Investigating Chemical Chaperones that can improve the stability of Lysozymes under high thermal temperature. Sabastine Aliyu Zubairu1, Joseph Oyepata Simeon2, Isaac Ralph Elon1, Mahdi Mohammed1, Sunday Blessing
Investigating Chemical Chaperones that can improve the stability of Lysozymes
under high thermal temperature.
Sabastine Aliyu Zubairu1, Joseph Oyepata Simeon2, Isaac Ralph Elon1, Mahdi
Mohammed1, Sunday Blessing
Dynamin I plays dual roles in the activity-dependent shift in exocytic mode i...Bryan Doreian
Under low stimulation, adrenal chromaffin cells release freely-soluble catecholamines through a restricted granule fusion pore while retaining the large neuropeptide-containing proteinacious granule core. Elevated activity causes dilation of the pore and release of all granule contents. Thus, physiological differential transmitter release is achieved through regulation of fusion pore dilation. We examined the mechanism for pore dilation utilizing a combined approach of peptide transfection, electrophysiology, electrochemistry and quantitative imaging techniques. We report that disruption of dynamin I function alters both fusion modes. Under low stimulation, interference with dynamin I does not affect granule fusion but blocks its re-internalization. In full collapse mode, disruption of dynamin I limits fusion pore dilation, but does not block membrane re-internalization. These data suggest that dynamin I is involved in both modes of exocytosis by regulating contraction or dilation of the fusion pore and thus contributes to activity-dependent differential transmitter release from the adrenal medulla.
PEPTIDOMIMETICS , HERE WE HAVE INCLUDED THE INTRODUCTION, CLASSIFICATION, ADVANTAGES , DISADVANTAGES, ITS METHODS PREPARATION, PRINCIPLES OD DRUG DESIGN, ITS CHEMISTRY. STEREOCHEMISTRY, SYNTHESIS AND APPLICATIONS
Dynamin I plays dual roles in the activity-dependent shift in exocytic mode i...Bryan Doreian
Under low stimulation, adrenal chromaffin cells release freely-soluble catecholamines through a restricted granule fusion pore while retaining the large neuropeptide-containing proteinacious granule core. Elevated activity causes dilation of the pore and release of all granule contents. Thus, physiological differential transmitter release is achieved through regulation of fusion pore dilation. We examined the mechanism for pore dilation utilizing a combined approach of peptide transfection, electrophysiology, electrochemistry and quantitative imaging techniques. We report that disruption of dynamin I function alters both fusion modes. Under low stimulation, interference with dynamin I does not affect granule fusion but blocks its re-internalization. In full collapse mode, disruption of dynamin I limits fusion pore dilation, but does not block membrane re-internalization. These data suggest that dynamin I is involved in both modes of exocytosis by regulating contraction or dilation of the fusion pore and thus contributes to activity-dependent differential transmitter release from the adrenal medulla.
PEPTIDOMIMETICS , HERE WE HAVE INCLUDED THE INTRODUCTION, CLASSIFICATION, ADVANTAGES , DISADVANTAGES, ITS METHODS PREPARATION, PRINCIPLES OD DRUG DESIGN, ITS CHEMISTRY. STEREOCHEMISTRY, SYNTHESIS AND APPLICATIONS
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
A seminar on the pharmacodynamic effects of drugs on enzymes along with their applications. Presented on 07/08/2019
Handout:
1) Introduction & history of enzymes
2) Nomenclature & classification of Enzymes (NC-IUBMB)
3) Structure of enzymes - Shape, active & allosteric sites
4) Mechanism of action of enzymes- Substrate binding, catalysis, dynamics, allosteric modulation
5) Role of enzymes
6) Enzymes as drug targets
7) Enzyme inhibition by drugs:
A) Targeted clinical effects by enzyme Inhibition
B) Enzyme kinetics
C) Types of enzyme inhibition - Competitive, Non competitive & uncompetitive inhibition
D) Adverse drug reactions due to enzyme inhibition
8) Enzyme activation by drugs
9) Microsomal enzymes as drug targets
10)Transmembrane receptors linked to enzymes:
A) Tyrosine Kinase pathway
B) JAK-STAT pathway
C) Serine Threonine Pathway
D) Toll like Receptors
E) TNF-α Receptors
11) Summary with system-wise drugs acting on enzymes
12) Newly Approved Drugs
13) Conclusion
14) References
Challenges in interpreting serum protein electrophoresis. Requires an approach to recognize pattern within the various protein fractions & differentiate systemic inflammatory response from abnormal antibody production due to neoplastic disorders.Presence of M-band does not always correlate with plasma cell disorders but can be seen some lymphomas, chronic leukaemias, systemic amyloidosis hence need further ancillary tests for diagnosis of aetiology for the M-band.
