Changes In Ultraviolet Absorption Research Articles

An Application of Ultraviolet Spectroscopy to Study Interactions in Proteins Solutions at High Concentrations

Studies of protein-protein interactions (PPIs), especially in high-concentration solutions, have become increasingly important from a pharmaceutical perspective. Analytical methods used to study protein interactions, however, rely primarily on the detection of nonideality in relatively dilute (<50 mg/mL) solutions. We present here an application of variable-pathlength ultraviolet (UV)-visible absorption spectroscopy to examine and better understand such interactions over a wide concentration range (5-240 mg/mL) using several representative proteins. In this study, the change in UV absorption (or extinction coefficient) was monitored by determining delta absorbance (ΔAbs), the difference between the measured absorbance and the corresponding theoretical absorbance (calculated from gravimetric dilution), over a wide range of protein concentrations. The ΔAbs, corrected for light scattering, was found to increase with protein concentration for three model proteins (bovine serum albumin, lysozyme, and monoclonal antibody). Because PPIs influence solution viscosity, we studied the correlation between ΔAbs measurements and viscosity as a function of protein concentration. The magnitude of ΔAbs and solution viscosity followed similar trends with increasing protein concentration, albeit to different extents for different proteins. These data support the use of such ΔAbs measurements as an alternative approach to monitor and evaluate interactions in protein solutions at high concentration.

Study on the Interaction of Bovine Serum Albumin with Ceftriaxone and the Inhibition Effect of Zinc (II)

The mechanism of the interaction between bovine serum albumin (BSA) and ceftriaxone with and without zinc (II) (Zn2+) was studied employing fluorescence, ultraviolet (UV) absorption, circular dichroism (CD), and synchronous fluorescence spectral methods. The intrinsic fluorescence of BSA was quenched by ceftriaxone in a static quenching mode, which was authenticated by Stern-Volmer calculations. The binding constant, the number of binding sites, and the thermodynamic parameters were obtained, which indicated a spontaneous and hydrophobic interaction between BSA and ceftriaxone regardless of Zn2+. Changes in UV absorption, CD, and synchronous fluorescence spectral data are due to the microenvironment of amide moieties in BSA molecules. In the BSA-ceftriaxone-Zn2+system, Zn2+must first interact with ceftriaxone forming a complex, which inhibits BSA binding to ceftriaxone. The present work uses spectroscopy to elucidate the mechanism behind the interaction between BSA and ceftriaxone in the presence and absence of Zn2+. The BSA and ceftriaxone complex provides a model for studying drug-protein interactions and thus may further facilitate the study of drug metabolism and transportation.

A RAPID METHOD FOR THE DETERMINATION OF GRISEOFULVIN IN FERMENTER BROTH.

Abstract A method has been devised for the determination of griseofulvin in fermenter broths. This is based on the conversion of griseofulvin to isogriseofulvin with methane sulphonic acid in methanol. The reaction is followed by measuring the resulting change in ultra-violet absorption. The method is rapid, accurate and suited to the routine examination of a wide variety of samples.

Change in the UV-VIS Absorbance of Amino Acids as a Result of Femtosecond Laser Irradiation

Problems such as genetic damage should be considered in the medical application of lasers. Pho- tochemical damage to living tissue is caused by high-photon energy light. Changes in the UV-VIS absorbance of amino acids by femtosecond laser irradiation are investigated in the present study. Ul- traviolet absorption spectra of amino acids were measured following their irradiation by femtosec- ond laser. The results indicate that changes in ultraviolet absorption appeared at a peak intensity above 70 GW/cm 2 , suggesting that a change in the chemical structure was caused by multiphoton absorption during the femtosecond laser irradiation.

The photosensitivity and ultraviolet absorption change of Sn-doped silica film fabricated by modified chemical vapor deposition

10.5 μ m thick Sn-doped silica films were prepared by the modified chemical vapor deposition followed by the solution-doping method. The films were exposed to 248 and 266nm laser light, respectively. Positive refractive index change up to 2×10−4 at 1550nm was observed by measuring the reflectivity based on Fresnel formulas. The data of UV absorption spectra suggest that the photosensitivity of the Sn-doped silica film under high energy density laser irradiation should be mainly due to the bond breaking of oxygen deficient defects, while under relatively low energy density laser irradiation, the refractive index change probably originates from photoconversion of optically active defects.

