Ligand-affinity sedimentation equilibrium using an air-driven ultracentrifuge

An air-driven ultracentrifuge has been used to study the distribution of radioactive ligands at sedimentation equilibrium. In the presence of a suitable acceptor and under conditions where the ligand is essentially all bound the distribution of ligand can be analyzed to yield the molecular weight of the acceptor molecule. Suitable conditions can be chosen either experimentally by measuring the ratio of bound ligand compared to unbound ligand or theoretically for systems in which the ligand-binding affinity and number of acceptor binding sites is known. The method is applicable to the molecular characterization of binding proteins in crude mixtures and results are presented for the binding of various fatty acids to serum albumin samples.     

Binding of metal ions to apoalkaline phosphatase from E. Coli: Effect of ionic radius

Binding of metal ions to $\text{E. Coli}$ apoalkaline phosphatase causes 1) chromophoric changes in tyrosine absorption, 2) changes in enzymatic activity and 3) the release of protons from the enzyme. Investigation of these effects for a selection of metal ions from the Group 11A, Group 11B and transition series revealed that only those ions having crystal ionic radii in the range of 0.72 – 0.99A are able to produce changes in these three properties. The hydrated ionic radii, which are in the range of 4.0 – 4.5A for the ions examined do not correlate well with the ability of the ion to affect the three properties studied here. The size requirement therefore would seem not to apply to the initial binding step which undoubtedly involves the hydrated ion. Rather, the size requirement reflects the size range of a binding site generated by the folding of the protein around the metal ion with concomitant displacement of water molecules from the coordination sphere of the metal.     

Preparative separation of DNA-ethidium bromide complexes by zonal density gradient centrifugation

Ethidium bromide-DNA complexes separated by rate-zonal sedimentation through a density gradient can be readily visualized and purified with little cross-contamination. The method is simple, rapid, and efficient.     

Purification of reticulocyte ribosomes using polyuridylic acid: sepharose chromatography.

Deoxycholate-KCl washed reticulocyte ribosomes are purified by affinity chromatography using Sepharose columns to which polyuridylic acid has been covalently bound. Upon passage through the column under nonenzymic conditions (high Mg²⁺:K⁺ ratio) approximately 10% of the ribosomes are retained. These ribosomes are then eluted with a buffer containing a high K⁺:Mg²⁺ ratio and are assayed for activity in various steps of the elongation process of protein synthesis. Approximately a 3- to 4-fold increase in the various activities compared with control ribosomes is obtained.     

Cu2+ probe of metal-ion binding sites in melanin using electron paramagentic resonance spectroscopy. II. Natural melanin

The isotope ⁶³Cu²⁺ has been used to probe the metal-ion binding sites of natural melanin from the choroid of bovine eyes using electron paramagnetic spectroscopy. Samples were in aqueous media over a wide range of pH values. At pH < 7, binding is to monodentate carboxyl complexes and to bidentate nitrogen-carboxyl complexes just as in synthetic melanin. At pH > 7 binding is to phenolic hydroxyl groups, but the number of such sites is much less than in synthetic melanin and there are indications of a superimposed spectrum of another site. At high pH, a signal unlike any found in synthetic melanin was observed with either three or four nitrogen ligands. A number of experiments indicate that natural melanin is 50% protein by weight. Metal-ion binding sites are the same with and without protein although with some differences in relative populations.     

Cu2+ probe of metal-ion binding sites in melanin using electron paramagnetic resonance spectroscopy. I. Synthetic melanins

The isotope ⁶³Cu²⁺ has been used to probe the metal-ion binding sites of synthetic (autoxidized) catechol and 3,4-dihydroxyphenylalanine melanins using electron paramagnetic resonance spectroscopy. Samples were in aqueous media over a wide range of pH values. Assignments of the structures of the melanin-copper complexes are based in part on model studies of the complexes formed with melanin precursors, catechol and 3,4-dihydroxyphenylalanine, and with phenanthroline. Nearly all complexes involve just one or two ligands from melanin. In catechol melanin below pH 5.0, complexes with carboxyl groups are formed; above 6.0, Cu²⁺ forms complexes with phenolic hydroxyl groups. These same complexes were found in 3,4-dihydroxyphenylalanine melanin and binding of Cu²⁺ at amino acid type sites also was detected. After partial reduction of copper ions bound to 3,4-dihydroxyphenylalanine melanin, a weak signal of copper with four melanin ligands (oxygen and nitrogen in various combinations) was observed.     

