivariate statistical analyses were applied to 16 physical and chemical properties of amino acids. Four of these properties; volume, polarity, isoelectric point (charge), and hydrophobicity were found to explain adequately 96% of the total variance of amino acid attributes. Using these four quantitative measures of amino acid properties, a structural discriminate function in the form of a weighted difference sum of squares equation was developed. The discriminate function is weighted by the location of each particular residue within a given tertiary structure and yields a numerical discriminate or difference value for the replacement of these residues by different amino acids. This resulting discriminate value represents an expression of the perturbation in the local positional environment of a protein when an amino acid substitution occurs. With the use of this structural discriminate function, a residue by residue comparison of the known mammalian myoglobin sequences was carried out in an attempt to elucidate the positions of possible deviations from the known tertiary structure of sperm whale myoglobin. Only 11 of the 153 residue positions in myoglobin demonstrated possible structural deviations. From this analysis, indices of difference were calculated for all amino acid exchanges between the various myoglobins. All comparisons yielded indices of difference that were considerably lower than would be expected if mutations had been fixed at random, even if the organization of the genetic code is taken into consideration. On the basis of these results, it is inferred that some form of selection has acted in the evolution of mammalian myoglobins to favor amino acid substitutions that are compatible with the retention of the original conformation of the protein.

A comparison of sulfonated phenylisothiocyanates for reducing losses of lysine-containing peptides during automated sequencing

Abstract A simple procedure has been devised to couple lysine-containing peptides with 3-sulfophenylisothiocyanate or with 4-sulfophenylisothiocyanate and then to subject them to automated Edman degradation. The better repetitive yields in all cases were obtained with the 3-sulfophenylisothiocyanate. The reasons for these observations are explained and an improved method of synthesis of the reagent is given.

Complete amino acid sequence of the myoglobin from the pacific spotted dolphin, Stenella attenuata graffmani

The complete amino acid sequence of the major component myoglobin from the Pacific spotted dolphin, Stenella attenuata graffmani, was determined by the automated Edman degradation of several large peptides obtained by specific cleavage of the protein. The acetimidated apomyoglobin was selectively cleaved at its two methionyl residues with cyanogen bromide and at its three arginyl residues by trypsin. By subjecting four of these peptides and the apomyoglobin to automated Edman degradation, over 80% of the primary structure of the protein was obtained. The remainder of the covalent structure was determined by the sequence analysis of peptides that resulted from further digestion of the central cyanogen bromide fragment. This fragment was cleaved at its glutamyl residues with staphylococcal protease and its lysyl residues with trypsin. The action of trypsin was restricted to the lysyl residues by chemical modification of the single arginyl residue of the fragment with 1,2-cyclohexanedione. The primary structure of this myoglobin proved to be identical with that from the Atlantic bottlenosed dolphin and Pacific common dolphin but differs from the myoglobins of the killer whale and pilot whale at two positions. The above sequence identities and differences reflect the close taxonomic relationship of these five species of Cetacea.

Identification of 2-thiohydantoins by gas chromatography and reconversion to the free amino acids

Abstract In this work the chemistry of identifying 2-thiohydantoins was investigated. The minimum conditions needed for silylating the 2-thiohydantoins were studied, and a new faster gas chromatographic temperature program was developed. Also, the conditions needed to reconvert the 2-thiohydantoins to the free amino acids were studied, and the severity of the hydrolysis conditions was minimized. Finally, some new 2-thiohydantoins were prepared, and their physical properties are reported.

Complete amino acid sequence of the major component myoglobin from the goose-beaked whale, Ziphius cavirostris.

The complete primary structure of the major component myoglobin from the goose-beaked whale, Ziphius cavirostris, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Over 80% of the amino acid sequence was established from the three peptides resulting from the cleavage of the apomyoglobin at its two methionine residues with cyanogen bromide along with the four peptides resulting from the cleavage with trypsin of the citraconylated apomyoglobin at its three arginine residues. Further digestion of the central cyanogen bromide peptide with S. aureus strain V8 protease and the 1,2-cyclohexanedione-treated central cyanogen bromide peptide with trypsin enabled the determination of the remainder of the covalent structure. This myoglobin differs from the cetacean myoglobins determined to date at 12 to 17 positions. These large sequence differences reflect the distant taxonomic relationships between the goose-beaked whale and the other species of Cetacea the myoglobin sequences of which have previously been determined.

Complete amino acid sequence of the major component myoglobin from Hubb's beaked whale, Mesoplodon carlhubbsi☆

The complete primary structure of the major component myoglobin from Hubbs beaked whale, Mesoplodon carlhubbsi, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. In all experiments a Beckman 890C automatic sequencer was used to degrade the peptides. A cyanogen bromide digest was used to fragment the protein at its two methionine residues into three fragments which were separated by gel filtration. In a similar pattern the protein was citraconylated to protect the lysine residues and the modified protein fragmented at its arginine residues with trypsin. The three peptides obtained here were also purified by gel filtration. Sequencer analysis of the whole apoprotein, cyanogen bromide fragments and the peptides cleaved at the arginine residues with trypsin provided 80% of the completed sequence. The remainder of the sequence was obtained by digesting the middle cyanogen bromide fragment with staphylococcal protease and by total tryptic digestion of the whole apoprotein and isolating the resulting peptides by ion-exchange chromatography. The primary structure of the Mesoplodon carlhubbsi myoglobin represents a sequence which may be closey related to the ancestral sequence of all cetacean myoglobins.