Changes in Ultraviolet Absorption of Sunscreens After Ultraviolet Irradiation

The incidence of skin cancer is increasing rapidly and sunscreens have been recommended in order to reduce damage from sunlight. In this investigation we have studied the change in the absorption spectrum of some photoactive organic species in sunscreens after ultraviolet A and ultraviolet B irradiation in a dose normally encountered during a full day in the sun. Samples of a number of photoactive compounds commonly used in sunscreens were irradiated with ultraviolet A and ultraviolet B light. A UVASUN 2000 MUTZHAS sunlamp was used for ultraviolet A irradiation and an Esshå Corona mini, equipped with two Philips TL12 20 W lamps, was used as the ultraviolet B source. The ultraviolet A dose was 100 J per cm2. The ultraviolet B dose corresponded to 20 minimal erythema doses. The absorption spectra of the compounds were recorded before and after irradiation. The absorbance of 2-ethylhexyl 4-methoxycinnamate was reduced significantly, whereas 3-(4-methylbenzyliden)camphor seemed to be rather stable. The benzophenones studied seemed to be relatively stable. In the case of 4-tert. butyl-4'-methoxy-dibenzoylmethane there was a rapid decrease in the ultraviolet A absorption leading to unsatisfactory protection in the ultraviolet A region. 4-Isopropyl-dibenzoylmethane also lost most of its ultraviolet protective capacity after irradiation with ultraviolet A. Ultraviolet B seemed to have a minor effect on all the samples. It is important for the clinician not only to know the initial absorption spectrum in the ultraviolet region for a specific sunscreen substance, but also whether it is altered during irradiation and in what way. This study including gas chromatography and mass spectrometry analysis indicates that some of the photoactive organic species commonly used today in sunscreens are unstable following ultraviolet irradiation.

Use of a one atmosphere uniform glow discharge plasma to kill a broad spectrum of microorganisms

The medical, industrial, and food processing industries are constantly in search of new technologies to provide improved methods of sterilization and pasteurization. Proposed techniques must deal with such problems as thermal sensitivity and destruction by heat, formation of toxic by-products, cost, and inefficiency in performance. We report results from a newly invented plasma source, a one atmosphere uniform glow discharge plasma (OAUGDP), which is capable of operating at atmospheric pressure in air and providing antimicrobial active species at room temperature. OAUGDP exposures have reduced log numbers of bacteria (Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa), bacterial endospores (Bacillus subtilis and Bacillus pumilus), and various yeast and bacterial viruses on a variety of surfaces. These surfaces included polypropylene, filter paper, paper strips, solid culture media, and glass. Experimental results showed at least a ⩾5 log10 colony forming units (CFU) reduction in bacteria within a range of 15–90 s of exposure, whether the samples were exposed in conventional sterilization bags or exposed directly to the plasma. An exception to these very short exposure times were experiments with solid culture media in which 5 min of plasma exposure was necessary to produce ⩾5 log10 CFU reduction in bacterial counts. The effects of plasma treatment on bacterial cell structures were investigated by exposing cells to plasma for various durations and examining them by transmission electron microscopy. The results showed that cell breakage (lysis) occurred with the release of cellular contents. These data were consistent with spectrophotometric results in which the release of cellular constituents was measured as a change in ultraviolet absorption at 260 nm. With all microorganisms tested, a biphasic survival curve (logarithmic number of survivors versus time) was observed in plots of dose-response data. Differences in susceptibilities of microorganisms observed on various surfaces suggested that the degree of lethality was dependent upon the time of diffusion of active species through the medium to the organism and the makeup of the microbial cellular surface.

