The effect of ultraviolet irradiation on acid-soluble collagen

1. A study has been made of the effect of ultraviolet irradiation on the conformational changes taking place in cooled solutions of thermally denatured acid-soluble calf-skin collagen. 2. The increase in negative rotation and viscosity at 15° for irradiated and thermally denatured collagen solutions becomes less as the irradiation dose is increased. 3. The principal effect of ultraviolet irradiation is the fission of the primary collagen chains, eventually yielding chain lengths incapable of stabilizing a helical structure. 4. The effects of ultraviolet irradiation on acid-soluble collagen may be closely correlated with similar effects on neutral salt-soluble collagen.     

The effect of γ-irradiation on soluble collagen

1. A study was made of the effect of gamma-irradiation on the sub-unit composition, as well as the conformational changes taking place in cooled solutions of thermally denatured neutral-salt-soluble and acid-soluble collagen. 2. The increase in negative rotation and viscosity at 15 degrees for irradiated and thermally denatured collagen solutions becomes less as the irradiation dose is increased. 3. The initial effect of gamma-irradiation is the depolymerization of the dimers found in both neutral-salt-soluble and acid-soluble collagen. 4. The principal effect of gamma-irradiation up to 10 Mrads is the fission of peptide bonds, yielding crystalline irradiation-resistant portions of the molecule incapable of associating to the native structure. 5. The effects of gamma-irradiation on both neutral-salt-soluble and acid-soluble collagen are very similar and bear a close resemblance to the effects induced by ultraviolet irradiation.     

The effect of low ionic strength on the radiation chemistry and physical properties of calf thymus DNA

Studies of ultraviolet and circular dichroism spectra of aqueous solutions of calf thymus (CT) DNA confirm the tendency of DNA to change conformation at low ionic strength. The qualitative shape and transition width of 260 nm melting curves below 1 mM NaCl differed significantly from those previously published for DNA solutions containing 1 mM NaCl and above. Neutral aqueous solutions of CT DNA at low ionic strengths (0.1 mM-10 mM NaCl) were irradiated with low doses of gamma-rays. The melting temperature, Tm, of irradiated DNA samples increased below 1 mM NaCl suggesting interstrand crosslinking of the denatured DNA or formation of regions of more thermally stable DNA conformation. The magnitudes of these radiation responses were found to be a function of the time elapsed between salt concentration changes and irradiation as well as time after irradiation. These results are consistent with the hypothesis that the purine and pyrimidine base chromophores in double stranded DNA are sheltered from radical attack by the sugar phosphate backbone. Low dose radiation studies (0.8-8.0 Gy) of CT DNA in 1 mM NaCl and below showed a split dose and dose rate dependence for the sample melting curves.     

Immunochemical studies on the correlation between conformational changes of DNA caused by ultraviolet irradiation and manifestation of antigenicity.

The conformational changes of double-stranded DNA induced during irradiation with ultraviolet, were immunologically investigated. These studies revealed that at least two distinct antigenic sites were induced in the irradiated DNA molecule, giving rise to two different antibodies specific for ultraviolet-irradiated (uv) DNA and thermally denatured DNA-like structure, and these were demonstrable using radioimmunoassay and double diffusion tests. A series of experiments, including melting profile, fluorescence intensity of the ethidium bromide complex and chromatographic behavior on hydroxyapatite, performed on the antigenically active uvDNA indicated that the duplex structure of DNA separated irregularly during irradiation. Furthermore, the data showed that the conformational determinants of uvDNA are located on the exposed single-stranded regions.     

1H-NMR relaxation studies of native and thermally denatured lysozyme solutions: an isotope dilution experiment

1H-NMR relaxation times are reported for native and thermally denatured lysozyme aqueous solutions measured as the function of the proton mole fraction in the sample. A two-exponential character of proton longitudinal relaxation function was observed for native lysozyme solutions: the fast component was attributed to the non-exchangeable protein protons, the slow one to water protons. Purely exponential decay of longitudinal magnetization was observed for the thermally denatured samples. This has been explained in terms of a fast spin exchange model. The contributions of the protein protons to the water proton relaxation rate in native and thermally denatured samples were determined, too.     

