Interaction of serum albumin with normal and sickle hemoglobins.

The stability of oxyhemoglobin S during mechanical shaking was enhanced by the addition of human serum albumin. The stabilizing effect was maximum when the concentration of serum albumin approached that of oxyhemoglobin, suggesting a molecular level interaction between them. The effects of serum albumin on oxyhemoglobin A were essentially similar to those on oxyhemoglobin S. Deoxy- and methemoglobins were also stabilized by serum albumin. The addition of human serum albumin to a solution containing sickle cell oxyhemoglobin slowly formed a compound which had an absorbance peak at 620 nm. After purification by Sephadex G-200 column chromatography, this compound was identified as methemalbumin. Comparison of the rates of formation of methemalbumin from hemoglobin with various ligand states and human serum albumin showed that the rate of formation from hemichrome was much faster than from met-, oxy- and deoxyhemoglobin. About 60% of the heme was transferred from hemichrome to albumin when the mixture was kept standing at room temperature for 5 min, in contrast to only 5% from methemoglobin. This result suggests that hemichrome, rather than methemoglobin, is the intermediate in the formation of methemalbumin from oxyhemoglobin and human serum albumin. This hypothesis is supported by the finding that the rate of formation of methemalbumin was faster at alkaline pH values than at acid pH values. Serum albumin from various animal sources showed different stabilizing effects. The formation of methemalbumin from these animal albumins was far less than that from human albumin.     

Spectrophotometric assay of long-chain unsaturated and hydroxy fatty acids in concentrated sulfuric acid

A spectrophotometric method has been developed for the determination of long-chain unsaturated and hydroxy fatty acids in concentrated sulfuric acid. The assay is based on the absorbance produced in the 290 to 300-nm range from their reaction with sulfuric acid at 100°C. α,β-Unsaturated aliphatic acids give absorption bands at 235–240 nm and thus can be easily differentiated from unsaturated fatty acids having the double bond(s) at positions not conjugated with the carboxyl group. A certain minimum chain length is required for full development of the absorption band at 300 nm. Position and intensity of the so-formed absorption band is independent on the position and number of the double bonds or hydroxyl groups. Carboxyl groups are not essential, as unsaturated hydrocarbons and higher alcohols likewise react with sulfuric acid to produce the absorbing species at 300 nm, providing a minimum chain length of 5 carbon atoms is present. The nature of the absorbing species at 300 nm is discussed.     

Determination of dipicolinic acid in bacterial spores by derivative spectroscopy

Dipicolinic acid was extracted from approximately 0.1 mg spores or 0.5 ml of sporulating culture with 20 mM HCl for 10 min at 100 degrees C. The suspension was diluted with 5 mM Ca2+, 100 mM Tris, pH 7.6, centrifuged, and the first derivative of the uv absorbance spectrum recorded from 275 nm to 285 nm. DPA concentration was determined from the difference between the maximum at 276.6 nm and the minimum at 280 nm. The use of the difference between two first derivative values removed possible interference from sloping baselines. Turbidity, nucleic acids, and bacteriological media did not interfere. Analysis time for four extracts was 4 min using a spectrophotometer reading at 0.1-nm intervals. Dipicolinate at 0.1 mM gave 0.184 absorbance/nm at 25 degrees C. The coefficient of variation was 1.5%, and the detection limit 1 microM.     

Initiation of lipid peroxidation as a result of hemoglobin transformation into hemichrome induced by free fatty acids

The effects of free fatty acids on hemoglobin conversion and lipid peroxidation were studied in hemoglobin-containing liposomes (hemosomes) formed from an equimolar mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). It was shown that in hemosomes oxyhemoglobin is converted into hemichrome by the interaction of saturated fatty acids (arachidic, stearic, palmitic, myristic and lauric). This is accompanied by accumulation of primary and secondary products of lipid peroxidation. All fatty acids, except for lauric acid, have a stabilizing effect on lipid peroxidation in liposomes prepared from an equimolar mixture of PC and PE. The formation of lipid peroxidation products is inhibited by superoxide dismutase, D-alpha-tocopherol, D-mannitol and thiourea. The relationships between hemoglobin conversion and lipid peroxidation in hemosomes under effects of fatty acids were studied. The mechanisms of these reactions are discussed.     