High temperature proton implantation induced photosensitivity of Ge-doped SiO2 planar waveguides

The possibility of using keV proton implantation at 800 °C to enhance the photosensitivity of Ge-doped silica has been investigated. Room temperature implantation induced defects indicated by absorption at ultraviolet (UV) (&amp;lt;200 nm) and visible wavelengths (&amp;gt;550 nm) were annealed during implantation at 800 °C to leave stable photosensitive neutral oxygen vacancy (NOV) centers with an absorption peak at ∼240 nm. The stable NOV defects were photochemically bleached after UV exposure, a process which is accompanied by a change in UV absorption. Positron annihilation spectroscopy demonstrated the effectiveness of implantation at 800 °C in annealing the implantation induced damage.

The new inhibitors of ribonucleotide reductase—comparison of some physico-chemical properties

Amidox (AX), didox (DX) and trimidox (TX), compounds synthetized as new ribonucleotide reductase inhibitors, have been investigated by ultraviolet (UV) spectrophotometry, polarography and high performance liquid chromatography (HPLC). The experiments have been performed at various pH values. The changes in UV absorption of the compounds studied were recorded and it was demonstrated that these changes are related to the pH and to structural features of the investigated molecules. From the compounds included in our series of experiments, only amidox and trimidox are reduced during polarographic experiments in Britton Robinson buffer. The reduction of both compounds proceeded in two one-electron steps in acidic pH. One two-electron diffuse irreversible wave was observed at basic pH. The values of the half-wave potential became more negative in accordance with the increasing pH. HPLC assay also showed changes in the retention of compounds investigated, particulary when the pH of the mobile phase was close to the dissociation constant of the particular drug. The changes of physico-chemical properties detected by the all used methods are related to different chemical structures (the most significant changes were observed in alkaline pH).

Continuous spectrophotometric assay of protein tyrosine phosphatase using phosphotyrosine

A continuous activity assay for protein tyrosine phosphatases (PTPs), employing phosphotyrosine (P-Tyr) as a substrate, has been developed and applied to measure the activities of two purified enzymes, namely, the full length T-cell protein tyrosine phosphatase (TC PTP) and its truncated form (TCΔC11 PTP). The reaction was followed by changes in ultraviolet absorption and fluorescence resulting from the dephosphorylation of P-Tyr. Both enzymes obey Michaelis-Menten kinetics, with K m = 304 μm, V max = 62,000 units/mg for TC PTP and K m = 194 μm, V max = 73,000 units/mg for TCΔC11 PTP. The d- and l-forms of P-Tyr are equally effective as substrates. The optimum pH for both enzymes is 4.75. The known effectors of PTPs have the predicted effects on catalytic activity.

An agreement on the quaternary oscillation of ultraviolet absorption changes accompanying the water splitting in isolated Photosystem II complexes from the cyanobacterium Synechococcus sp.

Authors of controversial publications with regard to the S-transition spectra of the water-splitting enzyme system S together reinvestigated the transitions in isolated Synechococcus Photosystem II complexes. It is confirmed that the spectrum of the S 0-S 1 transition is different from the spectra of the S 1-S 2 and S 2-S 3 transitions: at 355 nm, the S 0-S 1 transition is more than three times smaller. The S 0-S 1 transition is possibly due to an Mn(II) → Mn(III) change, the latter ones due to Mn(III) → Mn(IV) changes.

Studies on the nature of the water-oxidizing enzyme: II. On the functional connection between the reaction-center complex and the water-oxidizing enzyme system Y in Photosystem II

