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Proteine sind in Abhängigkeit von Temperatur und Druck durch ellipsenförmige Phasendiagramme charakterisiert. In dieser Arbeit wurden Druck- und Temperaturdenaturierungsexperimente an einem modifizierten Cytochrom c-Protein in einer Diamantambosszelle durchgeführt. Der Phasenübergang wurde anhand der Änderungen im laserinduzierten Fluoreszenzspektrum detektiert. Über die Änderungen in den Wechselwirkungen zwischen Chromophor und Umgebung, die sich in den Spektren widerspiegeln, konnten die unterschiedlichen Modellvorstellungen von Druck- und Temperaturdenaturierungen bestätigt sowie das vollständige Stabilitätsdiagramm von Zn-Mesoporphyrin-Cytochrom c ausgemessen werden. Zusätzlich wurde der Wechselwirkungsradius der Farbstoffsonde im Protein über Lochbrennspektroskopie abgeschätzt. The stability of proteins as a function of pressure and temperature is characterized by elliptic phase diagrams. In this work pressure- and temperature-induced denaturation experiments on a modified cytochrome c protein in a diamond anvil cell were carried out. The determination of the phase transition was possible by the change in the laser-induced fluorescence spectrum. The change in the interactions between chromophore and environment results in a characteristic spectral pattern, confirming different models for the explanation of pressure- and temperature-induced denaturation. The complete stability diagram of Zn-mesoporphyrin cytochrome c was determined. In addition, the interaction radius of the dye probe inside the protein was estimated by using pressure-tuning hole-burning spectroscopy.

This article presents the results of our initial clinical experience with the human growth factor FGF-1 as applied to the ischemic human myocardium. After the completion of extensive preliminary animal experiments, the human angiogenetic growth factor FGF-1, obtained from a genetically transformed strain of E. coli was introduced into aortocoronary bypass surgery as an additional therapeutic agent. A double-blind study was carried out in a total of 40 patients with coronary heart disease. The patients were randomized into growth-factor and control groups, each containing 20 patients. All patients underwent aortocoronary bypass surgery because of their coronary multivessel disease, in each case with an IMA bypass for the LAD and single venous bypasses for the RCX and/or RCA. In order to bridge over additional stenosis of the LAD or one of its branches, the human growth factor FGF-1 was injected into the myocardium during the operation. In the control group heat-denatured growth factor was substituted for FGF-1. After three months as well as three years postoperatively, the IMA bypasses were selectively depicted by intraarterial DSA. These angiographies were then quantitatively evaluated. After the application of the growth factor, the formation of new vessels could be demonstrated after three months as well as three years postoperatively. A capillary network initiating from the coronary artery could be found and the computer-supported evaluation of the angiographs revealed a significant increase in the blood supply of the region of the myocardium injected. According to the angiographic findings there was also a clinical improvement of the patients with FGF-1 application compared to the patients of control group, concerning the NYHA classification as well as the need for antiangina drug therapy. In the meantime, the results of other research groups support the evidence that the induction of neoangiogenesis by human growth factor could become a therapeutic approach especially in patients with diffuse coronary artery disease. Nevertheless further studies have to be carried out in order to prove the long-term clinical profit of those patients after the growth factor treatment.

The goal of this ex-vivo study was a controlled macroscopical and microscopical comparison of the immediate effects of surgical lasers on dense collagenous tissues. The investigation of the exact denaturizing effects following tissue interaction between lasers and collagenous fibers are of importance for a better understanding of the observed and described healing process. Partial tenotomies were performed ex vivo on sixty tendons of the flexor digitorum profundus muscle of New Zealand White Rabbits using four surgical lasers (Holmium:YAG-, Erbium:YAG-, Neodym:YAG-, and CO2-Laser). The tendons were evaluated macroscopically, histologically and observations were made on the surgical handling of the lasers during the procedure. The choice of lasers allowed the comparison of a wide spectrum of wavelengths (gamma = 1.0 to 10.6 microns). In addition, beam delivery modalities were compared to each other (focus vs. contact). The pulse (frequency and power) was varied within each laser. Other parameters including focus size, absorption, dispersion, and thermal tissue conductivity were maintained constant in this test arrangement in order to allow a later comparison between the observed areas of denaturated collagen. The macroscopical and histological results showed great differences in the effects of the four lasers. Even within each single laser group, the results varied greatly with the choice of variable parameters. Holmium:YAG- and CO2-Laser can produce similar areas of collagen denaturation. When comparing specimens with similar areas of collagen denaturation induced by different laser types, different amounts of charring were observed. Erbium:YAG-Laser tenotomies showed generally inhomogeneous denaturation areas, while tenotomies with Neodym: YAG-Laser used in continuous mode resulted in significant charring and tissue retraction in the area of interaction. These observations help in defining the combination of parameters with which Holmium:YAG-, Erbium:YAG-, and CO2-Lasers can be used for transsection of dense collagenous tissues. In addition, these histological results as well as already published observations on healing patterns following laser surgery allow for the formulation of a hypothesis on the effects of laser in the wound healing process.