Excitation spectra for photosystem I and photosystem II in chloroplasts and the spectral characteristics of the distributions of quanta between the two photosystems.

The parameters listed in the title were determined within the context of a model for the photochemical apparatus of photosynthesis. The fluorescence of variable yield at 750 nm at -196 degrees C is due to energy transfer from Photosystem II to Photosystem I. Fluorescence excitation spectra were measured at -196 degrees C at the minimum, FO, level and the maximum, FM, level of the emission at 750 nm. The difference spectrum, FM-FO, which represents the excitation spectrum for FV is presented as a pure Photosystem II excitation spectrum. This spectrum shows a maximum at 677 nm, attributable to the antenna chlorophyll a of Photosystem II units, with a shoulder at 670 nm and a smaller maximum at 650 nm, presumably due to chlorophyll a and chlorophyll b of the light-harvesting chlorophyll complex. Fluoresence at the FO level at 750 nm can be considered in two parts; one part due to the fraction of absorbed quanta, alpha, which excites Photosystem I more-or-less directly and another part due to energy transfer from Photosystem II to Photosystem I. The latter contribution can be estimated from the ratio of FO/FV measured at 692 nm and the extent of FV at 750 nm. According to this procedure the excitation spectrum of Photosystem I at -196 degrees C was determined by subtracting 1/3 of the excitation spectrum of FV at 750 nm from the excitation spectrum of FO at 750 nm. The spectrum shows a relatively sharp maximum at 681 nm due to the antenna chlorophyll a of Photosystem I units with probably some energy transfer from the light-harvesting chlorophyll complex. The wavelength dependence of alpha was determined from fluorescence measurements at 692 and 750 nm at -196 degrees C. Alpha is constant to within a few percent from 400 to 680 nm, the maximum deviation being at 515 nm where alpha shows a broad maximum increasing from 0.30 to 0.34. At wavelengths between 680 and 700 nm, alpha increases to unity as Photosystem I becomes the dominant absorber in the photochemical apparatus.     

Hydration-dependent conformational states of hemoglobin. Equilibrium and kinetic behavior

The equilibrium and kinetics of methemoglobin conversion to hemichrome induced by dehydration were investigated by visible absorption spectroscopy. Below about 0.20 g water per g hemoglobin only hemichrome was present in the sample; above this value, an increasing proportion of methemoglobin appeared with the increase in hydration. The transition between the two derivatives showed a time-dependent biphasic behavior and was observed to be reversible. The rates obtained for the transition of methemoglobin to hemichrome were 0.31 and 1.93 min-1 and for hemichrome to methemoglobin 0.05 and 0.47 min-1. We suggest that hemichrome is a reversible conformational state of hemoglobin and that the two rates observed for the transition between the two derivatives reflect the alpha- and beta-chains of hemoglobin.     

Induced hemichrome formation of methemoglobins A,S and F by fatty acids,alkyl ureas and urea

• 1.1. Spectral analysis of the Soret region (450-350 nm) has shown that saturated fatty acids, alkyl ureas and urea induce the conversion of methemoglobins A, S, and F to the hemichrome state.
• 2.2. In the presence of fatty acids (C8-C16), methemoglobin F is converted to the hemichrome state more readily than either methemoglobins A or S.
• 3.3. Using several alkyl ureas (methyl, ethyl, propyl, butyl), the extent of hemichrome formation was as follows: met Hb F > met Hb S > met Hb A. The ability of these compounds to induce hemichrome formation is related to their increasing hydrophobicity.
• 4.4. Conversion to the hemichrome state in the presence of urea (5M) led to the formation of molecular aggregates of hemoglobins S, F and A which may be initated by subunit dissociation and conformational changes, coupled to increased globin-globin interactions.
• 5.5. Similar aggregation occurred for methemoglobin S in the presence of octanoic acid; no significant aggregation was evident for methemoglobin A after 10 hr of exposure to octanoic acid.

