Derivative UV Spectroscopy Research Articles

Stabilization of proteins by recombinant human gelatins

Porcine gelatins have been widely used as stabilizers of macromolecular based pharmaceuticals but the mechanism by which they stabilize has not been precisely established. Their variability and immunogenicity, however, make them less than ideal excipients. In this work, we take advantage of the availability of recombinant human gelatins (rhGs) to explore the mechanism by which they may stabilize proteins. Three model recombinant proteins, human serum albumin (HSA), bovine granulocyte colony stimulating factor (bGCSF), and human fibroblast growth factor-20 (FGF-20) that display a range of isoionic points have been selected for this study. The interaction of these model proteins with four different molecular weight rhGs and porcine gelatin was studied using a variety of biophysical techniques including fluorescence, CD and second derivative UV spectroscopy to monitor tertiary and secondary structure as a function of temperature. The 8.5, 25, and 100 kDa rhGs had the greatest effect on conformational and colloidal stability of HSA. The 8.5, 25, and 50 kDa rhGs also increased the T(0) of aggregation of bGCSF and FGF-20. Experiments to probe the mechanism of interaction of model proteins with rhGs suggest that the rhGs might interact with the partially unfolded states of target proteins through a combination of electrostatic and other intermolecular mechanisms to inhibit aggregation.

Active dimer of Epratuzumab provides insight into the complex nature of an antibody aggregate

Understanding the intermolecular products of antibodies as a consequence of host-cell expression, aging, and heat-stress can be insightful especially when it involves the development of a stable biopharmaceutical product. The dimerized form of Epratuzumab (an IgG(1) antibody) with a molecular mass of approximately 300 kDa (twice the monomer antibody molecular weight of approximately 150 kDa) was examined to gain a better perspective of its properties pertaining to structure and activity. The nascent dimer was shown to partially dissociate upon incubation at 30 degrees C and 37 degrees C, exhibit no discernable alteration of structure (i.e., secondary or tertiary structure based on CD and 2nd derivative UV spectroscopy), have approximately 70% covalent forms (based upon CE-SDS results) and manifest twofold higher activity relative to the active monomer form (on a weight basis the dimer and monomer have equal activity). Interestingly, these properties were not attributed to a single dimer species, but rather to a more complex dimer assembly. The Epratuzumab dimer was digested with papain to reveal three uniquely dimerized aggregates. The relative molar distribution of Fab:Fab, Fc:Fc, and Fab:Fc was found to be 4:3:8, respectively. The data suggest that all three predominantly covalent dimer adducts are capable of full activity, shedding light on their complex nature and showing that their target specificity was unaltered. ESI-MS data indicated the presence of remnant levels of noncovalent dimers for all three dimerized forms. Material aged at 37 degrees C exhibited a similar papain digest molar distribution of the three dimerized forms, except with enhanced chemical heterogeneity and an increase in covalent forms to approximately 84%.

Role of Chloride Ions in Modulation of the Interaction between von Willebrand Factor and ADAMTS-13

The degradation of von Willebrand factor (VWF) depends on the activity of a zinc protease (referred to as ADAMTS-13), which cleaves VWF at the Tyr(1605)-Met(1606) peptide bond. Little information is available on the physiological mechanisms involved in regulation of AD-AMTS-13 activity. In this study, the role of ions on the ADAMTS-13/VWF interaction was investigated. In the presence of 1.5 m urea, the protease cleaved multimeric VWF in the absence of NaCl at pH 8.00 and 37 degrees C, with an apparent k(cat)/K(m) congruent with 3.4 x 10(4) M(-1) s(-1), but this value decreased by approximately 10-fold in the presence of 0.15 M NaCl. Using several monovalent salts, the inhibitory effect was attributed mostly to anions, whose potency was inversely related to the corresponding Jones-Dole viscosity B coefficients (ClO(4)(-) > Cl(-) > F(-)). The specific inhibitory effect of anions was due to their binding to VWF, which caused a conformational change responsible for quenching the intrinsic fluorescence of the protein and reducing tyrosine exposition to bulk solvent. Ristocetin binding to VWF could reduce the apparent affinity and reverse the inhibitory effect of chloride. We hypothesize that, after secretion into the extracellular compartment, VWF is bound by chloride ions abundantly present in this milieu, becoming unavailable to proteolysis by AD-AMTS-13. Shear forces, which facilitate GpIbalpha binding (this effect being artificially obtained by ristocetin), can reverse the inhibitory effect of chloride, whose concentration gradient across the cell membrane may represent a simple but efficient strategy to regulate the enzymatic activity of ADAMTS-13.