Two sequence-homologous alpha-amylases from B. amyloliquefaciens and B. licheniformis were studied with respect to their stability against heat denaturation and were compared with respect to common structure-stabilizing principles. The investigated alpha-amylases were isolated from culture broth of B. amyloliquefaciens and B. licheniformis. The molecular parameters (molecular weight and isoelectric point) are similar. The thermostability was determined by changes of the protein structure (changes of the fluorescence emission spectra). At pH 5.0 the thermostable alpha-amylase from B. licheniformis showed a rate of denaturation which was achieved by the thermolabile alpha-amylase from B. amyloliquefaciens at a temperature 15 degrees lower. The alpha-amylase from B. licheniformis exhibits a marked stability also at the alkaline pH-range in contrast to the alpha-amylase from B. amyloliquefaciens. From measurements in the presence of EDTA and Ca2+ follows that both enzymes are stabilized by binding of calcium ions. An analysis of preferred amino acid exchanges between the two sequence-homologous alpha-amylases showed correspondences and differences to the well-known diagram of ARGOS. Possibly an increased thermic stability can already be achieved by special amino acid exchanges without significant changes in the protein structure.

In view of their potential activity as antigen-specific immunosuppressive agents, the authors produced conjugates by treating the tri- and tetracyclic compounds 1-8 (with isothiocyanate structure) with human serum albumin (HSA) and bovine gamma globulin (BGG). In certain cases the conjugates of BGG were obtained only under conditions leading to protein degeneration. The corresponding products were devoid of antigen-specific immunosuppressive activity and must be considered to be merely repository forms of the respective low-molecular compounds. In contrast, the conjugates of HSA could be obtained under conditions under which no protein denaturation was detectable. These conjugates may be used for the planned investigation. After reaction of the isothiocyanates with the proteins, time dependent changes were observed in the ultraviolet spectra of some conjugates, the possible causes of which are discussed.

The influence of milieu factors on the thermal inactivation of peroxidase and lipoxigenase was investigated. Cationogenic, anionogenic, non-ionogenic and amphoteric tensides were more or less effective in inactivating horseradish peroxidase. Most effective in this respect were lecithine and monoglyceride, both capable of swelling in water. In presence of lecithine, peroxidase was inactivated already at 0 degrees C and pH 4.0. Linoleic acid was more efficient in an oxygen stream than in presence of nitrogen, in a stream of nitrogen its influence was comparable to oleic acid. This suggests an additional effect by lipid peroxides which are formed of linoleic acid under the heating process. Tensides prevented the regeneration of the heated peroxidase. In the case of lipoxigenase, the authors investigated the influence of lecithine and various fatty acids on the thermal inactivation at 60 degrees and pH 7.0. Lecithine accelerated the inactivation less distinctly than with peroxidase. The accelerated the inactivation less distinctly than with peroxidase. The accelerating effect of the fatty acids decreased in the order oleic acid, linoleic acid, palmitic acid, myristic acid and stearic acid.

The thermal reaction of the two components of the horseradish peroxidase--the apoenzyme and the prosthetic group--with that of the holoenzyme were compared. From this we conclude that the thermal inactivation of the peroxidase in aqueous solution is not caused by a change of the apoprotein alone, but rather by participation of the entire three-dimensional structure including the prosthetic group. Spectrophotometric studies of the inactivation process in the holoenzyme revealed that the absorbance of the Soret band changes parallely to a reduction of the enzyme activity during heating. Since it has been found that the Soret absorption increases again during storage and simultaneously the enzyme activity regenerates, the change of the Soret absorption during heating can be regarded as the result of both reversible and irreversible denaturation processes.

