Factors influencing fluorescence spectra of free porphyrins (original) (raw)
Related papers
Photodynamic and light independent action of 8 to 2 carboxylic free porphyrins on some haem-enzymes
The International Journal of Biochemistry & Cell Biology, 2001
Backgrounds and aims: skin lesions in cutaneous porphyrias appear to be determined by the structural properties of the porphyrins accumulated. To better understand the relationship between the structure and physicochemical properties of porphyrins and their specific effect on protein configuration, the action of a whole range of 8 to 2 carboxylic porphyrins has been studied. Materials and methods: d-aminolevulinic acid dehydratase (ALA-D) and porphobilinogen deaminase (PBG-D) partially purified from bovine liver, were exposed to 10 mM uroporphyrin (Uro), phyriaporphyrin (Phyria), hexaporphyrin (Hexa), pentaporphyrin (Penta), coproporphyrin (Copro) or protoporphyrin (Proto), either in the dark or under UV light. All experiments were performed in the enzyme solutions after removing the porphyrins. Results: under both illuminating conditions, all porphyrins inactivated the enzymes (20-70% under control values), indicating photodynamic action mediated by oxidative reactions and conformational changes due to direct binding of porphyrins to the protein. Total thiol content in ALA-D was not significantly changed by most porphyrins under UV light, while all porphyrins increase total sulfhydryl groups in PBG-D (23-52% over the control values) indicating changes in the redox status of SH residues. Free amino groups were reduced by all porphyrins in ALA-D (23-56% under controls), instead they were enhanced in PBG-D (23-51% over controls), suggesting protein fragmentation. The formation of molecular aggregates would be the consequence of cross-links between oxidation products, while fragmentation can be attributed to either rupture of disulphur bridges and/or enhancement of free amino groups on the protein enzyme. Conclusions: the effect of the porphyrins on enzyme activity, total SH groups and free amino groups content, was different for ALA-D and PBG-D, even under the same illuminating conditions. On the basis of these results, no correlation between enzyme alterations and the physicochemical properties of porphyrins could be established.
Clinical Biochemistry, 2001
The aim of our study was a) to optimize assays for measurement of total (T-) and pancreatic (P-)amylase at 37°C based on the principle recommended by the IFCC at 30°C, b) to evaluate the analytical performance of these assays in a multicentric study and c) to establish reference intervals for serum and urine for either method. Methods: Optimized conditions for 37°C were elaborated with regard to substrate concentration, pH, inorganic additives and glucosidase activity. The cleavage pattern of the EPS substrate was studied by HPLC. Liquid ready-to-use reagents for T-and P-amylase were provided to six European laboratories. Results: The assays showed good performance characteristics (median intraassay CVs 1.0% for T-and 1.3% for P-amylase, median interassay CVs 3.0% for either assay, dynamic range 15-fold URL for T-and 30-fold for P-amylase), high correlation with the previous EPS methods (r Ͼ 0.996, slope 0.43, intercept Ͻ 5 U/L) in serum, heparin plasma and urine and good analytical specificity of the P-amylase assay (residual S-amylase activity 2.4%). Serum reference ranges were found to be 28 to 100 U/L for T-and 13 to 53 U/L for P-amylase (n ϭ 775); URLs in urine were estimated as 490 U/L or 280 U/g creatinine for males and 450 U/L or 380 U/g creatinine for females with total amylase. Conclusion: We believe that these assays based on the 30°C IFCC recommendation represent a further improvement in amylase methodology at 37°C and merit broad application in clinical routine.
Assay of pancreatic amylase with use of monoclonal antibodies evaluated
Clinical chemistry, 1988
To evaluate a new method for measuring pancreatic amylase in serum, in which the salivary isoenzyme is inhibited with a specific monoclonal antibody, we determined the activity of pancreatic and salivary amylase in sera from 103 healthy subjects and from 114 hospitalized patients having a wide range of total amylase activities. CVs for the proposed method ranged from 0.8% to 5.1% (within day) and from 2.3% to 6.6% (day to day). Results correlated well with those obtained by the wheat-germ inhibition method (r = 0.998) and by electrophoresis on cellulose acetate. Analytical-recovery studies confirmed the good specificity of the monoclonal antibody for salivary amylase (97%) and its low cross-reactivity (0.6%) toward pancreatic amylase. The assay procedure presents a wide range of linearity (141-1817 U/L) and can easily be adapted to an automated kinetic system. We found the proposed method suitable for routine determinations of pancreatic amylase.