Abstract
Mitogen-Activated Protein Kinase (MAPK) pathway is a signal transduction pathway that functions in a wide range of physiological and pathophysiological cellular events including cell proliferation, differentiation, apoptosis, migration, inflammation, metabolic disorders and diseases. In skeletal muscle, it plays an essential role in muscle fiber specialization, muscle mass maintenance, damage induced muscle regeneration and muscle diseases. This review provides an overview of MAPK pathway and its pathophysiological role in skeletal muscle diseases with a primary focus on muscular dystrophy and atrophy.
Main simple and complex proteins along with their classification are presented. Simple proteins main features. Gluten disease. Nucleic acids. Chromatine. Participation in transcription and replication. Detailed explanation of DNA and RNA structure
Therapeutic Glycoproteins are budding medicine for the treatment of various life threatening diseases like cardiometabolic disorders, autoimmune diseases and cancer. Most of the approved therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cell line.
Its increasing demand has challenged the current production capacity. So as to increase the quality and quantity of these glycoproteins various attributes are being exploited such as the glycosylation patterns which is affected by the cell culture media components, manufacturing mode, culture system and operation, pH, temperature, different expression systems like mammalian, insect, plant, and animal expression system. The choice of a significant host system plays a crucial role in the resulting glycan produced. Glycosylation is highly regulated mechanism, a crucial protein quality attribute that can modulate the efficacy of therapeutic glycoprotein
Case studies, enzyme inhibition, factors affecting enzyme activity, regulation of enzyme activity, Isoenzymes, classification of enzymes, coenzyme and co-factors, the clinical significance of enzymes, and the mechanism of enzyme action
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
A seminar on the pharmacodynamic effects of drugs on enzymes along with their applications. Presented on 07/08/2019
Handout:
1) Introduction & history of enzymes
2) Nomenclature & classification of Enzymes (NC-IUBMB)
3) Structure of enzymes - Shape, active & allosteric sites
4) Mechanism of action of enzymes- Substrate binding, catalysis, dynamics, allosteric modulation
5) Role of enzymes
6) Enzymes as drug targets
7) Enzyme inhibition by drugs:
A) Targeted clinical effects by enzyme Inhibition
B) Enzyme kinetics
C) Types of enzyme inhibition - Competitive, Non competitive & uncompetitive inhibition
D) Adverse drug reactions due to enzyme inhibition
8) Enzyme activation by drugs
9) Microsomal enzymes as drug targets
10)Transmembrane receptors linked to enzymes:
A) Tyrosine Kinase pathway
B) JAK-STAT pathway
C) Serine Threonine Pathway
D) Toll like Receptors
E) TNF-α Receptors
11) Summary with system-wise drugs acting on enzymes
12) Newly Approved Drugs
13) Conclusion
14) References
Challenges in interpreting serum protein electrophoresis. Requires an approach to recognize pattern within the various protein fractions & differentiate systemic inflammatory response from abnormal antibody production due to neoplastic disorders.Presence of M-band does not always correlate with plasma cell disorders but can be seen some lymphomas, chronic leukaemias, systemic amyloidosis hence need further ancillary tests for diagnosis of aetiology for the M-band.
Abstract
Mitogen-Activated Protein Kinase (MAPK) pathway is a signal transduction pathway that functions in a wide range of physiological and pathophysiological cellular events including cell proliferation, differentiation, apoptosis, migration, inflammation, metabolic disorders and diseases. In skeletal muscle, it plays an essential role in muscle fiber specialization, muscle mass maintenance, damage induced muscle regeneration and muscle diseases. This review provides an overview of MAPK pathway and its pathophysiological role in skeletal muscle diseases with a primary focus on muscular dystrophy and atrophy.
Main simple and complex proteins along with their classification are presented. Simple proteins main features. Gluten disease. Nucleic acids. Chromatine. Participation in transcription and replication. Detailed explanation of DNA and RNA structure
Therapeutic Glycoproteins are budding medicine for the treatment of various life threatening diseases like cardiometabolic disorders, autoimmune diseases and cancer. Most of the approved therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cell line.