The effect of ultraviolet irradiation on soluble collagen

1. The effect of ultraviolet irradiation on acid-soluble and neutral-salt-soluble calf-skin collagen was studied by chromatography, gel filtration, amino acid analysis and sedimentation of the sub-units, and the reaction kinetics of degradation were obtained from viscosity and optical rotation measurements. 2. It was demonstrated that, whereas the structure of neutral-salt-soluble calf-skin collagen may be represented by the formula (alpha(1))(2)alpha(2), the acid-soluble extract has the formula alpha(1).(alpha(2))(2). The acid-soluble collagen is also unusual in containing a large amount of a component that could be beta(22). 3. Ultraviolet irradiation causes the progressive degradation of the collagen molecule into smaller molecular fragments that subsequently lose their helical nature. The rate constants show that the denaturation of soluble collagens by ultraviolet irradiation is much slower, under the conditions used, than denaturation by heat or enzymes.     

The electrical properties and stability of DNA to UV radiation and aromatic hydrocarbons

Studies of the d. c. conductive behavior of solid gels of DNA under high vacuum have shown that a reproducible energy gap of 2.4 eV and a photoelectric effect may be obtained following extensive evacuation procedures. Admission of air for short periods has a reversible effect on this photoelectirc effect and semiconductive behavior. After prolonged illumination in air the sample becomes permanently affected and will not reproduce the photoelectric effect or give rise to a semiconduction energy gap of 2.4 eV but give rise to a permanently lower energy gap of 1.6 eV. The effect of ultraviolet irradiation on the thermal denaturation of DNA in the presence of six aromatic hydrocarbons has been examined in order to determine the extent to which the presence of the hydrocarbon protects the DNA against ultraviolet damage.     

Fibronectin (cold-insoluble globulin), VI. Influence of heparin and hyaluronic acid on the binding of native collagen.

Fibronectin of human plasma associated readily with denatured collagen but gave only a weak reaction with the native protein. In the presence of heparin, however, solutions of native collagen type III, and fibronectin produced precipitates at an ionic strength of 0.2. In the presence of fibronectin and optimal additions of heparin, up to 60% of soluble native 125I-collagen type III, but only about 10% of native 125I-collagen type I, were insolubilized. Heparin also enhanced the formation of insoluble complexes from fibronectin and denatured collagen type I and type III. In the absence of collagen 125I-fibronectin was partially precipitated by heparin. Electron micrographs showed filamentous structures. Collagen did not increase the amount of 125I-fibronectin precipitated by heparin unless a critical collagen concentration was exceeded. It is suggested that heparin induced the transition of fibronectin from a globular to an elongated form, capable of forming filamentous precipitates which adsorb native collagen. Hyaluronic acid and putrescine prevented the insolubilization of native collagen type III, by fibronectin and heparin.     

The effect of microwave thermal denaturation on release properties of bovine serum albumin and gluten matrices

The purpose of this study was to compare the effects of denaturation by microwave irradiation on release properties of 2 physically different proteins. Matrices were prepared from water-soluble bovine serum albumin loaded with metoclopramide and sorbed with adequate amount of moisture were thermally denatured in a microwave oven. The release profile of the rather insoluble denatured albumin matrices followed the classical Fickian diffusion profile. The release rate was dependent on the degree of denaturation, which was highly dependent on the level of moisture originally absorbed by the albuminoidal matrices and the period of exposure to microwave energy. Consersely, attempts to reduce the rate of drug release through microwave irradiation of metoclopramide-loaded matrices prepared from water-insoluble gluten were futile. The denaturation process was shown to be limited to the relatively water-soluble protein core fraction, while aggregation between neighboring gluten proteins in the matrix was not achieved even in the presence of considerable amounts of sorbed water. Published: February 10, 2006     

