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Biomedical subjects

H L Schmidt

Publications and source records attributed to H L Schmidt.

At least 37 records · Page 2Linked to original sources
PubMed1987

Structure and electrochemistry of oxidoreductases.

The principles on which Nature has developed multifunctional redox centres, covering a large range of potentials, protected from water and oxygen and surrounded by highly specific proteins, are demonstrated. Structures and accessibilities of the active sites of iron-sulphur proteins, sulphur proteins, flavoproteins, cytochromes and copper proteins are correlated with their possibilities and modes of electron exchange with natural partners, artificial mediators and (modified) electrodes. The participation of charge-transfer and tunnelling processes in electron transport is demonstrated, and a suitable relative orientation of the partners is recognized as one of the most important requirements for electrochemical communication between large molecules and electrodes. The use of specifically modified electrode surfaces, for example those based on electroconductive polymers, is proposed as one of the aspects of future developments for direct electron transfer to proteins.

Cytochromes↗
PubMed1987

Carbon isotope effects on the pyruvate dehydrogenase reaction and their importance for relative carbon-13 depletion in lipids.

A method has been developed for the positional 13C isotope analysis of pyruvate and acetate by stepwise quantitative degradation. On its base, the kinetic isotope effects on the pyruvate dehydrogenase reaction (enzymes from Escherichia coli and Saccharomyces cerevisiae) for both of the carbon atoms involved in the bond scission (double isotope effect determination) and on C-3 of pyruvate have been determined. The experimental k12/k13 values with the enzyme from E. coli on C-1 and C-2 of pyruvate are 1.0093 +/- 0.0007 and 1.0213 +/- 0.0017, respectively, and, with the enzyme from S. cerevisiae, the values are 1.0238 +/- 0.0013 and 1.0254 +/- 0.0016, respectively. A secondary isotope effect of 1.0031 +/- 0.0009 on C-3 (CH3-group) was found with both enzymes. The size of the isotope on C-1 indicates that decarboxylation is more rate-determining with the yeast enzyme than with the enzyme from E. coli, although it is not the entirely rate-limiting step in the overall reaction sequence. Assuming appropriate values for the intrinsic isotope effect on the decarboxylation step (k3) and the equilibrium isotope effect on the reversible substrate binding (k1, k2), one can calculate values for the partitioning factor R (k3/k2: E. coli enzyme 4.67, S. cerevisiae enzyme 1.14) and the intrinsic isotope effects related to the carbonyl-C (k1/k'1 = 1.019; k3/k'3 = 1.033). The isotope fractionation at C-2 of pyruvate gives strong evidence that the well known relative carbon-13 depletion in lipids from biological material is mainly caused by the isotope effect on the pyruvate dehydrogenase reaction. In addition, our results indicate an alternating 13C abundance in fatty acids, that has already been verified in some cases.

Carbon Isotopes↗
PubMed1986

Isolation and properties of an H2O-forming NADH oxidase from Streptococcus faecalis.

An H2O-forming NADH oxidase from Streptococcus faecalis, recently described [Hoskins, D. D., Whiteley, H. R. and Mackler, B. (1962) J. Biol. Chem. 237, 2647-2651], has been isolated as a uniform protein with specific activity 690 U/mg in a total yield of 50% by a two-step affinity chromatography procedure. The enzyme is metal-free and has a molecular mass of about 51 000 Da and probably consists of a single polypeptide chain. As shown by fluorimetric titration, the prosthetic group is 1 mol FAD/mol protein. The affinity behaviour of the enzyme gives evidence for the existence of a dinucleotide-binding domain capable of binding NADH or FAD. The enzyme is specific for NADH (Km = 4.1 X 10(-5) M), NADPH is not oxidized. O2 is the preferred electron acceptor, in addition FAD and, very slowly, one-electron acceptors are reduced. It is not clear whether the reduction of FAD proceeds through the dinucleotide-binding site or by exchange of the prosthetic group. The stoichiometry of the reaction with O2 corresponds to the consumption of 2 mol NADH/mol O2, and only H2O is formed (2 NADH + 2 H+ + O2----2 NAD+ + 2 H2O). Neither H2O2 nor O2.- is detected as intermediate and H2O2 cannot replace O2 as an oxidant. The enzyme can, mainly in its reduced state, be inhibited by -SH reagents. Spectral data give no evidence for the existence of radical intermediates during reduction. The enzyme can obviously accept more than two electrons/mol. On the basis of these data two possible reaction mechanisms are discussed. A proposal for the biological purpose of the reaction is made.