Laser-flash-induced absorption changes have been measured with a time resolution of 1–2 μs in PS II membrane fragments. The following was found. (1) In the presence of 0.5 mM K 3 [Fe(CN) 6] at pH = 6.0 absorption changes at 830 nm induced by repetitive flash excitation in PS II membrane fragments pretreated (10 min) with trypsin at pH = 7.5 are dominated by 10–15 μs kinetics. Under the same conditions the relaxation kinetics at 325 nm are slower by at least two orders of magnitude. (2) In the same samples μs components contributing to the overall kinetics of transient ultraviolet absorption changes have the same half-life time as the 830 nm relaxation kinetics within the range of 4.0 ≤ pH ≤ 8.0. (3) At pH = 6.0 the extent of 10–15 μs kinetics of ultraviolet absorption changes as a function of wavelength is characterized by peaks at 260 nm, 300 nm, 360 nm (positive) and 340 nm (negative). It is vanishingly small in the range of 315–325 nm. (4) In the presence of 1 mM hydroxylamine dark-adapted samples illuminated with a few flashes (n ≤ 8) revealed the same pattern at 270 nm and 830 nm as PS II membrane fragments trypsinized at pH = 7.5. In contrast to that, after preillumination with 70 flashes the absorption changes at 270 nm and 830 nm exhibit slower kinetics in the microsecond range with half-life times of 200–300 μs. These results are interpreted as a light requiring disconnection of Z from P-680 by hydroxylamine. (5) After subtraction of the difference spectrum for the reduction of the primary plastoquinone acceptor Q A the extent of the separated initial amplitudes (limited by time resolution of 1–2 μs) as a function of wavelength exhibit characteristic features, depending on the functional integrity of the water-oxidizing enzyme system Y. If the samples were deprived of their oxygen-evolving capacity the data obtained resemble the difference spectrum reported for oxidation of chlorophyll a in solution (Borg, D.C., Fajer, J., Felton, R.H. and Dolphin, D. (1970) Proc. Natl. Acad. Sci. USA 67, 813–820). On the other hand, a markedly different spectrum is obtained in samples with a functionally competent water-oxidizing enzyme system Y. Based on kinetical arguments this difference spectrum is inferred to reflect Z-oxidation in oxygen-evolving PS II membrane fragments. The shape of the Z ox Z difference spectrum appears to be slightly affected by the functional integrity of systems Y. (6) No direct evidence was obtained for the existence of a kinetically and spectrally distinguishable redox component D x between Z and P-680 in samples that were deprived of their oxygen-evolving capacity. On the basis of the present data the mode of functional coupling between system Y and P-680 is discussed. Furthermore, implications of possible changes in the electrostatic interactions between the redox active chromophors and the surrounding protein matrices are considered.

Molecular recognition. II. The binding of the Lewis b and Y human blood group determinants by the lectin IV of Griffoniasimplicifolia

Using a radioimmunoassay to measure the relative potencies as inhibitors of a wide number of chemically modified structures related to the Lewis b human blood group determinant, it was found that derivatives of the Lewis b (αLFuc(1→2)βDGal(1→3)[αLFuc(1→4)]βDGlcNAc) and the Y (αLFuc(1→2)PDGal(1→4)[αLFuc(1→3)]βDGlcNAc) determinants are complexed by the lectin IV of Griffoniasimplicifolia through the recognition of a topographical feature that is common to both the tetrasaccharides. This surface provides a nonpolar region formed by the two methyl groups of the fucose units and extends along the C-1—O-5—C-5 side of the αLFuc(1→2) unit and terminates at one end by a polar grouping which is formed by OH-3 and OH-4 of the βDGal unit and OH-4 of the αLFuc(1→4) unit. Association constants were determined from changes in ultraviolet absorption that occur as the result of complex formation. For the reaction of the Lewis b-OCH3 tetrasaccharide, the thermodynamic parameters were found to be ΔH = −13 kcal/mol and ΔS = −22 cal/mol/K. The inhibition data for the relevant monodeoxy derivatives indicated that OH-2 and OH-3 of both of the αLFuc units are not directly involved in the binding reaction. The basis for drawing these conclusions was strengthened by finding that the reaction of the simple Lewis b analog, methoxymethyl (1→2)βDGal(1→3)[αLFuc(1→4)]βDGlcNAcOCH3 displayed very similar thermodynamic values; namely, ΔH = −14 kcal/mol and ΔS = −26 cal/mol/K, to those mentioned above for the Leb-OCH3 tetrasaccharide. The OH-4 of the αLFuc(1→2) unit may be bound in an intramolecularly hydrogen bonded form.