Silicone Oil Induced Aggregation of Proteins

Prior to delivery to the patient, protein pharmaceuticals often come in contact with a variety of surfaces (e.g., syringes and stoppers), which are treated to facilitate processing or to inhibit protein binding. One such coating, silicone oil, has previously been implicated in the induction of protein aggregation. We have investigated the propensity of model proteins to aggregate when silicone oil is present in solution and find significant induction of aggregation in four proteins of various molecular weights and isoelectric points in the presence of 0.5% oil. The ability of silicone oil to induce conformational changes that might be responsible for this aggregation was also examined by a combination of circular dichroism (CD) and derivative UV spectroscopy. Neither method produces evidence of large conformational changes or alterations in thermal stability although in a limited number of cases some small changes suggest the possibility of minor structural alterations. The most probable explanation for silicone oil induced aggregation is that the oil has direct effects on intermolecular interactions responsible for protein association through interaction with protein surfaces or indirectly through effects on the solvent.

Co-encapsulation of isoniazid and rifampicin in liposomes and characterization of liposomes by derivative spectroscopy

Taking into consideration the benefits of the combined therapy of isoniazid (INH) and rifampicin (RIF), this study focused on co-encapsulation of INH and RIF in the same liposome formulation. INH was incorporated in the aqueous phase and RIF in the lipid layer. Liposomes containing either INH or RIF were also prepared. All liposome formulations were compared for their loading capacity, encapsulation percentage and release properties. Drug amounts in the liposomes were estimated using peak-to-peak first-order derivative UV spectroscopy. Among the liposome formulations DPPC:chol liposomes showed the highest loading capacity (106.70±0.12 for INH and 18.17±0.06 (×10 −3) for RIF) and encapsulation percentage (73.84±0.78 for INH and 81.53±2.06 for RIF) compared to EPC:chol liposomes (loading capacity 93.36±0.58 for INH and 17.87±0.11 (×10 −3) for RIF; encapsulation percentage 64.61±0.51 for INH and 74.45±0.48 for RIF). Co-encapsulation of INH and RIF increased their individual encapsulation percentage and extended drug release compared to the formulations containing drug alone (Table 2). Results of this study support the conclusion that lipid and water soluble drugs can be successfully co-encapsulated in the same liposome formulation and also show that derivative UV spectroscopy is a sensitive method for direct and accurate quantification of these co-encapsulated drugs.

Second and Third Derivatives of UV Spectra as a Tool for Identification of Major Anthocyanins from Aronia melanocarpa Extract, Separated Using Reversed-Phase High-Performance Liquid Chromatography

The aim of the study was to propose a method for identification of major anthocyanins in black chokeberry: cyanidin 3-O-β-D-galactopyranoside and cyanidin 3-O-α-L-arabinopyranoside, using reversed-phase high-performance liquid chromatography on line with UV spectroscopy. The similarity indexes between standard spectra and spectra of major components of black chokeberry extract were calculated. The calculations were performed for zero-order spectra and first-fifth derivatives. Only the second and third derivatives of UV spectra provided correct identification of the compounds investigated. Third derivatives of spectra gave higher values of the parameters (calculated using Student's test) indicating statistical significance between similarity indexes to the derivatives of spectra of anthocyanin standards. Third derivative UV spectroscopy may be thus recommended as a tool to identify anthocyanins.

