Report on Mr. William Burk's plan for propelling Canal Boats

Abstract

The latest results of physicochemical techniques in protein chemistry have led to a better knowledge of the normal G-6-PD structure and also to a determination of the exact centre of the mutation for usual variants (Gd(+)A/variant) or variants with high activity (Hektoen variant).However these methods are still unapplicable when studying the unusual enzymes often with very weak activity. Yet in these cases the kinetic and thermodynamical study not only affords a better understanding of the functional abnormality of the enzymatic protein but sometimes allows to suggest a modification of some functional groups.We have investigated normal G-6-PD (Gd(+) B, Gd (+) A, a slow variant without deficiency (Gd (+) Madrona type); 10 deficient enzymes of the Gd (− A) type. By these methods, we showed three new types of G-6-PD: two deficient variants called Gd (−) Mali (in four people of that country) and Gd (−) Dakar (in two Senegalese); one variant without deficiency and with increased thermolability (in a subject from Martinique).Beyond the determination of usual kinetic constants (Michaelis constant for substrates and substrate analogues, activity versus pH, thermostability), and starch gel electrophoresis, the methods used by the authors to study G-6-PD were the following:o1.–The inactivation kinetics by parahydroxymercuribenzoate and the protective effect of various ligands on this inactivation, which occasionally allows the location of reactive sulfhydrile groups towards the fixation sites of these ligands. The inactivation which is normal for the Gd (+) A, Gd (+) B and Gd (−) A types, appeared to be slightly decreased for Madrona variant and strongly enhanced for the two new reported deficient types.2.–The activation energy of the heat inactivation reaction calculated from the velocity constant of this heat inactivation reaction at various temperatures: it goes from 80 to 100 K Cal per mole for normal enzymes and it increases for the other reported types in which it gives homogen groups without any overlap. It is always greater than 120 K Cal/mole in the Gd (−) A variants.3.–The thermostability in presence of substrates: the stability conferred by the substrates is a function of enzyme-substrate binding energy and must depend on the Michaelis constant. This has been checked for the variants with increased affinity for G-6P.4.–The «Log V max and pKm-G-6P = f (pH) functions calculated from Lineweaver and Burk's representation, between pH 5,5 and 10 at saturating concentrations in substrates and in organic buffers without inhibitor ions. These curves, up to now, were only plotted for Gd (+) B, Gd (+) A, Gd (−) A and Gd (+) Madrona type enzymes. They allow to study the ionization pK of one the ionizing groups in the catalytic site, found at pH 6.5 ± 0,5 and which may correspond to an imidazole radical. They also show the influence of the ionic strength of the concentration in MgCl2, and of other agents on the enzymic kinetics.Consequently, this kind of work requires small amounts of enzymic material and can bring meaningful informations about molecular mechanism of many enzymatic diseases even without any possibility of physicochemical and structural analysis.