Nitrous Acid Reactivation of Ultraviolet-Irradiated Transforming DNA from Hemophilus influenzae

Partial recovery of ultraviolet-damaged denatured or native transforming DNA from Hemophilus influenzae, has been obtained by exposing the irradiated DNA in the denatured form to nitrous acid. Some factors that affect this recovery are described. An erythromycin marker (E₂₀) was not reactivated. The UV damage reactivable by nitrous acid is different from that repaired by the photoreactivating enzyme from bakers' yeast. The pretreatment with nitrous acid affords a slight protection for denatured C₂₅ DNA and Sm₂₅₀ DNA against ultraviolet irradiation, but this pretreatment sensitized the E₂₀ DNA to this irradiation.     

Changes in the secondary structure of collagen after gamma-irradiation in an aqueous solution

The conformation of calf skin collagen at the secondary structure level changed after 60Co-gamma-radiation with doses ranging from 50 to 1,000 Gy in aerated aqueous solutions and in the presence of N2 and N2O. Despiralization of collagen at a dose of 1,000 Gy reached 90% in N2O, 70% in N2, and 60% in the aerated solution. The radiochemical yield of the despiralized collagen molecules was 0.007 mol/100 eV owing to OH(.). The presence of radioprotective agents upon irradiation of collagen solutions diminished considerably the injury to its secondary structure.     

Thermal Inactivation of Poliovirus in the Presence of Selective Organic Molecules (Cholesterol, Lecithin, Collagen, and β-Carotene)

Poliovirus type 1 strain LS-a exhibited the typical thermal inactivation pattern observed previously by other investigators for poliovirus strains sensitive to the temperatures used in these experiments. However, when the virus suspension was thermally treated at 121 C for 5 sec in the presence of 2% collagen, a stabilizing effect on the virus was observed. The stabilizing effect in the presence of other food additives, such as cholesterol, lecithin, or beta-carotene, was less dramatic or there was no effect at all. Pretreatment of the cells with the same additives before inoculation induced various changes in the susceptibility of the cells to infection by poliovirus. Lecithin and cholesterol treatment appeared to increase HeLa cell susceptibility to the invading virus, thereby enhancing infectivity. Ultraviolet examination of thermally inactivated virus (121 C) suspensions did not indicate any severe denaturation of the nucleic acid core. Subsequent phenol extraction of the infectious nucleic acid from the heat-inactivated virions revealed that infectious nucleic acid was still present in the denatured heat-treated (62 to 72 C) samples of virion. The immediate past history of treatment of the uninoculated cells appeared to be important, since pretreatment of the cells with cholesterol before inoculation resulted in a noticeable increase in infectivity. In addition, cholesterol-treated uninoculated cell sheets also exhibited an increase in longevity compared to the uninoculated, untreated controls.     

UV damage of collagen: insights from model collagen peptides

Fibrils of Type I collagen in the skin are exposed to ultraviolet (UV) light and there have been claims that collagen photo-degradation leads to wrinkles and may contribute to skin cancers. To understand the effects of UV radiation on collagen, Type I collagen solutions were exposed to the UV-C wavelength of 254 nm for defined lengths of time at 4°C. Circular dichroism (CD) experiments show that irradiation of collagen leads to high loss of triple helical content with a new lower thermal stability peak and SDS-gel electrophoresis indicates breakdown of collagen chains. To better define the effects of UV radiation on the collagen triple-helix, the studies were extended to peptides which model the collagen sequence and conformation. CD studies showed irradiation for days led to lower magnitudes of the triple-helix maximum at 225 nm and lower thermal stabilities for two peptides containing multiple Gly-Pro-Hyp triplets. In contrast, the highest radiation exposure led to little change in the T(m) values of (Gly-Pro-Pro)(10) and (Ala-Hyp-Gly)(10) , although (Gly-Pro-Pro)(10) did show a significant decrease in triple helix intensity. Mass spectroscopy indicated preferential cleavage sites within the peptides, and identification of some of the most susceptible sites of cleavage. The effect of radiation on these well defined peptides gives insight into the sequence and conformational specificity of photo-degradation of collagen.     