Identification of enzyme coupling sites with aromatic diazonium salts-a resonance raman study.

The coupling reaction of diazonium salts of aromatic compounds with the aromatic residues of proteins results in chromophoric covalent derivatives which yield strong resonance enhanced Raman spectra. The protein residues modified by these coupling reactions have been identified using the ν(NN) and ν(N-φ) vibrational bands in the resonance Raman spectra. Previous studies have established that diazoarsanilic acid couples with carboxypeptidase at tyrosine 248. The resonance Raman spectrum of arsanilazocarboxypeptidase was compared with spectra of arsanilazotyrosine and arsanilazohistidine model compounds; the results are consistent only with coupling at a tyrosine residue. This confirmation of the previously established site of modification establishes the utility of resonance Raman spectroscopy as a tool for identification of the site of covalent modification. To further investigate this approach, the diazonium salt of sulfanilamide (a site-specific reagent) was used to prepare a covalent coupling derivative of bovine carbonic anhydrase. The coupling reaction appears to have a stoichiometry of 1:1 and results in nearly complete loss of sulfanilamide binding capability and esterase activity. Comparison of the pH dependence of the resonance Raman spectra of sulfanilazocarbonic anhydrase with the spectra of sulfanilazotyrosine, sulfanilazohistidine, and sulfanilazotryptophan suggests that histidine is the site of modification of this new carbonic anhydrase derivative.     

Subsite mapping of enzymes. Use of subsite map to simulate complete time course of hydrolysis of a polymeric substrate.

In this paper we extend our earlier work on subsite mapping and show that our model for depolymerase action can be used to accurately predict product ratios vs the extent of reaction when a polymer is hydrolyzed. The experimental product ratios for Bacillus amyloliquefaciens α-amylase acting on reducing end-labeled ¹⁴C-maltodextrins ranging in chain length 3 to 10 are reported. These data and Michaelis parameters are used with a depolymerase computer model (J. D. Allen, 1977, Ph.D. thesis, University of Arkansas; J. D. Allen and J. A. Thoma, 1976, Biochem. J.159, 105) to compute an optimized subsite map. The depolymerase computer model generates a 10-subsite map for B. amyloliquefaciens α-amylase with the catalytic site located to the left of subsite 7. The binding affinities of the subsites are then used as the sole input in another computer program to quantitatively predict the mole fraction of products vs the extent of hydrolysis for substrates of varying chain length. Excellent agreement is obtained between the computed and experimental data for seven maltodextrins examined.     

Quantitative determination of myeloperoxidase using tetramethylbenzidine as substrate

Tetramethylbenzidine, a noncarcinogenic, nonmutagenic derivative of benzidine, has been used as a substrate to assay myeloperoxidase. The assay is sensitive to 0.1 μg of enzyme and can be used to quantitate myeloperoxidase over a pH range of 4.4 to 7.4.     

Violaxanthin de-epoxidase. Lipid composition and substrate specificity.

Violaxanthin de-epoxidase isolated from lettuce chloroplasts (Lactuca sativa var. Romaine) contained a single lipid component, monogalactosyldiglyceride (MG) at about 8 g per 100 g protein. The effects of MG on activation of solvent-extracted enzyme and on K_m suggest that MG has two roles, namely, as a functional component of the binding site and as a substrate-solubilizing agent whose structure satisfies binding site requirements. Substrate specificity examined with various naturally occurring and semisynthetic epoxy carotenoids with known chirality showed violaxanthin de-epoxidase to be stereospecific for 3-hydroxy, 5,6-epoxy carotenoids which are in a 3S, 5R, 6S configuration. Although monoepoxides with the above configuration were active, their rates varied, apparently due to the influence of structural differences in the nonepoxide end groups. Hence while all-trans neoxanthin showed low rates, the de-epoxidation rate of antheraxanthin was 5-fold higher than violaxanthin. Neoxanthin and violeoxanthin, both naturally occurring pigments with 9-cis configurations in the acyclic polyene chain, were inactive. These effects support the view that violaxanthin de-epoxidase is a mono de-epoxidase and that the stereospecific active center is situated in a narrow well-like cavity which favors an all-trans configuration of the polyene chain. The 3-hydroxy, 5,6-epoxy group of the naturally occurring pigments, diadinoxanthin, antheraxanthin, and β-cryptoxanthin epoxide are assumed to be the 3S, 5R, 6S configuration based on their reactivity with violaxanthin de-epoxidase.     