To inhibit the rapid deterioration of the production-technological properties of fish meat minced and frozen on board, it is necessary to become acquainted with the causes of protein denaturation under these conditions. It was found that the protein solubility in codfish is impaired by formaldehyde which develops from trimethyl-amine oxide during storage and also by the salt content. After 7 days at -20 degrees C, the solubility of the sarcoplasmic and myofibrillary proteins in minced fish meat added with 80 mg of formaldehyde per 100 g of total protein amounted to almost 70% and 35%, respectively, of the value determined in controls, i.e., samples without formaldehyde. After this period of storage, only 30% of the added formaldehyde were in the free state. After 1 month, the solubility of the myofibrillary proteins in waterleached fish meat was by 30% higher than in unleached controls, 80 mg of formaldehyde per 100 mg of total protein having been added in both cases. After 1 month at -5 degrees C and -20 degrees C, only 90 and 15 p.p.m. of formaldehyde, respectively, were found in minced fish meat, whereas leached samples contained no formaldehyde. The solubility of the myofibrillary proteins in leached fish meat (the initial salt content of which had been restituted) was by 30% lower than in the unleached controls.

Synthetic PZ peptide, synthesized by Wünsch and Heidrich, was used as substrate to determine PZ peptidase activities in the sera of man, rabbit, guinea pig and mouse. The PZ peptidase from rabbit serum was purified 200 fold and characterized. The enzyme has an isoelectric point of 5.0 and pH optima at pH 7.2 and pH 7.9. Its behaviour on gel filtration points to a molecular weight of 60.000 dalton. The peptidase is inhibited by heavy metal ions, SH reagents and serum. Ca ions are EDTA are without any effect. In contrast to collagen peptidases from micro-organisms, the enzyme from rabbit serum does not attack native, but only denatured collagen. The results suggest that PZ peptidases participate in collagen breakdown by cleaving the collagen fragments which are released by the collagenases.

Alkaline solutions of a mixture from rapeseed globulin and casein show plastic flow, which is influenced by protein concentration, relation of rapeseed globulin and casein, temperature and time of storage of the protein solution. The sodium hydroxide concentration with a great variability doesn't take effect on the rheological properties. With increasing content of casein in such alkaline solutions of rapeseed globuline and casein results an approach to NEWTONian flow. The spinnability of alkaline solutions of a mixture from rapeseed globulin and casein and the properties of the corresponding spun protein fibers are influenced by protein concentration, relation of rapeseed globulin and casein of the spinning solution and temperature of coagulating bath. The sodium hydroxide content of the spinning solution and the hydrochloric acid concentration of the coagulating bath with a great variability don't take effect. The spinnability of alkaline solutions of a mixture from rapeseed albumin starchsulphate and casein and the properties of the corresponding spun protein fibers compared to rapeseed globulin-casein mixtures are influenced additionally by sodium hydroxide concentration of the spinning solution and hydrochloric acid content of coagulating bath. The favourable influence on the properties of spun rapeseed albuminstarchsulphate-casein fibers isn't increased by a growth of the rapeseed albumin starchsulphate content more then 4% of the protein concentration of the spinning solution.

The thermal inactivation of lipase out of Geotrichum candidum and of lipoxigenase out of soybeans was investigated in phosphate buffer solution. Comparable to peroxidase, a sharp bend was observed in the inactivation curves of both enzymes. The z-value of 19 degrees C for lipase out of Geotrichum candidum was higher than the values indicated in the pertinent literature for pancreas lipase and milk lipase. The z-value for soy lipoxigenase was found to be 9.8 degrees C.

Soy-bean protein loses its immunogenicity by heat treatment through thermal denaturation. Incubation with 6m guanidine-hydrochloride restores the immunogenicity. This reagent causes dissociation of protein aggregates formed during autoclaving of soy protein. This seems to be the major step in the restitution of immunogenicity. These findings are discussed in connection with the quantification of heat-denaturated soy-bean protein by two independent immunologic methods: Solid-Phase Radioimmunoassay and Passive Hemagglutination (inhibition test).