     

An ultraviolet spectrophotometric assay for measuring lipase activity using long-chain triacyglycerols from Aleurites fordii seeds

In this study, we designed a specific, continuous, and sensitive UV spectrophotometric lipase assay using natural triacylglycerols (TAGs) from the Aleurites fordii seed oil (tung oil). alpha-Eleostearic acid (9,11,13-cis, trans,trans-octadecatrienoic acid) is the main fatty acid component (it accounts for up to 70%) of the TAGs from tung oil. The conjugated triene present in alpha-eleostearic acid constitutes an intrinsic chromophore, which confers strong UV absorption properties on both the free fatty acid and the TAGs from tung oil. The lipase assay is based on the difference between the apparent molar extinction coefficients of the two types of alpha-eleostearic acid present, that which is esterified into TAGs and that which is released into the reaction medium. This difference is responsible for the variations in the UV absorption spectrum of the reaction medium occurring upon enzymatic TAGs hydrolysis. Using the purified lipase from Thermomyces lanuginosa (TLL) and the detergent sodium taurodeoxycholate (NaTDC, 4 mM), it was established that the most suitable method of measuring lipolysis consisted of monitoring the decrease in the OD at 292 nm, which was linear with time and proportional to the amount of lipase added. In order to be able to estimate the specific activity of TLL, we determined an apparent molar extinction coefficient of alpha-eleostearic acid (epsilon = 13,900 M(-1) cm(-1)) under the assay conditions. Amounts of pure TLL as small as 1 ng can be easily detected in the presence of 4 mM NaTDC. Interestingly, the NaTDC concentration can be decreased as far as 0.05 mM. In comparison with other well-known methods of lipase assay, the detection limit of this new method is 100-fold lower than with the pH-stat method and similar to that of a fluorescent assay recently developed at our laboratory.     

A continuous spectrophotometric method for the determination of diphenolase activity of tyrosinase using 3,4-dihydroxymandelic acid.

A continuous spectrophotometric method for the rapid determination of diphenolase activity of tyrosinase is described. It uses 3,4-dihydroxymandelic acid (DOMA) as the substrate of tyrosinase and measures the final product, 3,4-dihydroxybenzaldehyde (DOBA). The spectrum of this product shows a bathochromic displacement of its absorbance maximum when the pH increases. The optimization of the method is described by using tyrosinase from several biological sources, whose enzymatic activities show different optimal pH. Thus, the enzymatic activity of mushroom tyrosinase was assayed at pH 7.5 and monitored at 350 nm (epsilon 350 pH 7.5 (DOBA) = 15,200 M-1 cm-1), whereas the spectrophotometric experiments with grape tyrosinase were carried out at pH 3.0 and monitored at 310 nm (epsilon 310 pH 3.0 (DOBA) = 9200 M-1 cm-1). The method for mushroom tyrosinase was found to be 50-fold more sensitive than the commonly used dopachrome assay, whereas for grape tyrosinase the method was found to be threefold more sensitive than the commonly used o-quinone production assay. The great solubility and stability of the chromophoric product, DOBA, as well as its high molar absorptivities at any pH, enable the method to be employed to determine the diphenolase activity of tyrosinase from different biological sources.     

Analysis of the acid and alkaline dissociation of earthworm hemoglobin, Lumbricus terrestris, by front-face fluorescence spectroscopy

The steady-state fluorescence properties of the multisubunit hemoglobin isolated from the earthworm, Lumbricus terrestris, were studied by front-face fluorometry. Acid and alkaline dissociation of this high-molecular-weight hemoglobin were examined over the pH range 3.7-12.5 using different liganded states (oxy, CO, met). The relative intensity of the emission maximum at 320 nm (exc. 280 nm) is ligand-dependent increasing as follows: oxy less than deoxy less than CO less than met at pH 7.0. The intensity of the emission maximum of oxyhemoglobin at the alkaline acid end point, pH 10.5 (333 nm), is significantly greater than that observed at the acid end point, pH 4.18 (320 nm), suggesting different subunit dissociation. The spectra of oxyhemoglobin at pH 4.18 and the spectrum of carbonmonoxy hemoglobin at pH 7.0 in the presence of 1 M magnesium chloride were almost identical, indicating similar subunit dissociation. Difference spectrum (pH 9.0-7.2) of fluorescence emission (exc. 305) resulted in a maximum at 341 nm, indicative of tyrosinate formation. This suggests that tyrosine(s) may also be located at the subunit interface(s) of this hemoglobin. These studies indicate that several aromatic amino acid residues are associated with the critical sites of subunit interactions within this molecule. Analysis of the fluorescence spectra also suggests that the formation of different subunit species resulting from acid and alkaline dissociation cannot be ruled out.     