Production of Yeast Cells from Methanol

Using immunochemical technique thermal denaturation of soybean 11S globulin, dissolved in different ionic strength solutions (µ=0~4.0) and heated at 100°C for 5 min, has been quantitatively studied. The curves of the percentage of antigenicity remaining were obtained as a function of salt concentration. The 11S globulin became strongly resistant to thermal denaturation with increasing both KCl and potassium phosphate. The stabilizing effect (in terms of percent antigenicity) was separated into three regions. At ionic strength below 0.7, potassium phosphate had no stabilizing effect while KCl had aslightly effect. The rise in stabilizing effect up to about 50%, near 1.0~1.5 µ, represented a second transition to a different denatured state which retains undissociated molecule. At rises up to 75~95%, near 2.5~3.5µ, a different conformational state resulted in which thermally denatured 11S globulin maintained almost intact native conformation after heating. The selection of an adequate ionic strength of protein solution has enabled preparation of thermally denatured 11S globulins which have desired-residual amounts of structured regions.     

Electron spin resonance studies of ultraviolet irradiation effects on some proteins

Summary Electron spin resonance signals of dry protein samples are enhanced after ultraviolet irradiation at wavelength 3650 Å, which corresponds closely to the energy gap between valence and conduction bands.Proteins denatured with short wave ultraviolet or 8 M urea fail to show the enhancement after irradiation at wavelength 3650 Å. This result supports the hypothesis that hydrogen bond structures are relevant in determining the semiconductor properties of proteins.     

In vitro phosphorylation of type I collagen by cyclic AMP-dependent protein kinase

Both the triple-helical and denatured forms of nonfibrillar bovine dermal type I collagen were tested as substrates for the catalytic subunit of cAMP-dependent protein kinase in an in vitro reaction. Native, triple-helical collagen was not phosphorylated, but collagen that had been thermally denatured into individual alpha chains was a substrate for the protein kinase. Catalytic subunit of cAMP-dependent protein kinase phosphorylated denatured collagen to between 3 to 4 mol of phosphate/mol of (alpha 1(I)2 alpha 2(I). Pepsin-solubilized and intact collagens were phosphorylated similarly, as long as each was in a nonhelical conformation. The first 2 mol of phosphate incorporated into type I collagen by the protein kinase were present in the alpha 2(I) chain. The alpha 1(I) chain was only phosphorylated during long incubations in which the stoichiometry exceeded 2 mol of phosphate/mol of (alpha 1(I)2 alpha 2(I). Phosphoserine was the only phosphoamino acid identified in collagen that had been phosphorylated to any degree by the protein kinase. The 2 mol of phosphate incorporated into the alpha 2(I) chain were localized to the alpha 2(I)CB4 cyanogen bromide fragment. The catalytic subunit of cAMP-dependent protein kinase phosphorylated denatured pepsin-solubilized collagen with a Km of 8 microM and a Vmax of approximately 0.1 mumol/min/mg of enzyme. Denatured, but not triple-helical, type I collagen was also phosphorylated by cGMP-dependent protein kinase, although it was a poorer substrate for this enzyme than for the cAMP-dependent protein kinase. Collagen was not a substrate for phospholipid-sensitive Ca2+-dependent protein kinase. These results suggest the potential for nascent alpha chains of type I collagen to be susceptible to phosphorylation by cAMP-dependent protein kinase in vivo prior to triple-helix formation. Such a phosphorylation of collagen could be relevant to the action of cAMP to increase the intracellular degradation of newly synthesized collagen.     