Its increasing demand has challenged the current production capacity. So as to increase the quality and quantity of these glycoproteins various attributes are being exploited such as the glycosylation patterns which is affected by the cell culture media components, manufacturing mode, culture system and operation, pH, temperature, different expression systems like mammalian, insect, plant, and animal expression system. The choice of a significant host system plays a crucial role in the resulting glycan produced. Glycosylation is highly regulated mechanism, a crucial protein quality attribute that can modulate the efficacy of therapeutic glycoprotein
Case studies, enzyme inhibition, factors affecting enzyme activity, regulation of enzyme activity, Isoenzymes, classification of enzymes, coenzyme and co-factors, the clinical significance of enzymes, and the mechanism of enzyme action
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Similar to Investigating Chemical Chaperones that can improve the stability of Lysozymes under high thermal temperature. Sabastine Aliyu Zubairu1, Joseph Oyepata Simeon2, Isaac Ralph Elon1, Mahdi Mohammed1, Sunday Blessing
Protein Protein Interactions Of Glycine Oxidase (Thi O)bturne
Project done as a final presentation for Experimental Biochemistry. The project was designed and proposed by me and performed by myself and Lauren Pioppo.
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Genetic Polymorphism is one of the factors that affects the Drug metabolism. Cytochrome P - 450, one of the prominent group of metabolizing enzymes. In this ppt, genetic polymorphism of cytochrome p 450 is discussed.
Effect of estradiol -17 β on arachidonic acid metabolism in sheep uterus: in ...iosrjce
The effect of estradiol-17 β on Arachidonic acid (AA) metabolism in non-pregnant sheep uterus was
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The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
Understanding the effects of steroid hormone exposure on direct gene regulati...Neil Raden
Similar to Investigating Chemical Chaperones that can improve the stability of Lysozymes under high thermal temperature. Sabastine Aliyu Zubairu1, Joseph Oyepata Simeon2, Isaac Ralph Elon1, Mahdi Mohammed1, Sunday Blessing (20)
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Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
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Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
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Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
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Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
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Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
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Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
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Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
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Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Investigating Chemical Chaperones that can improve the stability of Lysozymes under high thermal temperature. Sabastine Aliyu Zubairu1, Joseph Oyepata Simeon2, Isaac Ralph Elon1, Mahdi Mohammed1, Sunday Blessing
1. 1
Investigating Chemical Chaperones that can improve the stability of Lysozymes
under high thermal temperature.
Sabastine Aliyu Zubairu1
, Joseph Oyepata Simeon2
, Isaac Ralph Elon1
, Mahdi
Mohammed1
, Sunday Blessing3
.
1
Department of Pharmacology Gombe State University
2
Department of Pharmacology Bingham University
3
School of Medcine New Vision University Republic of Georgia.
Corresponding author: Sabastine A. Zubairu Department of Pharmacology, Faculty of
Pharmaceutical Sciences, college of Medicine Gombe State University, Gombe State
Nigeria. +234(0)8065201165, seb4christ@gmail.com sabastinezubairu@yahoo.com
Abstract
Keywords: Lysozymes, Chemical Chaperones, Type III galactosemia
Abstract
Conformational diseases such as, Type III galactosemia is a genetic disorder caused by mutations
of enzyme GALE, these mutations alter the stability of GALE by shifting the ligand UDP-
galactose and the cofactor NAD+
binding site in some variants, which are believed to improve the
stability of GALE, some disease-associated variants become more unstable than the wild type,
and some variants are more susceptible to proteolysis. There are no treatments for galactosemia,
and if left untreated complications could develop such as liver damage, early onset of cataract, it
can damage the ears, can cause mental retardation. In view of these challenges, we hypothesized
that Protein Stabilizing agents will increase the activity as well as the stability of Lysozymes and
diseaseassociated variants. We investigated the effect of glycerol and betaine on the rate of this
2. 2
reaction. by means of differential scanning fluorimeter (DSF) Thermal shift assay, we
investigated the melting temperature of Lysozymes (35µM) and have now studied the effect of
glycerol and betaine on the melting temperature of Lysozyme, comparative to control sample
without chaperone as assayed by differential scanning fluorimetry. The enzyme was stabilized by
glycerol at 50%v/v and betaine at 450mM. Significant effects were obtained by 50% glycerol
This innovative project gives insight into the possible treatment of Type III galactosemia and
other conformational diseases, as our results indicate that pharmacological chaperones aiming
the stability of Lysozymes could be used for the treatment of conformational disorders.