Time‐dependent ultraviolet absorption changes in proteins at high pH

The effect of alkali on the ultraviolet absorption of several proteins was examined by difference spectrophotometry. In addition to the well known generation of phenolate ions from tyrosine, time-dependent changes occurred. These were relatively slow in water, but arose more quickly and to a greater degree in 6 M guanidine hydrochloride. These time-dependent changes were attributed to modification of the sulfur-containing amino acids, cystine and cysteine. The magnitude of the changes depended on the number and accessibility to solvent of the cystine, cysteine or derivatized species present. The increase in absorption at 295 nm typically reached a maximum value for disulfide containing proteins after ca. 1 h exposure to pH greater than 12 in 6 M guanidine hydrochloride; thereafter the changes were at least partially reversible. Taken in conjunction with amino acid analysis data, the results lend support to the beta-elimination mode of action of alkali on proteins. However the reaction mechanism appears to be complex and more than one chromophore, arising from more than one reaction pathway, seems to be involved in the alkaline degradation of the sulfur-containing amino acids. Particularly for proteins containing large amounts of cystine or cysteine, caution must be exercised when performing tyrosine titration experiments in order to recognize and minimize potential interference from other non-tyrosine related chromophores.

Temperature-induced ultraviolet absorption changes of heavy meromyosin: An application of a computerized spectrophotometer system

The UV absorption of HMM (heavy meromyosin) was measured at various temperatures with a computerized spectrophotometer system. HMM showed temperature-induced absorption changes in the presence and absence of nucleotides. The temperature-induced absorption change at 293 nm, which is due to conformational changes around the tryptophan residues of HMM, was enhanced in the presence of nucleotides. The temperature-induced difference spectra of HMM + AMPPNP relative to HMM obtained by using a conventional spectrophotometer [(1977) J. Biochem. (Tokyo) 81, 313–320] could be reproduced by subtracting the temperature-induced spectral changes of HMM from those of HMM + AMPPNP.

Spectral characterization in the ultraviolet region of the precursor of photosynthetically evolved oxygen in isolated trypsinized chloroplasts

The reaction mechanism of the water-splitting enzyme system Y has been analyzed by measurements of ultraviolet absorption changes induced by a flash train in dark-adapted chloroplasts. In order to eliminate processes which gives rise to interfering absorption changes with a binary oscillation, mildly trypsizined chloroplasts were applied which are known to be disconnected from the secondary plastoquinone acceptor B, but are able to evolve oxygen, provided that K 3Fe(CN) 6 is used as electron acceptor (Renger, G. (1976) Biochim. Biophys. Acta 440, 287–300). It was found that: (a) The extent of the relaxation kinetics of the 320 nm absorption change accomplished within 5 ms oscillates with a periodicity of four in phase with the oxygen yield as a function of the number of the flash in the train. (b) The difference spectra of the difference between the initial amplitudes and those remaining after a 4.5 ms dark period, referred to as ΔA 4.5 n , exhibit pronounced bands peaking around 320 nm (positive) and 270 nm (negative). The extent oscillates in the flash train. It is at a maximum for the absorption change differences induced by the third flash, ΔA 4.5 3. (c) Taking into account the fact that the first flash of the train does not yield oxygen, whereas the oxygen production is at a maximum due to the third flash, the difference of ΔA 4.5 3 − ΔA 4.5 1 = ΔA 4.5 3,1 as a function of wavelength is interpreted as the difference spectrum of the precursor of molecular oxygen in system Y. The spectrum exhibits a band peaking around 320 nm. Accordingly, the precursor is referred to as Y-320. The data obtained are discussed in terms of a molecular model proposed for the mechanism of photosynthetic water oxidation (Renger, G. (1977) FEBS Lett. 81, 223–228).