Chromatography, Affinity↗
PubMed1986

[Biosensors].

By the combination of transducers (thermistors, selective electrodes, field-effect transistors, optical systems) with immobilized enzymes or antibodies specific sensors for biologically relevant substances are obtained. The construction, ranges, of linearity, response times and stability of biosensors are demonstrated. Examples are given for their application in clinical analysis and fermentation control. Finally limits and future possibilities are discussed.

Antibodies↗
PubMed1984

Investigation of enzyme-substrate complexes by affinity chromatography. Application to pig heart citrate synthase.

The relative affinities of Sepharose gels, to which coenzyme A (CoA-SH) and CoA-SH analogues were bound through a well defined site, for citrate synthase were determined. The relative eluting power of coenzyme derivatives for the CoA-SH-gel and the Matrex Gel Blue-bound enzyme was measured, and the influence of oxaloacetate on the binding of the enzyme investigated. From the results, the contributions of different parts of the coenzyme to its binding in the active site and kinetic concepts are derived and found to be in complete agreement with corresponding data for citrate synthase obtained from kinetic measurements reported in the literature. It is demonstrated for some other CoA-SH-specific enzymes that affinity chromatography is of value as an additional tool for the comparative investigation of binding sites of enzymes which depend on the same coenzyme.

Animals↗
PubMed1983

[Effect of natural isotope contents of nutrients on the background in 13C breath tests].

The long-term variation of the mean delta 13C-value of breath-CO2 of Europeans (base value -26%) was +/- 1% over a period of several months. However, the ingestion of products from corn, a C4-plant, caused remarkably higher shifts. In a fasted state and during rest a stability of the base value of +/- 0.3% could be attained for several hours. Longer fasting periods imply a shift of the CO2-delta-value due to the metabolism of body products. - By a change of the main energy source to corn products the delta 13C-base line could be shifted to -18% within one day. After this shift even the metabolism of products from C3-plants could be investigated. The degradation curve and the integral degradation of fats were largely dependent on the method of application, and the base value largely interfered with the results. The period of maximum disintegration for a naturally and uniformly labelled fat was different from that of an artificially labelled trilinoleate. The results shall be used for the adjustment and the correction of the base line in 13C-breath tests, especially with patients under parenteral nutrition.

Breath Tests↗
PubMed1982

Geographical variations in the carbon isotope composition of the diet and hair in contemporary man.

Geographic variations in the carbon isotope composition of the human diet and human hair were investigated. The carbon isotopic composition of common foodstuffs purchased in Chicago, USA, Tokyo, Japan and Munich, FRG, were determined by combustion and differential isotope ratio mass spectrometry. The dietary protein carbon for the United States (-18.1%) was calculated to be enriched in 13C relative to the Japanese (-21.2%) and the German (-23.6%) diets. To a large degree, the differences reflected the consumption of corn-fed animal products in the United States and Japan, as well as seafood in Japan. The carbon isotopic composition of hair (-16.4, -18.0 and -20.4%) for the three respective populations correlated with the calculated values of the dietary protein, but were 2-3% enriched in 13C. Changes in the isotopic composition of beard hair were shown to record the changes in dietary composition in travelers visiting the respective regions.

Animals↗
PubMed1982

[Direct determination of phenylalanine in serum by derivative spectrophotometry (author's transl)].