Excitation spectra for Photosystem I and Photosystem II in chloroplasts and the spectral characteristics of the distribution of quanta between the two photosystems

The parameters listed in the title were determined within the context of a model for the photochemical apparatus of photosynthesis.

The fluorescence of variable yield at 750 nm at −196 °C is due to energy transfer from Photosystem II to Photosystem I. Fluorescence excitation spectra were measured at −196 °C at the minimum, F_O, level and the maximum, F_M, level of the emission at 750 nm. The difference spectrum, F_M–F_O, which represents the excitation spectrum for F_V is presented as a pure Photosystem II excitation spectrum. This spectrum shows a maximum at 677 nm, attributable to the antenna chlorophyll a of Photosystem II units, with a shoulder at 670 nm and a smaller maximum at 650 nm, presumably due to chlorophyll a and chlorophyll b of the light-harvesting chlorophyll complex.

Fluorescence at the F_O level at 750 nm can be considered in two parts; one part due to the fraction of absorbed quanta, , which excites Photosystem I more-or-less directly and another part due to energy transfer from Photosystem II to Photosystem I. The latter contribution can be estimated from the ratio of F_O/F_V measured at 692 nm and the extent of F_V at 750 nm. According to this procedure the excitation spectrum of Photosystem I at −196 °C was determined by subtracting 1/3 of the excitation spectrum of F_V at 750 nm from the excitation spectrum of F_O at 750 nm. The spectrum shows a relatively sharp maximum at 681 nm due to the antenna chlorophyll a of Photosystem I units with probably some energy transfer from the light-harvesting chlorophyll complex.

The wavelength dependence of was determined from fluorescence measurements at 692 and 750 nm at −196 °C. is constant to within a few percent from 400 to 680 nm, the maximum deviation being at 515 nm where shows a broad maximum increasing from 0.30 to 0.34. At wavelengths between 680 and 700 nm, increases to unity as Photosystem I becomes the dominant absorber in the photochemical apparatus.     

Photoconversion of hemoglobin forms in buffer solutions under the effect of ultraviolet and visible radiation

Using spectrophotometric methods, the photochemical behaviour of hemoglobin in buffer solutions irradiated at 254-578 nm was studied. It was found that the hemoglobin photochemical activity is manifested in a cascade of photoreactions involving at least four protein forms and soluble oxygen. Hemoglobin photoconversions were observed both upon light absorptions by the globin and the heme. The efficiency of photoreactions decreased with an increase in the wavelength of the irradiating light. The values of quantum yields of the above photoreactions were determined.     

Electron paramagnetic resonance of Hb St Louis beta28 (B10) Leu replaced by Gln.

Hemoglobin St Louis beta28 (B10) Leu replaced by Gln is a new mutant which occurs as a natural valency hybrid (alpha2beta+2), or hemoglobin M (Cohen-Solal, M., Seligmann, M., Thillet, J. and Rosa, J. (1973) FEBS Lett. 33, 37-41). The electron paramagnetic resonance (EPR) spectrum of native Hb St Louis at pH 6.2 shows a mixture of three species. Two are high spin, one with tetragonal symmetry, like Hb+ A, the other with rhombic distortion. The third is a low-spin form corresponding to a hemichrome with the distal (E7) histidine as the sixth ligand of the ferric iron. The hemichrome is also found in red blood cells. After oxidation to the alpha+2beta+2 form, three EPR species are seen. Surprisingly, there remains only one high-spin signal, with almost tetragonal symmetry. Besides the low-spin hemichrome, a hydroxy signal is observed even at pH 6.2. These observations imply interactions between the alpha and beta hemes.     