Structural prerequisites for the stability of Sso7d from the archaeon Sulfolobus solfataricus versus high pressure and temperature

Sso7d is a protein purified from the thermoaddophilic archaeon Sulfolobus solfataricus. In this paper we present the effect of pressure and temperature on the stability of Sso7d and its mutants. Fourth derivative UV spectroscopy and ANS (I-anilinonaphtalene-8-sulfonate) binding were used to monitor the protein unfolding.

Stability of subtilisin and lysozyme under high hydrostatic pressure.

The stabilities of subtilisin and lysozyme under hydrostatic pressures up to 200 MPa were investigated for up to 7 days at 25 degrees C. Methods were chosen to assess changes in tertiary and secondary protein structure as well as aggregation state. Tertiary structure was monitored in situ with second derivative UV spectroscopy and after pressure treatment by dynamic light scattering and second derivative UV spectroscopy. Secondary structure and potential secondary structural changes were characterized by second derivative FTIR spectroscopy. Changes in aggregation state were assessed using dynamic light scattering. Additionally, protein concentration balances were carried out to detect any loss of protein as a function of pressure. For the conditions tested, neither protein shows measurable changes in tertiary or secondary structure or signs of aggregation. Lysozyme concentration balances show no dependence on pressure. Subtilisin concentration balances at high protein concentration (4 mg/mL and higher) do not show pressure dependence. However, the concentration balances carried out at 0.4 mg/mL show a clear sign of pressure dependence. These results may be explained by protein interaction with the vial surface and appear to be rate limited by the equilibrium between active and inactive protein on the surface. Pressure increases protein loss, and the estimated partial molar volume change between the two states is estimated to be -20 +/- 10 mL/mol.

Second-derivative UV spectrometric determination of simvastatin in its tablet dosage form

Simvastatin, a highly effective cholesterol-lowering agent, has been widely used for the treatment of hypercholesterolemia. During the development of simvastatin solid dosage form, formulation compositions were constantly varied to define a suitable matrix. A fast and reliable method for the dissolution and release testing of simvastatin was highly desirable to support formulation screening. A second derivative UV spectroscopic method was developed for determination of simvastatin in the tablet dosage form. After carefully choosing a zero-crossing technique of second derivative UV measurement at 243 nm, the selectivity and sensitivity of simvastatin was comparable to the previously developed HPLC method. In comparison with the direct UV method, second derivative UV spectroscopy eliminates the interference from UV absorbing excipients such as ascorbic acid, which often results in a bias of 2–10%. This method is also fast and economical in comparison to the more time-consuming HPLC method regularly used for formulation screening. Finally, this method has been validated to be precise and accurate, and is demonstrated to be an excellent alternative to HPLC method for the dissolution and release testing of simvastatin in the solid dosage form.

Determination of imidazole antimycotics in creams by supercritical fluid extraction and derivative UV spectroscopy

A supercritical fluid extraction (SFE) method was developed for the isolation of imidazole antimycotic drugs (miconazole, econazole, clotrimazole and bifonazole) from cream preparations. The SFE process involved static (1 min) and dynamic (4 min) extraction steps using pure and 10% methanol modified carbon dioxide. The SFE step was then followed by derivative UV spectrophotometric analysis. The method proved to be suitable for quality control assays of the examined antimycotics in commercial cream formulations.

Role of zinc in the structure and toxic activity of botulinum neurotoxin.