Introduction
There is increasing number of diseases that are hereditary in nature, most of these diseases
results from mutations that causes proteins to fold wrongly (1). Although rare, most of
these conformational diseases are detrimental and without cure (2, 3). Lately, groups of
minor molecules known as chemical/pharmacological chaperones are used to stabilize such
misfolded or mutant proteins and improve their transfer to their action site (1-3). Some
diseases such Alzheimer's, diabetes and non-neurogenic systemic diseases have turn out to
be serious public health challenges, they generally called neurodegenerative disorder.
These diseasesoccur because of deposition of abnormal proteins in tissues (4-7). They
canaccumulate both extracellularly, and intracellularly, consequently becoming
dysfunctional and are believed to have various pathogenic effects.
Certain types of Malignant polypeptides are often fashioned by folding errors, which are
enhanced by genetic mutations and numerous factors relating to the environment such as
oxidizing and toxic conditions, salt, heat, and molecular crowding (8-10). Misfolded
proteins are disposed to their hydrophobic regions that are suppressedwithin the interior
when the proteins are folded correctly. The visible hydrophobic surfaces facilitate
3. 3
theoligomerization and aggregation of proteins (11, 12)). There is normally acellular
response that copes with aberrant proteins to sustain cell homeostasis, thishas been
extensively investigated by a mechanism underlying a system known as the protein quality
control (14-17). A key aspect of the protein quality control system is located in the
endoplasmic reticulum (ER) (16). The ER obtains proteins that fail to undergo proper
folding, embraces them unto the step of ubiquitination and allocates them to further
processes like degradation by the system of the proteasome(18, 19). At times, stored
proteins in the cells can become a target of autophagy and eventually degradation (19).
Irregular proteins are sometimesisolated to a specific cellular compartment to produce an
intracellular inclusion body called the aggresome (23, 24). The most important factors in
the PQC system are the molecular chaperones such as heat shock proteins (Hsp) that
recognize abnormal proteins, prevent their aggregation, promote refolding and help to
reestablish the injured proteins [21]. It was stated that a reduction in the cell of molecular
chaperones and the loss of their function induced folding failure and an escalation in the
cell of aggregate proteins (22).
In a study, hereditary renal and non-neuropathic amyloidosis (23, 24), were recently
investigated using molecular and biochemical techniques, the pathogenesisof this type of
amyloidosis has been gradually elucidated.(24) To date, several types of amyloidosis,
including lysozyme have been revealed to cause this hereditary renal and non-neuropathic
amyloidosis (24). More recently, there are results indicating that renal amyloidosis (gene)
is associated with the newlydiscovered lysozyme mutation Phe57Ile (23, 24). During the
clinical courses of all patients in this family, the most noticeable clinical manifestation was
nephropathy caused byrenal amyloidosis (24). Our current work investigated the effect of
chaperones in restoring the stability of Lysozymes under very high thermal temperature
4. 4
(25-27). This provides a flat form to investigatepharmacological chaperones that can
improve the stability and folding of variant Lysozyme in any amyloidosis(27-28).
An example of chemical chaperone is Glycerol, it was used to correct the mutant TP53
conformation (29). The results suggest that glycerol is effective in reinstating several TP53
mutants to normal TP53 function, leading to normal CDKN1A expression after heat stress
(29).Betaine is very strong osmolyte (30), there was a study on betaine was carried out
with urea as a denaturising agent and it was discovered that betaine improved proteins
stability (30). This study is aim at Investigating pharmacological chaperones that have the
ability to improve the stability of Lysozymes
Materials/Methods
Differential Scanning Fluorimeter (DSF) Thermal Shift Assay of Lysozymes
This is a model set up to determine the melting temperature and the effect of chemical
chaperons (glycerol and Betaine) on lysozymes thermal stability (Noel et al., 2013).
Preliminarywork was carried out to determine the concentration of Lysozymes that will be
more sensitive to Sypro orange dye and to determine the melting temperature. The samples
include Lysozymes at a varying concentration of 2µM to 100µM, Spyro orange
fluorescence dye and PBS to a final volume of 20µl were loaded in a Rotor-Gene Q cycler
(7,8, 10).in a triplicate manner and a procedure was carefully chosen for the high-
resolution melting temperature (7,8). With increasing temperature from 25o
C to 95o
C, the
enzymes were subjected to a rise in temperature with 1o
C additions and a waiting period of
5seconds (9, 10). The fluorescence was subjected to an excitatory wavelength of 460 nm
and assessing emission at 510 nm (9, 10). The melting temperature (Tm) of each lysozymes
concentration was determined from the primary derivative of the melting curve to establish
5. 5
the right concentration of lysozyme with the best curve was used to investigate the effect
of chaperones.
