Hugo Theorell

Inhibitory effect on ethanol oxidation in man after administration of 4-methylpyrazole

Abstract The effect of 4-methylpyrazole on the metabolism of ethanol has been examined in 5 healthy male volunteers after oral administration of 5 μC ethanol-1- 14 C with a carrier dose of 9 g ethanol and measuring the 14 CO 2 excreted in the expired air. The degree of inhibition reached a maximum 1.5 – 2 hours after oral administration of 4-methylpyrazole and then the inhibitory effect decreased slowly. Thus 1000 mg of methylpyrazole inhibited the elimination of 14 CO 2 in expired with 49.6%, 1.5 hours after oral administration of the compound compared to the control experiment. The inhibitory effect decreased slowly to 28 per cent 6 hours after administration of methylpyrazole. The results of this investigation indicate a dose-response relationship between degree of inhibition and dose of 4-methylpyrazole. The results also suggest an individual response to 4-methylpyrazole. Experimental data with larger doses of alcohol to alcoholics suggest that 4-methylpyrazole acts as a competitive LADH-inhibitor. The data obtained in alcoholic volunteers suggest that 4-methylpyrazole might have an inhibitory effect on the ethanol induced rise in lactate/pyruvate ratio in blood.

Dissociation constants of the liver alcohol dehydrogenase coenzyme complexes

Abstract 1. 1. The fluorescence increase occurring when DPNH is bound to liver ADH has been used for determining the dissociation constant of ADH. DPNH at pH values from 5.3 to 10. The effects of formate and chloride were studied. 2. 2. The dissociation constant of ADH.DPN was determined under corresponding conditions, using as indicator the competition of DPNH and DPN for the same binding site(s). D ox , unlike D red , ascribes a univalent dissociation curve with p K ′ = 7.5. The implications of this are discussed. 3. 3. Whereas good agreement was found between the present values for D red and those calculated from previous kinetic measurements, discrepancies were observed in the case of D ox . Since formate competes with ethanol for a binding site at ADH.DPN, these discrepancies can be ascribed to the formation of a ternary ADH.DPN.ethanol complex, interfering with the kinetics particularly in acid solution.

Magnetic properties of some peroxide compounds of myoglobin, peroxidase and catalase.

Abstract The magnetic susceptibility has been determined for the compounds I, II, and IV of horse-radish peroxidase (HRP) and methyl hydrogen peroxide (MeOOH), the compounds of myoglobin (Mb) with H 2 O 2 and with MeOOH, the compound II of catalase (Cat) and MeOOH, and the compounds of catalase formed with azide and hydrogen peroxide in N 2 or CO atmosphere. In cases where oxygen (or another gas) was evolved, the gas was eliminated or a proper correction was applied. The previously described and since then further improved magnetic apparatus was used. The compounds Mb-H 2 O 2 and Mb-MeOOH are, by means of spectrophotometric data, classified as being of type III. The magnetic data obtained fits with the titrimetric data available: HRP-MeOOH-I has three odd electrons and two oxidizing equivalents; HRP-MeOOH-II has two odd electrons and only one oxidizing equivalent; Mb-III also has two odd electrons and one oxidizing equivalent. The structural difference between compounds of type II and III is not reflected in the magnetic data. They have different absorption spectra but the same number of odd electrons. The very active oxidizing agent HRP-II, however, has a definitely higher susceptibility than the spectrophotometrically very similar, but sluggishly oxidizing compound Cat-II. Mb-III, which is also sluggish, has the same susceptibility as Cat-II. The catalase-azide-peroxide compounds in N 2 or CO atmosphere are both found to be diamagnetic, and thus contain ferrous iron.

On the ternary complex of liver alcohol dehydrogenase with reduced coenzyme and isobutyramide. Effect of p-chloromercuriphenyl sulfonate and stability of the complex☆

Abstract The reaction between p -chloromercuriphenyl sulfonate and SH groups of the ternary complex of liver alcohol dehydrogenase, isobutyramide, and reduced coenzyme was studied by a new spectrophotofluorometric method. This is based upon the fact that reduced coenzyme is released from the ternary complex in proportion to the amount of mercurial added. Because of the high sensistivity of the method, SH groups of the enzyme in submicronormal (μ N ) concentrations can be readily determined. Since these determinations can be carried out in the same cuvette following coenzyme-binding site titrations, the ratio of SH groups to coenzyme-binding sites can be determined with great accuracy: the ratio was found to be 14.1 and was independent of the purity of the enzyme preparations. In a mixture of free-enzyme binary and ternary complexes, the mercurial reacts preferentially with the enzyme in forms other than the ternary complex, indicating that the latter is more resistant to the mercurial reaction than the others. A strongly increased stability of the ternary complex is also demonstrated in acid, heat, and alkali inactivation experiments. The complex is completely stable under conditions in which free enzyme or binary complexes are considerably destroyed.

On the microheterogeneity of horse myoglobin

Abstract A procedure for isolating three homogeneous myoglobins, Mb I, Mb II 1 , and Mb II 2 , from horse muscle is described. The iron and sulfur content were the same in all three myoglobins and no significant difference was found in the amino acid composition. The light-absorption curves were identical except for small differences in the region 235–380 mμ. The striking similarity between the three proteins indicated microheterogeneity, and investigation of tryptic digests of the myoglobins revealed the presence of two specific peptides, both missing in Mb II 1 and present in Mb II 2 , while Mb I contained one of them. Amino acid analyses of the specific peptides are given and the significance of the findings is discussed.