We characterize and describe for the first time the primary structure of a human porin with the molecular mass of 31 kDa derived from the plasmalemm of B-lymphocytes (Porin 31HL). Porin 31HL is shown to be a basic, channel forming membrane protein. The protein chain is composed of 282 amino acids with a relative molecular mass of 30641 Da without derivatisation. It is not a glycoprotein. The N-terminus is acetylated. Altogether the amino-acid sequence shows 56% hydrophilic or charged amino acids arranged in alternating regions of hydrophilic or hydrophobic character as it is typical for porins. In addition the 18 N-terminal amino acids of Porin 31HL can be arranged to an amphilic alpha-helix like in other porins. Porin 31HL shows approx. 29% or 24% identity to the primary structure of mitochondrial porins of Neurospora crassa and Saccharomyces cerevisiae. Partial data on mitochondrial porins from rat kidney and beef heart show sequence identity of about 90% to the human B cell porin elaborated here.

The thermal reaction of a lipid-acyl-hydrolase which seems to be important for the quality preservation of vegetable foods, was investigated in spinach. The authors applied a simple in-situ method using thin-layer chromatography which had been developed for the enzyme determination, to follow the thermal inactivation of the lipid-acyl-hydrolase by measuring the decomposition of lecithin, mono- und digalactosyl diglycerides. According to the inactivation curves, the enzyme is relatively little resistant to heat. Since the D- and z-values resulting from the inactivation curves for phospholipase, mono- and digalacto-lipase activities are almost the same, it can be assumed that the lipid-acyl-hydrolase is a multi-function enzyme in spinach.

The main fractions of the albumins from sunflower and rapeseeds (isolated by means of precipitation with ammonium sulphate or tannin + caffeine and subsequent gel chromatography) are low-molecular, very basic proteins. Their molecular weights range from 10 000 to 16 000, and their isoelectric points (determined by isoelectric focusing and free electrophoresis) are situated at pH greater than 10.0. From the circular dichroism in the wavelength range from 200 to 240 nm it is deduced that the main fraction of the rape albumin is a well-structured protein with 40-46% alpha-helix in aqueous and salt-containing solutions. Denaturation by heating is achieved only at temperatures about 100 degrees C and pH values greater than 9. 20% alpha-helix are left after the action of 8 M urea. This conformational stability is explained by the presence of disulphide linkages in the molecule.

A protein known as antigelatin factor (AGF) was isolated from human serum by affinity chromatography with immobilized denatured collagen. In biochemical and immunological assays AGF showed specificity to denatured, but not to native collagen of the types I, II and III. A close relationship to Cell Attachment Protein and Cold Insoluble Globulin was found in comparative studies.

To extend the knowledge about systemic glucagon metabolism, intact human erythrocytes were tested for association and degradation of glucagon. When erythrocytes were incubated at 37 degrees C in 0.15 M NaCl solution, glucagon and 125I-glucagon were degraded, depending both on time and hematocrit. At a hematocrit of 0.45, about 20% of 1 nM initial glucagon were degraded during 4 h of incubation. Furthermore, cellular association of glucagon took place amounting to about 10% of the initial glucagon. Nearly 3% of the initial glucagon, but 40% of the cell-associated hormone were found to consist of intracellularly accumulated glucagon degradation products. Despite of nondetectable hemolysis after 60 min of incubation at 37 degrees C, a glucagon-degrading activity was eluted from the cells into the medium. This degrading activity was quantitatively inhibited by Contrykal, EDTA, and o-phenanthroline. Both glucagon degradation and association by human red cells were quantitatively inhibited by bacitracin. These results provided evidence for a participation of human erythrocytes in glucagon metabolism in vitro.

Oxidized lipids react with proteins to form lipoproteid complexes in which the lipids are bound to the proteins in part by physical forces, in part by covalency. The free radicals resulting from the cleavage by hydroperoxides are the major precursors of the lipoproteid complexes. The interaction is associated with protein denaturation and oligomer formation. The lipids contained in the lipoproteid complexes are only in part extracted by chloroform-methanol; in part not until after acid or alkaline hydrolysis. The nutritive value of the protein moiety is diminished by the reaction of the hydroperoxides with methionine and cysteine and by the reaction of the peroxidic radicals and aldehydes with lysine and other basic amino acids. Secondary reactions of the lipoproteid complexes lead to brown coloured, only partly soluble compounds which often impair the organoleptic value. The rancidity products of the fats are neutralized by the reaction with proteins. The action of highly unsaturated oxidized lipids on proteins results in the development of a fishy aroma.