Tabtoxin-resistant protein: overexpression, purification, and characterization

One of the self-protection mechanisms in Pseudomonas syringae pv. tabaci, a pathogen of tobacco wildfire, is thought to be due to its tabtoxin-resistance gene (ttr). In this study, the ttr gene was inserted into an expression vector, pQE30, and successfully expressed in Escherichia coli M15 at high levels. The purified recombinant tabtoxin-resistant protein (TTR) had an apparent molecular mass of about 21 kDa on SDS-PAGE as well as by mass spectroscopy and had a pI of 6.6 on isoelectric focusing-PAGE. Spectral analysis showed that TTR possesses a maximum fluorescence wavelength (lambda(max)) of 325 nm upon excitation at 282 nm and a positive band with a maximum at 195 nm and a broad negative band with a minimum at 215 nm in the far-UV CD spectrum. The spectrophotometric assay demonstrated the strong detoxification activity of TTR. These results are the first report of the characterization of the purified tabtoxin-resistant protein. Its capacity to detoxify tabtoxinine-beta-lactam shows that it must be one of the self-protection mechanisms in pv. tabaci.     

A chromogenic oxidative coupling reaction of laccase: applications for laccase and angiotensin I converting enzyme assay

A sensitive chromogenic assay for p-hydroxybenzoic acid, laccase activity, and angiotensin I converting enzyme activity is described. The method relies on the oxidative coupling of 2,2'-azino-di(3-ethylbenzothiazoline-6-sulfonic acid) and p-hydroxybenzoic acid. Lacase catalyzes the formation of a deep-purple compound, which shows a broad absorption between 530 and 630 nm with a maximum at 593 nm (the molar absorption coefficient was calculated to be 26,900). By means of this chromogenic coupling reaction, a spectrophotometric method for the assay of laccase activity and estimation of the amount of p-hydroxybenzoic acid was developed; laccase activity in the range 1-10 pmol protein could be estimated with a 10-min incubation time. Angiotensin I converting enzyme was also assayed by the laccase-catalyzed indicator reaction, using p-hydroxybenzoyl-glycyl-histidyl-leucine as the substrate, and N alpha-carbobenzoxy amino acid urethane hydrolase as the coupling enzyme.     

Interaction of sodium dodecyl sulfate with human native and cross-linked hemoglobins: a transient kinetic study

The interaction of sodium dodecyl sulfate (SDS) at a concentration range (0-515 microM) below the critical micelle concentration (CMC approximately 0.83 mM) with human native and cross-linked oxyhemoglobin (oxyHb) and methemoglobin (metHb) has been investigated by optical spectroscopy and stopped-flow transient kinetic measurements. It is observed that the interaction of SDS with human native and cross-linked oxyHb shows the disappearance of the bands of oxyHb at 541 and 576 nm and the appearance at 537 nm. The resultant spectra are characteristic of low spin (Fe(3+)) hemichrome. Similarly SDS has been found to convert human native and cross-linked high spin (Fe(3+)) metHb to low spin (Fe(3+)) hemichrome. The interaction of SDS with oxyHb suggests a conformational change of the protein in the heme pocket, which may induce the binding of distal histidine to iron leading to the formation of superoxide radical. The formation of hemichrome from metHb is found to be concentration-dependent with SDS. The stopped flow transient kinetic measurements of the interaction of SDS with metHb show that at least four molecules of SDS interact with one molecule of metHb. The interaction of SDS with human cross-linked oxy and met hemoglobin shows results similar to those for human native oxy and met hemoglobin indicating that the covalent modification does not alter the interaction of SDS with cross-linked hemoglobin.     

Effects of detergent on substrate binding and spin state of purified liver microsomal cytochrome P-450LM2 from phenobarbital-treated rabbits