Zn2+-protease activity of botulinum neurotoxin causes the blockage of neurotransmitter release resulting in botulism disease. We have investigated the role of Zn2+ in the biological activity of type A botulinum neurotoxin by removing the bound Zn2+ by EDTA treatment, followed by monitoring its structure in terms of secondary and tertiary folding (second derivative UV, FT-IR, and circular dichroism spectroscopy) and function in terms of its effect on the release of norepinephrine from PC12 cells. The single Zn2+ bound to each neurotoxin molecule was reversibly removed by EDTA treatment, whereas the biological activity of the neurotoxin was irreversibly lost. Based on the Amide III IR spectral analysis, the alpha-helical content of neurotoxin increased from 29% to 42% upon removal of Zn2+, which reverted to 31% upon treatment with 1:5 molar excess of exogenous Zn2+. Second derivative UV spectroscopy revealed no change in surface topography of Tyr residues with removal of Zn2+. However, near-UV circular dichroism signals suggested significant alterations in the topography of Phe and Tyr residues that could be buried in the protein matrix. Thermal unfolding experiments suggested that removal of Zn2+ results in the formation of the molten globule-like structure of type A botulinum neurotoxin. Tertiary structural changes introduced by Zn2+ removal were irreversible, which correlated well with the irreversibility of the biological activity of the neurotoxin. On the basis of these results, we suggest that Zn2+ plays a significant structural role in addition to its catalytic role in Zn2+-protease activity of type A botulinum neurotoxin.

Interaction of glucagon with dimyristoylphosphatidylcholine in vesicular and discoidal complexes.

Glucagon fragments dimyristoylphosphatidylcholine liposomes into discoidal complex under appropriate conditions. The concentration of glucagon required to fragment the vesicles increases with increasing pH, which appears to be related to the glucagon binding. It was also observed that the fragmentation is facilitated by NaCl, which is also due to increased glucagon binding. From the quenching of Trp fluorescence by doxyl group located at various positions of the acyl chain of the lipid, Trp of glucagon was found to be located close to the bilayer surface in the vesicular complex. However, the Trp fluorescence was quenched by the doxyl group in the discoidal complex to an equal extent regardless of the position of this spin label in the acyl chain. This and the results of second derivative UV spectroscopy of Tyr suggested that segments including Tyr-13 and Trp-25 are involved in the discoidal complex formation and that the orientation of glucagon is not normal to the bilayer surface.

Analysis of ACE inhibitors in pharmaceutical dosage forms by derivative UV spectroscopy and liquid chromatography (HPLC)

Derivative UV spectroscopy and high performance liquid chromatography (HPLC) were applied to the determination of angiotensin-converting enzyme (ACE) inhibitors in their pharmaceutical dosage forms. For spectrophotometric determinations, the more appropriate derivative order was selected for each drug: ramipril (third-order), benazepril (second-order), enalapril maleate (second-order), lisinopril (first- and second-order) and quinapril (first-order). Reverse phase HPLC procedures (ODS column) were developed able to provide a single, symmetric peak for each drug; mixtures A–B, where A is 20 mM sodium heptansulphonate (pH 2.5) and B is acetonitrile–THF (95:5 v/v), proved to be suitable mobile phases to obtain selective separations of the cited ACE inhibitors. At ambient temperature, a low pH value (2.5) was found to be critical to avoid peak splitting and band broadening.

Characterization of ciprofloxacin liposomes: Derivative ultraviolet spectrophotometric determinations

Neutral, (-) and (+) charged CF encapsulated liposome formulations prepared with EPC:Chol and DPPC:Chol lipids were investigated for their loading capacity, encapsulation % and release properties. CF amounts in the liposomes were estimated using derivative UV spectroscopy. Among the liposome formulations DPPC:Chol and EPC:Chol neutral formulations had a signifi cantly higher loading capacity and slowest release rate compared with (-) and (+) charged liposomes. The derivative UV spectroscopy was found to be an easy and sensitive method for direct estimation of CF in liposomes.