Liver alcohol dehydrogenase as a 3β-hydroxy-5β-cholanic acid dehydrogenase

Abstract Horse liver alcohol dehydrogenase (LADH) is one of the most extensively studied enzymes with respect to kinetics, inhibitors, and reaction mechanism, but its main physiological function is still unknown. To determine if LADH could catalyze the oxidationreduction of bile acids a study of thirty-six bile acids as possible substrates was conducted. The only common feature in the reactive bile acids was the presence of a 3β-hydroxyl or 3-keto group. It was clearly established that LADH is stereospecific for 3β-hydroxy-5β-cholanic acid. It was also found that LADH could oxidize the primary alcohol group of several sterols. A kinetic study of the 3β-hydroxy-5β-cholanic acid ⇌ 3-keto-5β-cholanic acid indicated that the Theorell-Chance mechanism was not applicable to this dehydrogenation reaction.

Methanol activity of alcohol dehydrogenases from human liver, horse liver, and yeast☆

Abstract Formaldehyde and NADH are formed from methanol and NAD in the reaction catalyzed by horse liver ADH and yeast ADH. In this reaction the above enzymes resemble closely human liver ADH, which has been known for some time to catalyze methanol dehydrogenation. The formation of formaldehyde from melhanol has been followed by chromatography of 2:4-DNPH derivatives of the reaction products and quantitation by the highly specific chromotropic acid method. Kinetic results have shown that turnover numbers × sec −1 × active site −1 are very similar for all three enzymes. The specificity of all three enzymes with respect to methanol is therefore identical. Human liver ADH, however, has the lowest K m for methanol, and on this basis only may be considered to be the best catalyst of this reaction.

On the inhibitory power of some further pyrazole derivatives of horse liver alcohol dehydrogenase

Abstract We found in 1969 (1) that the inhibitory power of pyrazole on LADH was greatly increased by methyl substitution in the 4-position. In this paper we have studied the effect of increasing the size of this side chain. The inhbitory power was found to increase by a factor of two for each methyl group added in a normal side chain. Some other side chains were tested. Already the 4-butyl and 4-pentyl pyrazoles were so active that for the calculation of their true inhibition constants these had to be corrected for the concentration of the enzyme (0.0005 μN). To our knowledge this never happened before.

Excitation transfer in complexes of horse liver alcohol dehydrogenase

Abstract The protein fluorescence of LADH 1 was quenched upon coupling with NADH, NAD + , o -phenanthroline, or thyroxine and its related compounds, while AMP, ADP, ADPR, or NMN did not quench the fluorescence. Addition of isobutyramide or pyrazole to E 2 R 2 or E 2 O 2 did not alter the degree of quenching. The coupling of two molecules of NADH to one molecule of E 2 I 2 caused an equal fluorescence enhancement for both molecules of NADH when excited in its 340-mμ absorption band. However, with excitation in the protein absorption range, it was found that the binding of the first NADH molecule to LADH caused a larger fluorescence change than the binding of the second one. This was ascertained by following the increase of the fluorescence caused by addition of excess E 2 to E 2 I 2 R 2 , whereby the complexes E 2 I 2 R and E 2 I 2 were formed. This seemed to indicate that excitation energy could be transferred from one subunit to the other in the same LADH molecule. Similar results were obtained for the binding with NAD + or o -phenanthroline by measuring the quenching of the protein fluorescence. Direct determinations of the “on” and “off” velocity constants for the reactions of LADH with NADH, NAD + , or o -phenanthroline were also made using the quenching of the LADH fluorescence upon binding of the ligands.

Plasmaeiweiß, Blutlipoide, Erythrocyten und Senkungsreaktion

ZusammenfassungDa Unsicherheit darüber herrschte, in welchem Maße die Senkungsreaktion wirklich als Ausdruck quantitativer Verschiebungen in den Eiweißfraktionen des Plasmas betrachtet werden kann, wurden unter Anwendung möglichst exakter Methoden 100 Analysen an 84 Fällen vorgenommen (heterogenes klinisches Material). Eine statistische Bearbeitung der 62 Analysen, bei welchen eine solche zulässig war, zeigt eine besonders starke und deutliche Korrelation der SR. zum Fibrin (r=0,82±0,04), eine schwächere zum Globulin (r=0,50±0,10) und eine negative Korrelation vom selben Grad zum Albumin (r=−0,46±0,10). Als genereller Korrelationskoeffizient zwischen SR. einerseits, Fibrin, Globulin und Albumin anderseits ergibt sich 0,87±0,03. Von dem Eiweißbilde ausgehend, läßt sich SR. ausrechnen, und beim Vergleich mit der beobachteten wurde dabei ein Durchschnittsfehler von 13,6 mm erhalten.Wenn man die SR. und das Eiweißbild mit dem Zellvolumen (dem Hämoglobingehalt des Blutes) vergleicht, zeigt es sich, daß bei Anämie nicht selten eine relativ erhöhte Senkung zu beobachten ist, noch öfter fehlt dieser Einfluß aber. Die statistischen Korrelationen zum Eiweißbilde werden auch durch eine gemäß dem in-vitro-Einfluß von Veränderungen im Zellvolumen vorgenommene Korrektur der SR. nicht besser, sondern im Gegenteil etwas schlechter. Eine generelle solche Korrektur der SR. wird deshalb abgelehnt.In sämtlichen Fällen wurden auch umfassende Lipoidanalysen vorgenommen. Eine sichere Korrelation zum gesamten Cholesterin- und Lecithingehalt in den Blutkörperchen, bzw. im Plasma, existiert nicht. Weitere Untersuchungen über die Bedeutung der Lipoide für die Senkungsgeschwindigkeit sind in Vorbereitung.