Spectral changes accompanying the binding of the nonionic detergent n-octyl beta-D-glucopyranoside (n-octyl glucoside) to cytochrome P-450LM2 purified from liver microsomes of phenobarbital-treated rabbits have been compared to changes in catalytic activity obtained in a reconstituted system consisting of various levels of detergent, P-450LM2, and NADPH-cytochrome P-450 reductase. In the absence of substrate and reductase, addition of n-octyl glucoside to 2-3 mM resulted in a difference spectrum (detergent-bound minus detergent-free cytochrome) characterized by a small maximum at 390 nm and a minimum at 410 nm, suggestive of a slight stabilization of the high-spin (S = 5/2) state of the cytochrome. As the detergent concentration was increased to 4-8 mM (corresponding to maximal activity and pentameric or hexameric P-450), a new peak appeared at 427 nm while the minimum remained at 410 nm. Between 10 and 30 mM n-octyl glucoside (conditions which produced catalytically inactive and monomeric P-450) the minimum in the difference spectrum shifted to 390 nm and the maximum to 425 nm, characteristic of a shift in spin equilibrium toward low-spin (S = 1/2) cytochrome. At low and high detergent concentrations, substrate [d-benzphetamine with n-octyl glucoside or cyclohexane with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)] was bound to P-450LM2 with formation of high-spin P-450, although the increase in high-spin cytochrome was less at high detergent levels than at low. The affinity of P-450 for substrate decreased by 2-3-fold at high detergent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resolution of two forms of cytochrome P-450 from liver microsomes of rabbit treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Two forms of cytochrome P-450 with different substrate specificities were isolated from liver microsomes of rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). A specific antibody was produced toward the major form of the cytochrome. The antibody inhibits microsomal acetanilide hydroxylation (80%). It does not cross-react with the minor fraction of the cytochrome or inhibit the hydroxylation of 3,4-benzpyrene or coumarin, the N-demethylation of aminopyrine or the O-deethylation of 7-ethoxycoumarin catalyzed by rabbit liver microsomes. The major form has an estimated Mr = 54,000 and displays an n-octylamine difference spectrum with an absorption maximum at 426 nm and a minimum at 391 nm. When reconstituted, this cytochrome catalyzes acetanilide hydroxylation at a higher rate than microsomes or the minor fraction. The n-octylamine difference spectrum of the minor fraction displays an absorption maximum at 431 nm and a minimum at 410 nm. When reconstituted, this fraction catalyzes the hydroxylation of 3,4-benzpyrene and the O-deethylation of 7-ethoxycoumarin. The two cytochromes appear to be distinct entities and function in different catalytic pathways.     

Phage T4 lysozyme. Physical properties and reversible unfolding.

Phage T4 lysozyme has been used extensively in studies of the genetic code. However, little work has been done on the characterization of the purified enzyme. Therefore, we determined the spectral properties of native T4 lysozyme and used these properties to follow the unfolding transition. The ultraviolet absorption spectrum and solvent perturbation difference spectrum indicate that the aromatic amino acids are extensively exposed to solvent. The CD and ORD spectra are characteristic of a high fraction of helix. Guanidine hydrochloride denaturation results show that over a T4 lysozyme concentration range of 0.07-1 g/l the c-m equals 2.7 M guanidine hydrochloride at pH 5 and that the transition is 100% reversible as judged by enzymatic assay and four different spectrophotometric criteria: CD at 295 nm, CD at 223 nm, fluorescence intensity at 350 nm and wavelength of maximum fluorescence. Guanidine hydrochloride denaturation at pH 2.5 was followed using fluorescence emission and has a c-m equals 1.7 M guanidine hydrochloride, indicating a strong pH dependence of chemical unfolding. Reversible thermal denaturation conditions were located at acid pH, 0.2 M NaCl, 10-4 M dithiothreitol and 10-6 M T4 lysozyme. The CD signal at 223 nm was used to measure the unfolding. Thermodynamic analysis of the thermal data showed an increase in T-m, increment H-unf and increment S-unf with increasing pH.     

Spectrophotometric titration of tyrosyl residues in alkaline mesentericopeptidase.

At pH 7.0 the alkaline mesentericopeptidase has ultraviolet absorption spectrum with a minimum at 251 nm and a maximum at 280 nm and no visible absorption. From the tyrosine to tryptophan ratio a value of 3 tryptophyl residues per mole of protein is obtained. The molar extinction coefficient at 280 nm is 3.55 X 10(4)M-1cm-1. Spectrophotometric titration studies show that the molecule of mesentericopeptidase contains seven phenolic groups with a pKapp - 9.92 and four to five groups with a pKapp = 11.96. Denaturing agents, such as 5 M guanidine hydrochloride or alkali, normalize the ionization of the tyrosyl residues. There is a good correlation between the spectrophotometric titration data and the results for the reactivities of the tyrosines in mesentericopeptidase towards tetranitromethane. The correlation is explained by the mechanism of nitration. Conclusions about the state of the tyrosyl residues and the three-dimensional structure of mesentericopeptidase are made.