Charles J. Gray

β-d-glucosidase chemically bound to microcrystalline cellulose☆

Abstract Almond β- d -glucosidase reacts with cellulose carbonate to give an enzymically active, insoluble, enzyme derivative. The coupling reaction is pH-dependent, the optimum being at pH ∼7.8. The bound enzyme is more stable than the free form to incubation at 37°. The pH-activity profile for the bound is similar to that for the free enzyme.

Photooxidation of glucoamylase I from aspergillus niger

Abstract Photooxidation of glucoamylase I in the presence of methylene blue results in the abolition of the enzymic activity. The process is pH- dependent, being more rapid at high pH. Loss of activity appears to be associated with destruction of tryptophan residues. The pH- dependence of the process is interpreted in terms of a conformational change undergone by the enzyme resulting in exposure of tryptophan residues which may be essential either directly for the activity or for the maintenance of the active three-dimensional structure.

Tryptophanyl and carboxylic acid residues in the a centre of glucoamylase I from Aspergillus niger

The pH-dependence of the photo-oxidation of L-tryptophan, in the presence of Rose Bengal and Methylene Blue, has been investigated. True, initial rate constants were determined in order to circumvent errors due to secondary processes. Photo-oxidation of glycoamylase I from A. niger in the presence of Methylene Blue or Rose Bengal resulted in a pH-dependent loss of enzymic activity, which was analogous to the destruction of free L-tryptophan during photo-oxidation. The loss of enzymic activity was closely associated with the destruction of tryptophan residues in the enzyme. Significant protection of both enzymic activity and tryptophanyl residues in the enzyme molecule was achieved by performing the photo-oxidation in the presence of maltose, which is a substrate for the enzyme. The tryptophanyl residues of glucoamylase I, which had been inactivated by reaction of its carboxylic acid residues with glycine methyl ester in the presence of a water-soluble carbodi-imide, were also substantially protected by maltose. It is concluded that the active centre of glucoamylase I is a cleft lined with tryptophanyl residues that participate in the binding of the substrate. One or more carboxylic acid residues are involved in bond cleavage.

A new and convenient method for enzyme insolubilisation using diazotized m-diaminobenzene

Abstract A convenient method for enzyme insolubilization is described, which involves diazotization of m- diaminobenzene in the presence of a solid support and exposure of the resultant material to a solution of the enzyme. Optimum conditions for the attachment of β- d -glucosidase (EC 3.2.1.21) to cellulose have been established. The method is successful for a wide range of enzymes and a number of different solid supports.

Preparation and properties of a conjugate containing dextranase and concanavalin A

Abstract Conditions for the reaction of concanavalin A and dextranase with glutaraldehyde have been established to give a soluble, intermolecularly cross-linked conjugate possessing both dextranase and concanavalin activities. Evidence is presented that the dextranase and concanavalin molecules are linked to each other in the conjugate. The conjugate gives a different pattern of hydrolysis products on incubation with dextran than does dextranase.

Action of almond β-d-glucosidase on fluorogenic substrates derived from 4-substituted 7-hydroxycoumarins

Abstract A number of fluorogenic 4-substituted derivatives of 7-β- d -glucopyranosyloxycoumarin have been synthesized, and their hydrolysis by almond β- d -glucosidase has been examined. Comparisons have been made with the enzymes action on the β- d -galacto-epimers of these fluorogens. The carboxylic acid derivative 7-β- d -glucopyranosyloxycoumarin-4-acetic acid is recommended as the substrate of choice for the fluorogenic assay of β- d -glucosidase because of its higher solubility and higher V max /K m ration than the other substrates. The results also suggest that the enzyme possesses a large hydrophobic aglycone-binding site, at the edge of which is a positively charged group that binds firmly to the carboxylate group of this optimum substrate.

Absorption of a dextranase-concanavalin a conjugate on to hydroxyapatite

Abstract Dextranase was only weakly absorbed on to hydroxyapatite while concanavalin A was strongly absorbed. A conjugate of the two proteins was absorbed strongly and while absorbed had substantial dextranase activity. Thus, the absorption of dextranase was enhanced by incorporating it into a conjugate with concanavalin A.

Quantitative injections or calibrations in gas chromatography: Anomaly associated with syringe type

When syringes with open needles are used for quantitative injections, e.g. to calibrate gas chromatographic peak areas against the volume of liquid injected, the resultant plot may display an intercept. Current studies on cyclohexane have shown that this effect is so severe that it can make the major contribution to the total signal. Since the magnitude of the anomaly increases with the volatility of the liquid, this means that calibrations of liquid mixtures, or the use of an internal standard, will be unreliable when moderately volatile components are injected with syringes of the above type. The problem is shown to be caused by volatilisation from the needle whilst it is inserted in the heated injection port, and is exacerbated by increasing the temperature of the port or increasing the residence time of the needle. The anomaly is totally avoided if the calibration is performed using a syringe of the type in which the piston passes down the needle. Somewhat similar effects to the ones described can also arise for other reasons, and literature references to such situations are given.

7-Hydroxycoumarin-4-acethydrazide: A fluorescent derivatizing reagent for aldehydes and ketones

Abstract This paper describes a simple synthesis of 7-hydroxycoumarin-4-acethydrazide and use of the compound as a convenient and sensitive fluorophoric derivatizing reagent for aldehydes and ketones. Experiments were carried out to study the absorbance and emission characteristics, and the stability in alkaline solution, of these derivatives. Restricted torsion was observed in some of the hydrazone derivatives and was studied by variable temperature NMR spectroscopy.

Chemico-biological relationships of follicle stimulating hormone-releasing hormone and luteinizing hormone-releasing hormone.

Abstract Photo-oxidation studies of FSH-RH/LH-RH showed that at least one of the aromatic residues in the structure was essential for hormonal activities. A theory of internal sensitisation is invoked to explain the observation that only oxygen, and not added sensitiser, was essential for the photo-oxidative reaction to proceed. From the results of derivatisation of the hormone molecule with formaldehyde and t-butylazidoformate, it was shown that the guanido group is essential for biological activity. That these derivatisations could have involved a free amino group generated from the pyroglutamyl N-terminus was discounted in view of the failure of the hormone to react with maleic and citraconic anhydrides.