Effect of Glycerol on the Differential Scanning Fluorimetry (DSF) of Lysozymes
Lysozyme enzyme (Sigma UK) at a concentration of 35µM, Sypro orange (Sigma UK) a
fluorescent dye protein was mixed in serial dilution as described in previous articles of
50X solution into a PBS 50mM, pH 7.5, each stock solution was thoroughly mixed before
use.The same protocol was followed as the above.
Effect of Betaine on the Differential Scanning Fluorimetry (DSF) of Lysozymes
Lysozyme enzyme (Sigma UK) at a concentration of 35µM, Sypro orange (Sigma UK) a
fluorescent dye protein was mixed in serial dilution as described in previous articles of
50X solution into a PBS 50mM, pH 7.5, each stock solution was thoroughly mixed before
each use. Sample of lysozyme (35µM) were prepared using 50mM PBS, betaine (at
concentrations of 50Mm, 100Mm, 150Mm, 200mM, 250mM, 300mM, 350mM, 400mM,
450mM and 500mM) were prepared using PBS. Same as the above.
Statistical analysis and data evaluation
One-way ANOVA used to determine the level of significance difference between glycerol
and control as well as the effect of various concentration of glycerol. Turkey`s post hoc test
was used to determine the significance of the of the difference in Tm due glycerol binding.
Results and Discussion
Differential Scanning Fluorimetery
6. 6
Previous studies described methods on ways to establish and carry out protein stability
measurement by means of a Rotor-Gene Q cycler (9, 10). There are couples of applications
data signifying the importance of discovering drugs using this technique. An initial model
was set up using Lysozymes, first a preliminary determination of the right concentration of
Lysozyme that correspond to the melting temperature from 5µM, 10µM, 15µM, 20µM,
25µM, 30,µM, 35µM 40µM, 45µM and 50µM. The effect of glycerol and betaine as
chaperones were tested as shown below
Effect of Glycerol on the Melting Temperature of
Lysozymes
Lysozymes 15µM
Lysozymes 20µM
Lysozymes 25µM
Lysozymes 30µM
Lysozymes 35µM
Temperature (o
C )
Fig. 1: Thermal shift assay, transition curves make available valuable information. Effect of temperature on
the stability lysozyme were measured by DSF at varying concentration of lysozyme and at a scan rate of
1o
C/min, the graph represent the lysozyme (15µM, 20µM, 25µM, 30µM and 35µM), each well contain 5µM
of spyro orange dye, 10µl of PBS 50mM (pH 7.5) to a total volume of 20µl.
Preliminary fluorescence of lysozymes at various concentrations; this model is to
determine the best concentration of lysozyme and melting temperature, each sample
contains the enzyme at various concentration in 50mM PBS (pH=7.5), Spiro orange dye at
50X to a final concentration of 5µM (2.5µL) to a final volume of 20μL. The test sample
and control were loaded into a 36 well plates. Thermal denaturation was then monitored,
by following the ANS fluorescence emission with excitation wavelength of 395nm, and
0 20 40 60 80 100
0
10
20
30
40
50
Flu
ore
sce
nce
7. emission wavelength of 500 nm. A: represents Lysozyme concentration from 2µM, 5µM,
10µM, 15µM and 25µM.
Fig. 2:Effect of glycerol and betaine on
concentration and at a scan rate of 1o
C/min. each well was set up in a triplicate manner containing lysozyme
only (without any ligand) as the control, then in the presence of glycerol at 5%
orange dye 5µM, PBS 50mM (pH 7.5). The melting temperature of each was compared to the control.
7
emission wavelength of 500 nm. A: represents Lysozyme concentration from 2µM, 5µM,
:Effect of glycerol and betaine on the stability of lysozyme measured by DSF 35µM lysozyme
C/min. each well was set up in a triplicate manner containing lysozyme
only (without any ligand) as the control, then in the presence of glycerol at 5%- 50% concentration. Spyro
orange dye 5µM, PBS 50mM (pH 7.5). The melting temperature of each was compared to the control.
emission wavelength of 500 nm. A: represents Lysozyme concentration from 2µM, 5µM,
the stability of lysozyme measured by DSF 35µM lysozyme
C/min. each well was set up in a triplicate manner containing lysozyme
ntration. Spyro
orange dye 5µM, PBS 50mM (pH 7.5). The melting temperature of each was compared to the control.