Radiolytic and enzymatic dimerization of tyrosyl residues in insulin, ribonuclease, papain and collagen

Insulin, ribonuclease, papain and collagen solutions saturated with nitrogen, N2O or air were irradiated with doses of 10 to 640 Gy of gamma rays. Protein solutions were also oxidized enzymatically in a system of horse-radish peroxidase: hydrogen peroxide. Column chromatography (Sephadex G-75 or Sephacryl S-200) of treated protein solutions revealed that they contain protein molecular aggregates. Nitrogen saturation of solution before irradiation was most favourable for radiation-induced aggregation of proteins. Fluorescence analysis of protein solutions resulted in detection of dityrosyl structures in irradiated as well as in enzymatically oxidized proteins. Concentration of dityrosine in proteins studied was determined fluorimetrically in their hydrolysates separated on BioGel P-2 column. In irradiated proteins, dityrosine was present almost exclusively in their aggregated forms. In proteins oxidized enzymatically, dityrosine was also present in fractions containing apparently unchanged protein. Mechanisms which could account for differences in the yield of dityrosine formation in radiolysis and in enzymatic oxidation of proteins are suggested.     

Digestion of native collagen, denatured collagen, and collagen fragments by extracts of rat liver lysosomes.

Extracts of highly purified lysosomes from rat liver were examined for their ability to degrade native collagen and thermally denatured collagen at pH values between 3.5 and 7.0. After a 24-h digestion at 36 degrees with the lysosomal extract at a pH of 5.5 or lower (collagen/lysosomal protein; 2/1 or 8/1), both native and denatured collagen were degraded to an extent equivalent to 60 to 70% of that observed upon total acid hydrolysis in 6 N HCl as measured by the ninhydrin reaction (570 nm). At a pH of 6.0, native collagen and denatured collagen were degraded by the mixture of lysosomal proteinases to 11% and 40% of total acid hydrolysis, respectively. At pH 6.5 AND 7.0, the corresponding values were 3% versus 33% and 0.3% versus 11%, respectively. Fragments of collagen (TCA and TCB) are produced when mammalian collagenase degrades native collagen at 25 degrees. These fragments were degraded by the lysosomal extract at 36 degrees to an extent equivalent to 28% and 8% of total acid hydrolysis at pH 6.5 and 7.0, respectively. The experiments at pH 6.5 and 7.0 were done using a collagen/lysosomal protein ratio of 2/1. At pH 5.0 (a pH which is found within secondary lysosomes), the lysosomal extracts degraded collagen to a mixture of free amino acids and small peptides. Amino acid analysis established that approximately 30% of the amino acid residues of the collagen appeared in the lysosomal hydrolysate as free amino acids. Hydroxyproline and perhaps hydroxylysine were the only amino acids found in collagen which did not appear at least to some extent as the free amino acid in this hydrolysate.     

Tolerance of 5,10-methenyltetrahydrofolate to ultraviolet radiation

Aeration of aqueous solutions of 5,10-methenyltetrahydrofolic acid (MTHF) during exposure to ultraviolet irradiation (lambda 300-390 nm, 240 W/m2, 30 min) slowed down photolysis in comparison with deaerated solutions. The rate of photolysis in the presence of oxygen depended on buffer composition. It did not exceed 6% of the starting amount of MTHF. Photolysis of MTHF included opening of the imidazoline ring, dehydrogenation of the tetrahydropterin portion, and elimination of the p-aminobenzoylglutamate moiety. 6,7-Dimethyltetrahydropterin was used as a model compound to show that protonation of the reduced pterin heterocycle increased its tolerance to oxidation, and UV irradiation did not accelerate this process. The stabilizing effect of protonation of the pterin portion and the presence of the positively charged imidazoline moiety are assumed to hamper MTHF oxidation and photolysis. It is assumed that these factors favored the choice of MTHF molecules as photosensors in light-sensitive proteins in the course of evolution.