Crude preparations

Crude preparations of mescaline (61) from peyote were first reported by the Spanish as they learned of its use from the natives of Mexico during the Spanish invasion of that country in the sixteenth century. The colorful history (44) of mescaline has drawn attention to its use as a hallucinogen and even today it is in use among natives of North and South America. Although in connection with dmg abuse complaints, mescaline is considered dangerous, it has been reported (45) that it is not a narcotic nor is it habituating. It was also suggested that its sacramental use in the Native American Church of the United States be permitted since it appears to provoke only visual hallucination while the subject retains clear consciousness and awareness. 

Luciferase-catalyzed luminescence of luciferin. Odontosyllis luciferin emits light in the presence of Mg2+, molecular oxygen and luciferase. The relationship between the luminescence intensity and the pH of the medium shows a broad optimum (Fig. 7.2.8). The luminescence reaction requires a divalent alkaline earth ion, of which Mg2+ is most effective (optimum concentration 30 mM). Monovalent cations such as Na+, K+, and NH have little effect, and many heavy metal ions, such as Hg2+, Cu2+, Co2+ and Zn2+, are generally inhibitory. The activity of crude preparations of luciferase progressively decreases by repeated dialysis and also by concentrating the solutions under reduced pressure. However, the decreased luciferase activity can be completely restored to the original activity by the addition of 1 mM HCN (added as KCN). The relationship between the concentration of HCN and the luciferase activity is shown in Fig. 7.2.9. Low concentrations of h and K3Fe(CN)6 also enhance luminescence, but their effects are only transient. 

Enzyme-mediated chiral sulfoxidation has been reviewed comprehensively in historical context [188-191]. The biotransformation can be mediated by cytochrome P-450 and flavin-dependent MOs, peroxidases, and haloperoxidases. Owing to limited stability and troublesome protein isolation, a majority of biotransformations were reported using whole-cells or crude preparations. In particular, fungi have been identified as valuable sources of such biocatalysts and the catalytic entities have not been fully identified in all cases. 

Rapidase C-80 (Gist -brocades) was used as enzyme source. The fractionation procedure of the crude preparation included chromatography on Bio-Gel PIO (100-200 mesh), DEAE-Bio-Gel A, and Bio-Gel HTP (Bio-Rad, Richmond, CA, USA). Other column materials used were cross-linked alginate (degree of cross-linking 2.34, prepared in our laboratory). Phenyl Superose HR 5/5 and Mono Q HR 5/5 (Pharmacia Biotech, Uppsala, Sweden). 

Editor s Note This was a crude preparation probably containing many other... 

Hernandez, L., Luna, H., Rulz-Teran, F. and Vazquez, A. (2004) Screening for hydroxynitrile lyase activity in crude preparations of some edible plants. Journal of Molecular Catalysis B-Enzymatic, 30, 105-108. 

Hofrichter, M., and Fritsche, W., Depolymerization of Low-Rank Coal by Extracellular Fungal Enzyme Systems. 3. In Vitro Depolymerization of Coal Humic Acids by a Crude Preparation of Manganese Peroxidase From the White-Rot Fungus Nematoloma Frowardii B19. Applied Microbiology and Biotechnology, 1997. 47(5) pp. 566-571. 

Procedure Cholinesterase activity was measured according to the modified biochemical methods developed for crude preparations (Gorunef ah, 1978), using Ellman reagent 5,5"-dithio-bis(p-nitrobenzoic acid) or its red analogue 2,2-dithio-bis-(p-phenyleneazo)-bis-(l-oxy-8-chlorine-3,6) -disulfur acid in the form of sodium salt, which interact with thiocholine salt (Roshchina 2001). Water extracts of vegetative microspores of horsetail (Equisetum arvense) or Hippeastrum hybridum microspores (150 mg of microspores in 30 ml for 1 h) were used. 

Precautions The yellow colour of the crude preparation may interfere with the Ellman reaction product, that need the appropriate special control. 

Disadvantages Dark coloured materials, due to the presence of anthocyanins, in particular are not suitable for the assay without strong dilution of the crude preparation... 

Wellferon was the tradename given to one of the first such approved products. Produced by large-scale mammalian (lymphoblastoid) cell cultures, the crude preparation undergoes extensive chromatographic purification, including two immunoaffinity steps. The final product contains nine IFN-a subtypes. 

After initial cell fermentation and product extraction from the producer cells, the crude preparation is subject to multiple chromatographic steps, including ion-exchange, hydrophobic interaction chromatography and gel-filtration chromatography. The purified product is presented in lyophilized form in vials (1 mg active/vial) and excipients include a phosphate buffer, sodium chloride and serum albumin. 