A simple and precise method is described for the determination of phenylalanine in serum or plasma, using derivative spectrophotometry. After the removal of protein and uric acid, 100 microliters serum or plasma are analysed directly for phenylalanine. The magnitude of the difference between the peak minimum at 254 nm and the peak maximum at 257 nm of the derivative spectrum (2nd derivative of the absorption spectrum) is directly proportional to the phenylalanine content. The precision was 2.1-10.3%, depending on the concentration of phenylalanine. Recovery was between 97.1 and 101.1%. Sensitivity was 5 mg/l serum. The method was compared with a column chromatographic determination of phenylalanine.

Humans↗
PubMed1981

A study of oxygen isotope scrambling in the enzymic and non-enzymic oxidation of linoleic acid.

Lipoxygenases-1 and -2 isolated from soybeans were incubated with linoleic acid in the presence of a mixture of 16O2 and 18O2. The formation of 16O/18O-molecules which is indicative for a head-to head reaction of peroxy radicals was determined and compared with that produced during autoxidation of a linoleic acid emulsion in the presence of ferric ions. Lipoxygenase-1 was much less active in scrambling than lipoxygenase-2 which was comparable to that found in the autoxidation reaction.

Isoenzymes↗
PubMed1976

Coenzyme properties of NAD+ bound to different matrices through the amino group in the 6-position.

A method for the synthesis of N6-(2-aminoethyl)-NAD+ is given. The binding of this NAD+ derivative to different soluble and insoluble supports and the direct coupling of NAD+ to epoxyactivated Sepharose are described. Proofs are given that NAD+ is bound through the amino group in 6- position and the NAD+ derivative through the aliphatic amino group of the side chain. Non-enzymic reduction of the bound coenzyme to an almost quantitative extent is possible in all cases, but the enzymic reduction is largely influenced by the support. While N6-(2-aminoethyl)-NAD+ coupled to soluble dextran is nearly completely reducible by different dehydrogenases with a velocity of about 40% of that for free NAD+, the coenzyme bound to different insoluble matrices is very slowly reduced. Only 5% of the coenzyme derivative bound to BrCN-activated Sepharose are reducible, but 40% when it is bound through a spacer. From capacity determinations evidence is given that, even in this coenzyme gel, only those coenzyme molecules are useful in affinity chromatography which are on the surface of the gel grains; it is supposed that this may be due to the slow diffusion of an enzyme into the inner parts of an affinity gel.

Binding Sites↗
PubMed1976

Microcalorimetric methods for substrate determination in flow systems with immobilized enzymes.

The enthalpy of processes catalyzed by immobilized enzymes in the reaction cell of a LKB-flow calorimeter is used for determination of urea (0.5-5 mumol) and glucose (0.03-0.5 mumol). Accuracy is 2-5% and the time needed for one analysis is 20 min. A sensitive "enzyme thermistor" consisting of a flow through cell with an immobilized enzyme and two thermistors is described, which permits glucose determinations (0.05-1 mumol +/- 0.03 mumol) by means of temperature difference caused by reaction heat. Coupling of enzyme reactions for increasing reaction heat and consequently sensitivity in calorimetric determinations is demonstrated.

Binding Sites↗
PubMed1976

[Coenzym properties of some Ade-1- and Ade-N6-substituted NAD derivatives (author's transl)].

By reaction of NAD with different oxiranes or with aziridine, derivatives of the coenzyme are obtained with substituents in position 1 or on the amino group in position 6 of the adenine ring. While the Ade-1-substituted derivatives show high Km values with different dehydrogenases and are reduced only very slowly by these enzymes, the coenzyme derivatives substituted at the amino group behave very similarly to NAD. Correlations were found between coenzyme efficiency of the compounds and the lipophilic character of their substituents. The results can be interpreted from the structure of the active site of the dehydrogenases investigated.

Coenzymes↗