Measurement of human placental 5′-AMP deaminase activity by radiometric assay

The level of 5′-AMP deaminase in homogenates of human term placenta has been measured by means of a simple radiometric assay. The assay uses 14C-labeled AMP as substrate and incorporates conditions of pH and K + concentration, which optimize the 5′-AMP deaminase activity, and inhibitors of 5′-nucleotidase and adenosine deaminase to reduce interference from these enzymes. Assay products are separated by descending paper chromatography and quantitated by liquid scintillation counting. The activity of 5′-AMP deaminase in human term placenta determined by this assay was 474 ± 37 nmol min −1 g −1 at 30°C and was less than the 5′-AMP phosphatase activity evident under the same assay conditions. The assay is suitable for measurement of 5′-AMP deaminase in extracts of other tissues in which high levels of phosphatases and adenosine deaminase preclude assay of 5′-AMP deaminase by such techniques as ultraviolet absorption changes or ammonia estimation.

Photoreactive derivatives of corticotropin. 1. Preparation and chracterization of 2,4-dinitro-5-azidophenylsulfenyl derivative of corticotropin.

A photoaffinity probe for corticotropin (ACTH) receptors was prepared by selective modification of the single tryptophan residue in ACTH. A new photoreactive agent, 2,4-dinitro-5-azidophenylsulfenyl chloride, was synthesized and used for introducing the photoreactive group into ACTH. 2,4-Dinitro-5-azidophenylsulfenyl-Trp9-ACTH (DNAPS-ACTH) was also prepared by thiolysis of 2,4-dinitrophenyl-sulfenyl-Trp9-ACTH to form 2-thiol-Trp9-ACTH and reaction of this with 2,4-dinitro-5-azidofluorobenzene. DNAPS-ACTH was characterized by ultraviolet spectra, peptide mapping, and amino acid analysis. Covalent attachment of DNAPS-ACTH to a pituitary protein fraction FI by photolysis was demonstrated by ultraviolet absorption changes as well as by the use of tritiated DNAPS-ACTH.

γ Irradiation of Bowman-Birk Soybean Proteinase Inhibitor

WANDELL, J. L., AND KAY, E. 7 Irradiation of Bowman-Birk Soybean Proteinase Inhibitor. Radiat. Res. 72, 414-426 (1977). Radiation damage to Bowman-Birk soybean proteinase inhibitor was studied in dilute aqueous solutions containing: (a) air, (b) N20 plus KBr, and (c) NO20 plus KCNS. The degradation of tyrosines, monitored by changes in ultraviolet absorption and circular dichroism (CD) spectra of irradiated inhibitor, led to the loss of chymotrypsininhibitory activity without affecting antitrypsin activity. Of two tyrosyl residues in the inhibitor, Tyr 45, located next to the antichymotrypsin site, was shown to be essential to chymotrypsin-inhibitory activity. Radiation damage to Tyr 59 had no effect on either of the antiproteinase activities. The loss of trypsin-inhibitory activity paralleled decrease in disulfide CD. The breakage of disulfide bonds was confirmed by the p-chloromercuribenzoate assay for sulfhydryl groups. However, the exact role of disulfide bonds in antitryptic activity, other than the maintenance of conformational integrity, is not apparent. It seems more likely that the loss of trypsin-inhibitory activity is due to damage in other amino acid (s), although the degradation of tyrosine (2 residues) and histidine (1 residue) did not affect anti

42] Modification of tryptophan to oxindolylalanine by dimethyl sulfoxide-hydrochloric acid

The high reactivity of the indole nucleus of tryptophan toward electrophilic reagents has evoked much interest in finding techniques for the selective chemical modification of tryptophan residues in proteins. However, in spite of the variety of reagents introduced over the years for this purpose, only a few are specific for tryptophan residues and yield a single well-defined product. The procedure described in this chapter allows for various permutations because different halo acids and sulfoxides can be used in combination. Effective bromination and iodination systems can be produced by using HBr or HI with a sulfoxide. The treatment of a protein with dimethyl sulfoxide/hydrochloric acid (DMSO/HCl) under the above conditions brings about negligible overoxidation to the dioxindole level or fission of the tryptophanyl bond, whereas with DMS0/HBr both of the secondary reactions occur to a considerable extent. The oxidation of tryptophan to oxindolylalanine residues in peptides and proteins by DMSO/HCl can be followed by determining the ultra-violet absorption changes that accompany the oxidation of the indole nucleus to the oxindole chromophore.