By use of a crude preparation obtained after the cultivation of Aspergillus niger,104 pectinesterase was purified by repeated chromatography on DEAE-cellulose, using gradient elution. The homogeneity of the product was checked by free electrophoresis, sedimentation analysis, and determination of the N-terminal amino acid (phenylalanine). 

Effective and simple immobilization of enzymes can be obtained by the cross-linking of enzyme aggregates, so-called CLEAs [55]. In this way, essentially any enzyme, including crude preparations, can be transformed into a heterogeneous type of material, insoluble in both water and organic solvents, that is stable and recyclable with high retention of the enzyme s original activity [56], These enzyme preparations are, therefore, of special value for both bio-bio and bio-chemo cascade processes. 

The first of these new, electron transferring components was coenzyme Q (CoQ). Festenstein in R.A. Morton s laboratory in Liverpool had isolated crude preparations from intestinal mucosa in 1955. Purer material was obtained the next year from rat liver by Morton. The material was lipid soluble, widely distributed, and had the properties of a quinone and so was initially called ubiquinone. Its function was unclear. At the same time Crane, Hatefi and Lester in Wisconsin were trying to identify the substances in the electron transport chain acting between NADH and cytochrome b. Using lipid extractants they isolated a new quininoid coenzyme which showed redox changes in respiration. They called it coenzyme Q (CoQ). CoQ was later shown to be identical to ubiquinone. 

In pharmaceutical and biochemical research, crude preparations of material will often exhibit significant desired biological activity. However, that activity will sometimes be lost when the material is purified. Comment. 

In various runs, specific activity went from an average of 2.75 lU/mg protein (based on the Bio-Rad protein assay with bovine serum albumin standard) for Ae centrifuged crude preparation after alcohol precipitation, to 8 lU/mg after DEAE-cellulose chromatography, to 100 lU/mg after DEAE-Fractogel chromatography, to 1000 RJ/mg after Sephadex G-50 chromatography, and to 3200 lU/mg after CM-Trisacryl chromatography. 

In our laboratory crude preparations of aphantoxins and anatoxin-a(s) are extracted similarly except at the final stages of purification (Fig. 2). A Bio-gel P-2 column (2.2 x 80 cm) is used for aphantoxins gel filtration and a Sephadex G-15 (2.6 x 42 cm) column for ana-toxin-(s). Both toxins are eluted with 0.1 M acetic acid at 1.5 ml/ min. Fractions of aphantoxins from Bio-gel P-2 run are spotted on thin-layer chromatography plates (Silica gel-60, EM reagents) and developed according to Buckley et al. (1976) (31). The Rf values for the aphantoxins, saxitoxin and neosaxitoxin standards (Table 1) indicates that two of the aphantoxins (i.e. I and II) are similar to saxitoxin and neosaxitoxin. 

The neurotoxins isolated from Aph. flos-aquae were shown to have similar chemical and biological properties to paralytic shellfish poisons (PSP) (25,29,38) Sawyer et al. in 1968 (25) were the first to demonstrate that the crude preparation of aphantoxins behave like saxitoxin, the major paralytic shellfish poison. They showed that the toxins had no effect on the resting membrane potential of frog sartorius muscle blocked action potential on de-sheathed frog sciatic nerve and also abolished spontaneous contractions in frog heart. Sasner et al. (1981) (29) using the lab cultured strain reported similar results. 

Further confirmation of the similarities in biological activities between aphantoxin and PSP was shown by Adelman et al. (1982) (30). They showed that crude preparations of aphantoxins blocked the Na channel of giant squid axon with equal potency as saxitoxin. 

The metalloprotease thermolysin, obtained from Bacillus thermoproteolyticus, a strain of B. stearothermophilus, is used as a crude preparation in an aqueous medium. The enzyme is recovered from the reaction mixture by ultrafiltration with a yield of >95%. 

Efficient kinetic resolution of chiral unsaturated secondary alcohols by irreversible enzyme-mediated acylation (with vinyl acetate as acylating agent, a crude preparation of Pseudomonas AK, and hexane as solvent) is possible, provided one relatively large and one small substituent are attached to the carbinol carbon. However, the method can be used to resolve substrates that are not amenable to asymmetric epoxidation (see examples 23, 25, 27, 29, where the double bond is either deactivated by an electron-withdrawing substituent, or is of the propargyl alcohol type). Acylation of the / -enantiomer consistently proceeds faster than that of the 5-enantiomer. An example of an allenic alcohol was also reported248. 

Fig. 2.—Effect of Concentration of Substrate on the Rate of Hydrolysis4 of p-Nitro-phenyl a-D-Mannoside in Acetate Buffer, pH 3.5, at 37° by a Crude Preparation of Limpet a-D-Mannosidase in the Presence of , 0.1 mM ZnS04 and 0.1 M NaCl A, 0.1 M NaCl only and O, 0.1 mM ZnS04 only.

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