Studies of canine-liver amylase
Biochimica et Biophysica Acta (BBA) - Specialized Section on Enzymological Subjects, 1964
Some properties of canine-liver amylase (a-I,4-glucan 4-glucanohydrolase, EC 3.2. i. i) were studied both in homogenates and in partially purified extracts of isolated washed liver cells and compared with the properties of amylase in pancreatic extracts prepared from the same animals. Canine-liver amylase appears to be almost entirely in solution. Extracellular amylase appears to be confined largely or entirely to plasma. Iodometric amylase determinations were found to be unreliable when applied to tissue homogenates or suspensions of subcellular particles, indicating the necessity of reexamining previous reports on tissue amylases which were based on iodometric assays. The temperature responsiveness (Q10) of amylase in pancreatic extracts was noted to increase with the age of the preparation, possibly representing the influence of a labile amylase inhibitor. Indirect evidence for the presence of an amylase inhibitor in dog serum was also noted. Preliminary observations suggest that there are no differences between pancreatic amylase and liver amylase on the basis of fluoride inhibition, temperature coefficients or pH-activity curves.
Production and certification of an enzyme reference material for pancreatic α-amylase (CRM 476)
Clinica Chimica Acta, 1996
We describe the preparation of a lyophilized material containing purified human pancreatic a-amylase and the certification of its catalytic concentration. The enzyme was purified from human pancreas by ammonium sulphate precipitation and chromatography successively on DEAE-Sephacel, CM-Sepharose and Sephadex G-75. The purified enzyme Nonstandard abbreviations: IFCC, International Federation had a specific activity of 52.9 kU/g protein and was >99% pure on polyacrylamide gel electrophoresis. Only trace amounts of lipase and lactate dehydrogenase were detected in the purified fraction. The purified pancreatic a-amylase had a molar mass of 57 500 g/mol and an isoelectric point at 7.1. The material was prepared by diluting the purified or-amylase in a matrix containing PIPES buffer 25 mmol/1, pH 7.0, sodium chloride 50 mmol/l, calcium chloride 1.5 mmol/1, EDTA 0.5 mmol/1 and human serum albumin 30 g/l, dispensing in ampoules and freeze-drying. The ampoules were homogeneous and the yearly loss of activity on the basis of accelerated degradation studies was less than 0.01% at -20°C. The certified value for or-amylase catalytic concentration in the reconstituted reference material is 555 U/1 _+ 11 U/I when measured by the specified method at 37°C. The material can be used to verify the comparability of results from different laboratories, for intra-laboratory quality control or for calibration of a-amylase catalytic concentration measurements.
CLEARANCE TIMES OF PORPHYRIN DERIVATIVES FROM MICE AS MEASURED BY in vivo FLUORESCENCE SPECTROSCOPY
Photochemistry and Photobiology, 1984
AbstractThe clearance times of 17 different porphyrin derivatives from SKH:HR-1 mice have been measured using the technique of in vivo fluorescence spectroscopy. This technique monitors the in vivo porphyrin fluorescence observed from the external skin surface. Most hydrophilic porphyrin derivatives show relatively short clearance times, in the order of 2.5–6 h. The dicarboxylic acid porphyrins, proto-, hydroxyethylvinyldeutero-and hematoporphyrin IX have clearance times of 7.8, 12.2 and 14.7 h respectively. The mixture hematoporphyrin derivative has an intermediate clearance time of 12.6 h. N-methylated porphyrins show clearance times in the vicinity of 15–22 h. Monoaspartyl chlorin e6 shows the longest clearance time of all porphyrin derivatives measured (30.3 h).