Fourth derivative UV-spectroscopy of proteins under high pressure I. Factors affecting the fourth derivative spectrum of the aromatic amino acids

A tunable fourth derivative UV absorbance method based on a variable spectral shift has been developed and compared to the Savitzky-Golay method and the analytical derivative. The parameters of the method were optimised for the analysis of the UV absorbance spectra of the aromatic amino acids to quantify the effect of decreasing solvent polarity on their fourth derivative spectra. The wavelength of the highest maximum (λmax) (for tyrosine and phenylalanine) or the amplitude of the highest maximum (Amax) (for tryptophan), were shown to depend linearly on the dielectric constant of the solvent, ranging from water to cyclohexane. The only effect of pressure in the 1 to 500 MPa range is a small decrease in the fourth derivative amplitude. This method appears therefore as a suitable tool to evaluate changes of the dielectric constant in the vicinity of the aromatic amino acids in proteins which undergo pressure induced structural changes.

Fourth derivative UV-spectroscopy of proteins under high pressure II. Application to reversible structural changes

The structural basis and the thermodynamics of pressure induced reversible spectral transitions in the fourth derivative ultraviolet absorbance spectra of proteins were analysed as described in the preceding paper. Three proteins were studied: adrenodoxin (a small iron-sulphur protein that serves as an electron donor for cytochrome P450scc), ribonuclease A, and methanol dehydrogenase (a tetrameric protein). Fourth derivative spectroscopy is used to probe important mechanistic aspects of these proteins. For adrenodoxin, the results suggest that one or two phenylalanines interact with the iron-sulphur redox centre. High pressure denaturation of ribonuclease leads to a molten globule like structure that also occurs as an intermediate in the high temperature induced denaturation process. This state is characterised by the local dielectric constant in the vicinity of tyrosines. Methanol dehydrogenase was found to be very stable towards pressure. High pressure appears to strengthen the interaction between the two α-subunits possibly through the increased interaction of four tryptophans with other aromatic amino acids.

Spectroscopic characterization of tick anticoagulant peptide

Tick anticoagulant peptide (TAP) is a disulfide rich potent inhibitor of factor Xa. Although this peptide is of potential clinical utility, very little is known about its higher order structure. Therefore, the secondary structure of recombinant TAP (rTAP) has been examined by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. Both techniques suggest that rTAP is rich in β-sheet structure. Disulfide bonds play a significant role in the folding and structural stability of rTAP. This is apparent from the resistance of rTAP to fluorescence-detected unfolding by guanidinium chloride (Gdn-HCl), unless disulfides are first reduced. The protein's tryptophan and tyrosine residues exhibit greater solvent exposure upon reduction of the cystines as indicated by fluorescence spectra and second derivative UV spectroscopy. A considerable amount of β-structure appears to be retained after reduction of disulfides, although the CD spectrum manifests an increased amount of disordered structure in the reduced peptide. While rTAP does not bind the hydrophobic fluorescence probe 2- p-toludinylnaphthalene-6-sulfonate (TNS) at neutral or acidic pH, the reduced peptide binds TNS at pH 2.0 but not at pH 7.0. The secondary structure of the reduced peptide at pH 2 is, however, similar to that at pH 7 as judged by CD spectroscopy. The reduced form of rTAP at acidic pH thus resembles a molten globule-like state.

Analysis of dithiocarbamate fungicides. Reaction products of the thiuram disulphide fungicide thiram (TMTD) during acid hydrolysis

The acid hydrolysis products (CS2, COS, and H2S) of thiram (tetramethylthiram disulphide, TMTD) absorbed in a methanolic amine reagent (ethylenediamine, piperidine) were investigated by second derivative UV spectroscopy. When the hydrolysis temperatures are below the boiling point the formation of the side products COS and H2S at the expense of CS2 is unavoidable. Failure to carefully scrub the liberated gases with a lead acetate solution leads to the absorption of H2S also in the amine reagent and, like COS, causes an absorption at 230 nm that is erroneously attributed to COS. With the second-derivative technique it has been undoubtedly proved that, under the conditions of the Deutsche Forschungsgemeinschaft method S 15, thiram liberates CS2 and COS in a molar ratio of 1.92 to 0.06 (average of eight determinations) but not in a molar ratio of 1.5 to 0.5, as previously reported by some workers. Under these conditions it is impossible, contrary to earlier claims, to differentiate thiram from other dithiocarbamates based on the ratio of the hydrolysis products in residue analysis.