The dissociation and reassociation behavior of the hexameric leucine aminopeptidase (LAP) in solutions of 0.5 to 6 M guanidine-HC1 (Gu-HC1) was investigated by means of thin layer chromatography on Sephadex G-200 superfine. Up to 0.5 M Gu - hc1 the hexameric LAP-structure remains intact. In 0.75 to 2 M Gu - HC1 the enzyme dissociates nearly completely into its half-molecules (LAP-trimers, MW 160000 +/- 10,000). In 2.5 and 2.75 M Gu - HC1 a mixture of LAP-monomers (subunits) (MW 60000 +/- 5000) and trimers was to be found. Treatment with beta-mercapto ethanol increases the portion of monomers. Only monomers occurred in 3 to 6 M Gu - HC1. Parallel to the dissociation, a loss of the essential zinc (half life: 10.5 and 3.5 hours in 1 M and 4 M Gu - HC1 resp.) and a decrease of the enzymatic activity (30 min after treatment with 1 M and 4 M Gu - HC1 50% and 1% resp. residual activity) of the observed. On the other hand, the enzyme was activated by dilute solutions of Gu - HC1 (0.1 to 0.3 M: max. increase 50%). Removal of the denaturant causes reassociation of all types of fragments to hexameric LAP which was indistinguishable from the native LAP in Ouchterlony immune diffusion test. The reassociation was accompanied by a small increase in activity only.

The thermal inactivation and storage behaviour for horseradish and spinach peroxidases were investigated in defined systems, in spinach also within its natural environment. The inactivation curves of either enzyme show a sharp bend which is clearly visible at low, but not at higher temperatures. The D-values were taken from the inactivation curves. z-values resulting from the D-values were 25.5 degrees C for horseradish peroxidase, 13 degrees C for isolated peroxidase and 18 degrees C for peroxidase in spinach extract. Horseradish peroxidase was relatively heat-resistent at pH 6.0, spinach peroxidase at pH 5.0-6.0; both enzymes were found to be highly susceptible to heat at pH 4.0. Peroxidase isolated from spinach responded differently to heating than the enzyme in spinach extract or suspension. This discrepancy indicates that certain model experiments cannot be transferred to foods. Heated peroxidase from horseradish and spinach were found to regenerate during storage; the extent of regeneration depended on the pH.

The amount of plasma proteins associated to erythrocytes was determined or calculated by using three different techniques. Thus, there are 3.48 X 10(12) g of protein in a single erythrocyte or 94.12 g of protein respectively in the total of all erythrocytes in a healthy man with a body weight of 70 kg. The mass of plasma protein associated to erythrocytes will decrease in case of immunocomplex aggregates or otherwise denatured plasma proteins being fixed to the erythrocyte surface so that from a purely calculating point of view protein amounts on a scale of up to 46% of all plasma proteins may enter the free blood plasma under extreme conditions. In this, an immediately efficient possibility of compensation is seen by the authors in case of an acute consumption of blood proteins.

When applying various concentrations of NaC104 (0.15-7 M) to tendon fibres from rat tails, we found denaturation beginning at 2M, and with increasing concentration a linear increase of the maximal contraction tension and an expoential shortening of the contraction time down to a minimal value of 7.5 min. In a second group of experiments we investigated the weight changes of tendons of old (26-30 months) and young (8 months) rats treated with 5M NaC104 and compared the weight curves and the tension curves. We found an increase in weight up to 900% as the tension decreased. This indicates a connection between the swelling and the relaxation of collagen fibres. Other solutions of neutral salts (5M NaC103, NaC1; 0.15 M NaC104, NaC103, NaC1) tested on young and old tendons did not produce any tension nor other visible changes. The weight curves show an immediate increase up to about 200% which remains constant and which is low compaired with the effect of 5M NaC104. The reason for the contraction of tendon fivres seems to be the denaturation caused by the increased lipophilia of the aqueous salt solutions, while the relaxation is merely the result of the subsequent swelling of the collagen fibre.