8. 8
Fig. 3: Effect of glycerol binding on the stability of lysozyme measured by DSF 35µM lysozyme
concentration and at a scan rate of 1o
C/min. each well was set up in a triplicate manner containing lysozyme
only (without any ligand) as the control, then in the presence of glycerol at 5%- 50% concentration. Spyro
orange dye 5µM, PBS 50mM (pH 7.5). The melting temperature of each was compared to the control.
This result indicates the melting temperature of Lysozyme in the presence of glycerol comparing
it with Lysozymes only as control and 5%, 10%, 15%, 20%, 30%, 35%, 40%, 45% and 50%
Glycerol with corresponding melting temperature 62.5o
C, 62.5o
C, 61.5o
C, 63.5o
C, 62.5o
C,
62.5o
C, 64.5o
C, 65.5o
C and 68.5o
C respectively,
One-way ANOVA analysis of the melting temperature of lysozymes plus glycerol, it
revealed that the higher the concentration of glycerol the more it stabilizes the enzymes;
there is a significant different between the melting temperature of Lysozymes only and in
the presence of glycerol.
9. 9
Figure 4: This represents the effect of betaine on the melting temperature and stability of 35µM Lysozymes:
Each sample contains the enzyme at 35µM concentration in 50mM PBS, Spiro orange dye at 50X to a final
concentration of 5µM (2.5µL) to a final volume of 20μL. The test sample and control were loaded into a 36
well plates. Thermal denaturation was then monitored, by following the ANS fluorescence emission with
excitation wavelength of 395nm, and emission wavelength of 500nm shows the effect of betaine on the
melting temperature of Lysozymes and there appears to be no respond of betaine on the melting temperature
of lysozyme and statistical analysis revealed no significant difference between the control and betaine with p-
value <0.05 except for 400mM.
Preliminary to Determine Melting Temperature of Lysozyme
Lysozyme at low concentration of 2µM to 25µM does not appear to fluorescence. In the
preliminary, we investigated the response of lysozymes at different concentration, and we
did observe the melting temperature of lysozyme, at various concentrations of 5µM, 10
µM, 15µM, 20µM, 25µM, 30µM, 35µM, 40µM, 45µM and 50µM of lysozyme.
Interestingly, a melting curve appeared only from 35µM. pharmacological chaperones
effects were tested to see if they could improve the melting temperature of lysozyme. In
10. 10
fig. 4. Above, lysozyme melted at 65o
C average. The use of Lysozyme was to function as a
model to study the melting temperature and the stability of GALE.
Here, we demonstrated what concentration of Lysozyme can show a level of sensitivity to
temperature and because Lysozyme like most enzymes is supposed to denature at extreme
temperature and thus loss of activity. Therefore, if a temperature is significantly risen
above the physiology the protein tends to denature. Here, the melting temperature of
lysozyme was affected by 5%, 10%, 15%, 20% 25%, 30%, 35%, 40%, 45% of glycerol, as
the melting temperature remained even after these concentrations were added. However,
glycerol at 50% v/v tend to be improved the melting temperature of lysozyme as shown in
fig.7, this means that a very high concentration of glycerol can be used to improve the
stability of lysozyme.
On the other hand, betaine did not indicate any benefit in improving the stability and the
melting temperature of lysozyme. When compared with the control, betaine showed no
significant difference as a stabilizer of lysozyme, although there some effect is seen, but
the effect is not significant at these concentrations. This could be because the concentration
of betaine was too low, per haves a higher concentration should be use or that fact that
betaine has no stabilizing effect on the melting temperature of lysozyme. Surprisingly, at
400mM of betaine some effects were observed but this effect disappeared with higher
concentration of 500mM betaine, this is quite interesting and it suggest that there could
some error with 40mM of betaine or it could be that lysozyme does not response to betaine
at a higher concentration.
Conclusions
11. 11
• Pharmacological chaperones can improve the stability and activity of Lysozymes.
• Glycerol at high concentration can improve the melting temperature of enzymes.
• This innovative project discovered Pharmacological chaperones and other protein
stabilizing-agents that will possibly treat type III galactosemia by increasing enzyme
catalytic activity, increase protein stability, increase cofactor binding and decrease
proteolytic activity of the mutants. However, glycerol revealed some level of effect at a
very high concentration (50%v/v).
• Thermal shift assay is a potential technique for drug discovery.
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