William H. Scouten

N-dansylaziridine: A new fluorescent modification for cysteine thiols

A new fluorescent reagent, N-dansylaziridine (I), which reacts selectively with protein thiols, has been synthesized. This reagent is readily prepared from the reaction of dansyl chloride (1-dimethyl aminonaphthalene-5-sulfonyl chloride) with an equimolar quantity of ethyleneimine in the presence of triethylamine. N-Dansylaziridine reacts at pH 6.5–8.6 with cysteine, mercaptoethanol and other sulfhydryl-containing compounds, but shows little or no reactivity toward weaker nucleophiles such as amines and alcohols. When proteins containing free sulfhydryls are reacted with 5% N-dansylaziridine suspended on cellulose, fluorescent dansylated proteins result, while proteins containing no free sulfhydryl groups are not derivatized. When N-dansylaziridinyl proteins are subjected to mild acid hydrolysis (105 C, 4 h, in 6.6 M HCl), the only hydrolysis product formed is S-(2-dansylamino-ethyl)-cysterine. Upon vigorous hydrolysis (110 C, 18 h) another product, dansyl taurine, results. The latter is formed by oxidative degradation of the former.

Purification of lipoamide dehydrogenase by affinity chromatography on propyllipoamide-glass columns

Abstract Lipoamide dehydrogenase (EC 1.6.4.3) from pig heart, yeast and Escherichia coli has been purified by chromatography on columns of propyllipoamide-glass beads. Chromatography of crude or partially purified lipoamide dehydrogenase on these columns has affected up to 100-fold purification of the enzyme in less than 30 min.

Photooxidation of methionine with immobilized methylene blue photooxidizer

Methylene blue immobilized on porous glass beads was used to catalyze the photooxidation of methionine alone and the methionine residues of lysozyme. A solution of 2 mM methionine in 50% acetic acid was oxidized to methionine sulfoxide in the presence of immobilized methylene blue after 6 h of photooxidation at 37 degrees C. Selective photooxidation of the methionyl residues in lysozyme was achieved after 26 h of reaction in 84% acetic acid at 4 degrees C. The specific activity of lysozyme exposed to light in the presence of methylene blue decreased by 94%, while that of a lysozyme solution in the presence of methylene blue not exposed to light decreased by 21%. The lysozyme solution exposed to light but not containing the methylene blue beads lost 33% of its specific activity after the same period of photooxidation. It was shown that the decrease in enzyme activity was not caused by adsorption of the enzyme onto the beads.

N-Propyldihydrolipoamide glass beads An immobilized reducing agent

Abstract N -Propyllipoamide-controlled pore glass has been synthesized and utilized as a potential immobilized reducing agent. It has been shown to be effective in reducing glutathione, cysteine and thiolated gelatin. N -Propyllipoamide glass can also be employed to activate papain (EC 3.4.4.10). However, after activation a substantial percentage of the papain activity remains non-covalently bound to the N -propyllipoamide glass. The attraction between the glass derivative and papain appears to be hydrophobic and can be reduced by the addition of non-ionic detergents. N -Propyllipoamide glass has also been used for the activation of mercuripapain and as an immobilized chelation agent with specificity toward heavy metal ions, e.g. Hg 2+ , Pt 2+ , Au 3+ .

Immobilized Protein Modification Reagents

Solid phase biochemistry is a rapidly growing area including immobilized enzymes, affinity chromatography, protein synthesis and protein sequencing. The research here describes a new potential application of solid phase reactions to biochemistry, namely the use of immobilized protein modification reagents. These reagents offer the same increased ease of handling and improved yields of most other solid phase applications in biochemistry. In addition, they may, in many cases, be used as topology probes, since the reaction of immobilized reagents with proteins is often sterically restricted to the surface of the protein. A small number of such immobilized protein reagents have appeared in the literature. Rose Bengal (1) and methylene blue (2,3) have been employed as immobilized photocatalysts, while immobilized α-keto halides (4) have been employed in the selective cleavage of protein methionyl residues.

Lipoamide dehydrogenase immobilized on porous glass

Lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) isolate from pig heart and Escherichia coli was covalently coupled by both diazonium and amide bonds to controlled pore glass beads (96% silica). When the enzyme was immobilized in the presence of NAD+, the enzyme no longer exhibited its normal requirement for NAD+ for full activity. If the immobilized enzyme was then treated with NADase, the requirement for NAD+ was restored. Enzyme immobilized in the absence of NAD+ exhibited normal NAD+ dependence both prior to an after NADase treatment. These results are discussed in terms of co-immobilization of NAD+ at or near the allosteric site of the enzyme.

Immobilized Modifiers for Proteins (IMPs)

A wide series of insolubilized protein modification reagents have been synthesized and applied to the modification and/or purification of a variety of amino acids, model proteins, and cell membranes. These materials possess sterically hindered protein modification reagents which have potential applicaiton as protein topology probes.