Two experiments lasting 10 vs. 8 weeks respectively were conducted in order to determine the effects of feeding heated proteins (casein + glucose, 65 degrees C for 4 days) on histopathological alterations in the kidneys of rats. Histological examinations showed enlarged epithelial cells and cell nuclei in the pars recta of the proximal tubules (inner stripe of the inner kidney cortex zone) beginning after two weeks of the test. The differences in the average size of the nuclei were significant after a feeding period of 6, 8 or 10 weeks (44 rats in the test group vs. 36 rats in the control group respectively). In this way the alterations are very similar to that which are induced by lysinoalanine. The substance causing the lesions seems not to be fructoselysine although it was present in the heat damaged diets in concentrations of 16,000-23,000 ppm but is suggested among the substances formed in the early and advanced stage of Maillard reaction.

The interaction of magnesium ions with inorganic pyrophosphatase from baker's yeast was studied by means of heat denaturation. The heat inactivation of this enzyme is a biphasic process. The velocities in the initial range and in the subsequent slower part of inactivation are diminished with rising Mg2+ concentration in the inactivation assay. A model is proposed which describes this behavior. It is assumed that two enzyme conformations exist in equilibrium whose conversion rates correspond to the inactivation rate in its order of magnitude. The equilibrium is shifted by Mg2+. The two enzyme species differ in their Mg2+ binding behavior as evidenced by differences in the half-saturation constants and the cooperativity of the binding. The same conclusions are drawn from the fluorimetric measurement of denaturation of inorganic pyrophosphatase. Besides, an additional Mg2+ binding site is demonstrable, the saturation of which obviously leads to stabilisation of part of the enzyme structure without protecting it against loss of enzymatic activity. With the same method the labilizing effect of Zn2+ on the structure of the inorganic pyrophosphatase from baker's yeast was studied.

Peracids are a class of substances inactivating the largest spectrum of viruses in comparison to the other known disinfectants. The mode of action is unknown however. For the detection of the reaction mechanism we studied the inactivating kinetics of poliovirus and polio-RNA as well as the virus- and RNA-inactivation in dependence on the peracid concentration. In addition the sedimentation of the virus and the virus-RNA were determined. The inactivation curves of virus particles and virus-RNA respectively showed a similar, not linear course. The sedimentation of the virion and the RNA were altered after treatment with peracid and showed a disrupted capsid and a fragmented RNA as a result of incubation with the disinfectant.

The specific demand of sodium hydroxide is determined for the dissolution of sunflower seed globulin, casein and a mixture of them to equal parts. In low protein-containing solutions it depends for sunflower seed globulin very much on the sodium chloride concentration. From sunflower seed globulin, casein and a mixture of them to equal parts are prepared with sodium hydroxide high protein-containing alkaline solutions. Sunflower seed globulin forms temporally a gel phase. After this phase the solution of sunflower seed globulin shows like casein and a mixture of sunflower seed globulin/casein (I:I) pseudoplastic flow. The flow curves of the pseudoplastic solutions are described mathematically with the OSWALDian power statement. By alkaline solutions of casein and sunflower seed globulin/casein (I:I) the flow exponent n is distributed statistically about 0.9, by solutions of sunflower seed globulin a distribution exists about the mean values n = 0.85 and n = 0.50. lg k depends in all protein solutions on the concentration of protein, sodium chloride, sodium hydroxide and on the temperature and time. For all protein solutions exists a linear relation between the logarithm of viscosity and the reciprocal temperature for lg k and I/T, which is derived normally for NEWTONian flow behaviour. In a suitable scope of spinning for all protein solutions are carried out complete factorial experiment, which guide to regression equations of lg k; in the case of sunflower seed globulin are calculated also a regression equation of the flow exponent n. Going out from the parameters of the spinning process the properties of the spun sunflower seed globulin/casein (I:I) fibers are described.

Admixture of starchsulphate to an alkaline spinning solution of proteins increases the thermostability of the corresponding spun protein fibers.

By electron microscopic studies collagenase, hyaluronidase, HCl, ascorbic acid, and iron ions have been found to attack the collagen fibers of bovine vitreous. Because of the possible role of ascorbic acid in collagen synthesis and the ability of ascorbic acid to degrade hyaluronic acid and collagen we suggest that the ascorbic acid of the vitreous essentially participates in construction and metabolism of the vitreous body.