Showing 4 results for Circular Dichroism
Azadeh Azizi, Bijan Ranjbar, Khosro Khajeh, Tayebeh Ghodselahi, Soraya Hoornam,
Volume 3, Issue 2 (11-2012)
Abstract
The stability of enzymes with no reduction in their catalytic activity still remains a critical issue in industrial applications. Naturally occurring osmolytes are commonly used as protein stabilizer. Apart from increasing the stability and catalytic activity, these osmolytes do not change the structure of enzyme. There are a few general schemes about the stabilization mechanism of these osmolytes but the details of their mechanism have not been found so far. In this study, we investigated the simultaneous effects of sorbitol and trehalose on the activity and structural stability of Pseudomonas cepacia lipase (PCL) using UV–visible, fluorescence and circular dichroism (CD) spectroscopy. In order to trace the refractive index and dielectric constant alterations upon the addition of osmolytes, microenvironment of the enzyme (PCL) was studied by means of SPR technique.The results revealed that osmolytes increased catalytic activity and intrinsic fluorescence intensity of PCL. In the presence of both osmolytes the activity of enzyme is greater than when each of the osmolytes is used individally. Far-UV CD spectra indicated that the secondary structural content of protein has been some what increased upon interacting with these osmolytes.The results of SPR technique indicated none of the above osmolytes could change the dielectric constant of medium considerably. This study revealed the synergy of two osmolytes toward increasing the activity and stability of enzyme.
Mehdi Sadeghi, Bijan Ranjbar, Mohammad Reza Ganjalikhany,
Volume 7, Issue 3 (11-2016)
Abstract
The Cu dependent restriction deoxyribozyme is the unique example of known deoxyribozymes. The uniqueness of this deoxyribozyme is originated from specific cleaving of single stranded DNA and formation of triple helix DNA structure which is necessary for substrate recognition and binding. The most established method for measuring the kinetic parameters of deoxyribozyme is based on use of radiolabeled substrates which have several difficulties. In this study we present accurate, fast and inexpensive methods for kinetic study of the deoxyribozyme which is based on extrinsic fluorescence and UV-visible spectroscopy techniques. As mentioned above, DNA triple helix formation is necessary for substrate identification and also enzyme activity. Circular dichroism spetropolarimetery is used for structural study of enzyme. Analysis of spectrum results from this technique indicates structural changes which is a direct evidence for the triple helix formation in enzyme-substrate complex. Extrinsic fluorescence experiment is based on high affinity of SYBR gold to double stranded DNA compared to single stranded DNA. Enzyme activity can be measured by SYBR gold fluorescence emission upon addition of cofactor to the enzyme-substrate complex. Continuous hyperchromicity assay method which is based on UV-visible spectroscopy was used for measuring of enzyme activity by hyperchromicity of the enzyme-substrate complex after addition of cofactor. Comparison of the results show that the continuous hyperchromicity assay is more accurate than the extrinsic fluorescence method, because of this method is based on intrinsic physicochemical properties of DNA without interference of external factors.
Z. Solgi, Kh. Khalifeh , S. Hosseinkhani, B. Ranjbar ,
Volume 9, Issue 3 (9-2018)
Abstract
Aims: The probability of establishing electrostatic interactions due to the abundance of charged hydrophilic residues and especially arginine is considered the most important thermal stabilizing factor of thermophilic enzymes. The current study was conducted with the aim of comparing thermodynamic stability and kinetic refolding of Lampyris turkestanicus and some of its mutants.
Materials and Methods: In the present experimental thermal stability and the way of refolding Lampyris turkestanicus and 3 mutations, including ERR, ERR/I232R, ERR/Q35R/I182R/I232R were investigated by various spectroscopic techniques. In order to high expression of proteins, a single clone of each sample was selected and inoculated into 10ml of LB culture medium, containing Kanamycin at a concentration of 50μg/mg and incubated at 37°C with an ideal aeration for 12-15 hours. The culture medium was centrifuged for 5 minutes at 5000g at 4°C to provide the cellular contents of the bacteria. The results were obtained through spectroscopic methods of remote and near circular dichroism, intrinsic fluorescence, differential scanning calorimetry, and kinetics experiments, using fluorescence-stopped flow technique.
Findings: Along with the increase in the number of arginine residues at the protein level, the stability and structural compression of the mutated enzymes in comparison with the wild enzyme were increased and the thermograms obtained from differential scanning calorimetry showed a slight increase in Tm and calorimetric enthalpy of mutated proteins in comparison with wild protein.
Conclusion: The rate constant of refolding mutated enzymes has increased compared with the wild type. The improvement of thermodynamic and kinetic parameters results from the improvement of electrostatic interactions, which results in a higher degree of compression and structural density.
Seyedeh Maryam Hosseini, Sadegh Hasannia, Bijan Ranjbar,
Volume 11, Issue 3 (10-2020)
Abstract
Human alpha1-antitrypsin is a glycoprotein comprised of 394 amino acids and 52 kDa molecular weight, which is mostly synthesized and secreted by hepatocytes, diffuses to interstitial lung tissues, and has an essential function to protected tissues against neutrophil elastase. One of the significant challenges in dealing with alpha-1-antitrypsin is the structural instability of the folded form of protein and, consequently, the accumulation of polymers in lung tissue. This makes patients vulnerable to chronic obstructive pulmonary disease, severe asthma, and emphysema. Intravenous augmentation therapy of alpha 1-antitrypsin is one of the most prevalent therapies. Moreover, patients who are candidates for that have respiratory symptoms, and the use of bronchodilator (Salbutamol) is the first recommended standard treatment. In this study, protein purification was performed by using high-performance affinity chromatography and, its purity was confirmed by gel electrophoresis. The effect of different concentrations of salbutamol on heat-induced polymerization at 60 ° C was investigated by non-denaturing polyacrylamide gel electrophoresis, dynamic light scattering (DLS), and circular dichroism (CD) techniques. Protein activity was measured by trypsin inhibitor capacity (TIC) assay. The results indicated that salbutamol decreases the rate of polymerization by reducing the flexibility of the reactive center loop, thus decelerate protein activity reduction. Therefore, salbutamol can be an appropriate supplementary for alpha 1-antitrypsin and a proper option for the treatment of protein polymerization associated diseases.