The formation of unhydrated propionaldehyde by dioldehydrase.

When dl-1,2-propanediol is converted to propionaldehyde by dioldehydrase, an enzyme which requires B₁₂-coenzyme, the product is unhydrated propionaldehyde. Its formation was demonstrated by measuring the rate of reduction of the enzymically formed aldehyde by NADH, catalyzed by yeast alcohol dehydrogenase.     

Study of x-irradiated DNA using formamide gradients

The results of these studies have revealed no differences in the level of the cyclic-3′,5′-AMP (cAMP) -dependent or independent protein kinases, using calf thymus histone as substrate, in normal and feline sarcoma virus transformed cells. Similarly, the degree of responsiveness of the basal protein kinase activity to cAMP was also identical in the two cell types. These experiments have been carried out in normal, bovine-derived (thymic) fibroblasts and confirmed in feline-derived, embryonic mixed cell cultures. Thus, these results are consistent with the conclusion that one of the major amplification mechanisms for cAMP is not altered following viral transformation.     

Kinetics of reaction zone formation with radial diffusion of ligands over a receptor-coated surface

Theoretical calculations are presented, describing the kinetics of reaction zone formation with radial diffusion of ligands over a receptor coated surface. Calculated concentration distributions of ligands diffusing radially over a receptor-coated surface are combined with different types of receptor-ligand reactions, taking place at the surface, in order to obtain theoretical relations between the initial concentration of ligand in the source, the diameter of the receptor-ligand reaction zone and reaction time. These relations are compared to experimental data, using bovine serum albumin (BSA) as immobilized receptor and anti-BSA antibodies as diffusing ligand. The theory predicts how the diffusion constant of the ligand and the detection level of the visualization method may be determined and how to discriminate between different kinetics of the receptor-ligand reaction. The practical use of the theory in experimental studies of receptor-ligand interaction is discussed.     

A sensitive assay of galactocerebroside beta-galactosidase by high-performance liquid chromatography using galactosyl-N-dansyl-sphingosine as substrate

A rapid and sensitive method was devised for determining β-galactosidase activity specific for galactocerebroside. A fluorescent derivative of galactocerebroside, 1-O-galactosyl-2-N-1-dimethylaminonaphthalene-5-sulfonyl-sphingosine, was used as substrate, and the product, 2-N-1-dimethylaminonaphthalene-5-sulfonyl-sphingosine, was taken into organic solvent phase. Quantitative analysis of 2-N-dimethylaminonaphthalene-5-sulfonyl-sphingosine was carried out fluorometrically by use of high-performance liquid chromatography on silica gel column.     

The production of oxyluciferin during the firefly luciferase light reaction.

The luciferase-product complex (E · P) was isolated from the reaction mixture after light emission had occurred. The spectral properties of the product in the E · P complex are similar to those of oxyluciferin, with a broad absorption at 385 nm. The enzyme from the complex regains full activity upon the addition of substrates. The product is not covalently bound to the enzyme and readily dissociates in the presence of 6 m urea. The isolated E · P complex was found to have 1 mol of oxyluciferin per 100,000 daltons of luciferase. No AMP could be detected in the E·P complex unless inorganic pyrophosphatase was present during the reaction. In that case 1 mol of AMP per 100,000 daltons was found.Stopped flow studies showed that an increase in 385 nm absorption occurred concomitant with light emission. Measurement of the initial rate of product formation and the rate of photon emission showed they were identical, suggesting that oxyluciferin is indeed the light-emitting product. In the initial burst of the reaction two oxyluciferin moles per 100,000 daltons of luciferase are formed. A plot of the log of the initial rate of product formation was biphasic, indicating that the first mole of product is formed at a faster rate than the second. These results are consistent with previous experiments. However, they do not resolve the question of the molecular weight of the catalytically active species.