Aspects of the inhibition of post-cure photo-yellowing of novel amine-diacrylate-terminated ultraviolet- and electron-beam-cured TMPTA-based coatings

The post-ultraviolet (UV) and -electron-beam (EB) cure photo-yellowing of four novel amine cosynergist terminated diacrylate monomers has been compared with that of a standard commercial diethylamine diacrylate monomer based on trimethylol propane triacrylate (TMPTA) using second-order derivative UV spectroscopy. Inhibition of photo-yellowing was also studied using four acrylated hindered piperidine stabilisers as well as an acrylated hindered phenol. Of the amine co-synergists the diethylamine diacrylate system gave the greatest degree of photo-yellowing in both UV- and EB-cured coatings whilst those based on piperazine gave the least yellowing. The presence of readily abstractable hydrogen atoms alpha to the nitrogen atom and subsequent ease of amine oxidation appears to be a key feature in the yellowing mechanism. The incorporation of acrylated and methacrylated hindered piperidine stabilisers as well as that of a hindered phenol are effective in impairing the photo-yellowing of both the UV- and EB-cured diethylamine diacrylate system. Of the stabilisers studied the acrylated hindered phenol was the most effective in both. UV- and EB-cured coatings. Inhibition of hydroperoxidation of the tertiary amine functionality in the amine diacrylate coating is considered to be of primary importance in the overall mechanism. Significantly lower levels of photo-yellowing are observed in the non-amine TMPTA. Here UV curing of the stabilised coatings gave rise to an initial yellowing which was photodecomposed on subsequent irradiation, the rate depending upon the stabiliser structure. The EB-cured coatings however, exhibited no initial yellowing followed by only a minor growth in photo-yellowing on subsequent irradiation, again with a rate dependent upon the stabiliser structure.

The purification and characterization of an extremely thermostable alpha-amylase from the hyperthermophilic archaebacterium Pyrococcus furiosus.

The alpha-amylase from Pyrococcus furiosus, a hyperthermophilic archaebacterium, has been purified to homogeneity. The enzyme is a homodimer with a subunit molecular mass of 66 kDa. The isoelectric point is 4.3. The enzyme displays optimal activity, with substantial thermal stability, at 100 degrees C, with the onset of activity at approximately 40 degrees C. Unlike mesophilic alpha-amylases there is no dependence on Ca2+ for activity or thermostability. The enzyme displays a broad range of substrate specificity, with the capacity to hydrolyze carbohydrates as simple as maltotriose. No subtrate binding occurs below the temperature threshold of activity, and a decrease in Km accompanies an increase in temperature. Except for a decrease in Asp and an increase in Glu, the amino acid composition does not confirm previously defined trends in thermal adaption. Fourth derivative UV spectroscopy and intrinsic fluorescence measurements detected no temperature-dependent structural reorganization. Hydrogen exchange results indicate that the molecule is rigid, with only a slight increase in conformational flexibility at elevated temperature. Scanning microcalorimetry detected no considerable change in the heat capacity function, at the pH of optimal activity, within the temperature range in which activity is induced. The heat absorption peak due to denaturation, under these conditions, occurred within the temperature range of 90-120 degrees C. When the pH was increased, a change in the shape of the heat absorption peak was observed, which when analyzed thermodynamically shows that the process of heat denaturation is complex, and includes at least three stages, indicating that the protein structure consists of three domains. At temperatures below 90 degrees C no excess heat absorption or change in the CD spectra were observed which could be associated with the cooperative conformational transition of the protein. According to the thermodynamic characteristics of the heat denaturation, the cold denaturation of this protein can be expected only at -3 degrees C. Therefore, the observed inactivation of this enzyme is not caused by the cooperative change of its tertiary structure. It can be associated only with the gradual changes of protein domain interaction.