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IDENTIFICATION OF MEMBRANE PROTEINS DIFFERENTIALLY

EXPRESSED IN ERYTHROCYTES FROM CML PATIENTS AND

CONTROLS

Dissertation submitted to the University

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

(Biomedical Genetics)

By

SUNITHA PULIKKOT

(Reg.No. 06MSG079 )

DIVISION OF BIOMOLECULES AND GENETICS

SCHOOL OF BIOTECHNOLOGY, CHEMICAL AND BIOMEDICAL

ENGINEERING

VIT UNIVERSITY, VELLORE-632014

-Hemoglobin

-Cathepsin G7/94/9kDa

No Id6/95/9Da

No Id8/94/9Da

Not done1/96/9Da

No Id9/97/9Da

Chain C,cryogenic crystal structure of human

myeloperoxidase C8/96/9Da

No Id0/99/9Da

spectrin0/99/9kDa

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JUNE-2008

Certificate of the External Guide

This is to certify that the dissertation entitled IDENTIFICATION OF

MEMBRANE PROTEINS DIFFERENTIALLY EXPRESSED IN ERYTHROCYTES

FROM CML PATIENTS AND CONTROLS submitted by SUNITHA PULIKKOT,

06MSG079 to the VIT University, Vellore-632014, for the degree of Master of Science

in Biomedical Genetics is her work, based on the results of the experiments and

investigations carried out by her during the period (December-June 2008) of study under

my guidance.

This is also to certify that the above said work has not been previously submitted

for the award of any degree, diploma, fellowship in any Indian or foreign University.

Date;May 29, 2008 Signature of the Guide

Place: ACTREC, Navi Mumbi Dr.Rukmini.B.Govekar

Scientific officer,ACTREC, Navi Mumbai.

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Certificate

This is to certify that the dissertation entitled IDENTIFICATION OF

MEMBRANE PROTEINS DIFFERENTIALLY EXPRESSED IN ERYTHROCYTES

FROM CML PATIENTS AND CONTROLS submitted by SUNITHA PULIKKOT,

06MSG079 to the VIT University, Vellore-632014, for the degree of Master of Science

in Biomedical Genetics is her work, based on the results of the experiments and

investigations carried out by her during the period (December-June 2008) of study under

my supervision.

This is also to certify that the above said work has not been previously submitted

for the award of any degree, diploma, fellowship in any Indian or foreign University.

Date: Signature of the Internal Guide

Division Leader /Dean

Internal Examiner External Examiner

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ACKNOWLEDGEMENTS

I express my heartfelt gratitude to Dr.Radha Saraswathy, Associate professor &

Division leader, Biomolecules and Genetics, School of Biotechnology, Chemical and

Biomedical Engineering, Vellore Institute of Technology, Vellore, for her constant

guidance and valuable suggestions during my project work.

I acknowledge my sincere thanks to Dr.Rukmini.B. Govekar.Ph.D., Scientific

Officer, Advanced Centre for Treatment Research and Education in Cancer, Tata

Memorial Centre, for her valuable guidance without which my study would not have

been possible.

I extend my heartfelt gratitude to Dr. S.M. Zingde, Deputy Director, ACTREC

for allowing me to work in her laboratory and giving me access to all required material

during the course of my study.

I extend my thankfulness to Mrs. Poonam Kawle, Mr Parag Madankar, Ms

Siddhi , Mr. Peter Sequira, Mr. Sanjeev Shukla, Mr. Amit Fulzele, Mr Atul Pranay andMiss Florine Cynthia, ACTREC, who rendered whole hearted help all the way to

complete my work. I would like to especially thank Mrs Poonam for teaching me all the

techniques and allowing me to use her silver stained gels for mass spectrometry. I thank

Amit for his friendly advise at every occasion.

I am grateful to my classmates for rendering their hands, encouragement and

timely help during the period of my work.

I acknowledge with fondness and respect the enormous support and encouragement

given by my parents and brother

Sunitha Pulikkot

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INDEX

CONTENTS PAGE NO.

1. Introduction

2. Review of literature

3. Objectives

4. Materials and methods

5. Results and Discussion

6. Summary and Conclusion

7. References

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Introduction

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Chronic myelogenous leukemia (CML) is a hematological malignancy which

accounts for 15-20% of adult leukemias. Its annual incidence is 1-2 per 100,000 people

and slightly more men than women are affected. The only well described risk factor for

CML is exposure to ionizing radiation. The disease is characterized by the expansion of

predominantly myeloid cells in bone marrow and accumulation of these cells in blood.

(23).

The disease often progresses through three clinically and cytologically discernible

phases. In the initial stage of CML called the chronic phase, the main clinical findings

include enlarged spleen, fatigue and weight loss. The peripheral blood shows

leukocytosis (approximately 150 x.109/L white blood cells (WBCs)), predominantly

owing to neutrophils in different stages of maturation, as well as basophilia and

eosinophilia. Blasts usually represent 20% circulating basophils,

persistent thrombocytopenia and/or the appearance of new clonal cytogenetic

abnormalities. The final stage of CML, which may or may not be preceded by an

accelerated phase is the blast crisis. Patients experience worsened performance status,

and symptoms related to thrombocytopenia, anaemia and increased spleen enlargement.

There is absence of mature forms of myeloid cells in circulation and the hematological

picture resembles that of acute leukemia. (18)

Molecular basis of CML pathogenesis

CML is an acquired abnormality that involves the hematopoietic stem cell. It is

characterized by a cytogenetic aberration consisting of a reciprocal translocation between

the long arms of chromosomes 22 and 9; t(9;22). The translocation results in a shortenedchromosome 22, an observation first described by Nowell and Hungerford and

subsequently termed the Philadelphia (Ph) chromosome after the city of discovery.

This translocation relocates an oncogene called abl, from the long arm of

chromosome 9 to the long arm of chromosome 22 in the BCR region (Fig 1). The

resulting BCR/ABL fusion gene encodes a chimeric protein with strong tyrosine kinase

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activity. The expression of this protein leads to the development of the CML phenotype

through processes that are not yet fully understood.

Fig 1

Hematopoietic cells affected in chronic myeloid leukemia

CML is initiated by expression of the BCRABL fusion gene product in self-

renewing, haematopoietic stem cells (HSCs). HSCs can differentiate into common

myeloid progenitors (CMPs), which then differentiate into granulocyte/macrophage

progenitors (GMPs; progenitors of granulocytes (G) and macrophages (M)) and

megakaryocyte/erythrocyte progenitors (MEPs; progenitors of red blood cells (RBCs)

and megakaryocytes (MEGs), which produce platelets). HSCs can also differentiate into

common lymphoid progenitors (CLPs), which are the progenitors of lymphocytes such as

T cells and B cells (Fig 2).

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Fig 2

The initial chronic phase of CML (CML-CP) is characterized by a massive

expansion of the granulocytic-cell series. Acquisition of additional genetic mutations

beyond expression of BCRABL causes the progression of CML from chronic phase to

blast phase (CML-BP), characterized by an accumulation of myeloid (in approximately

two-thirds of patients) or lymphoid blast cells (in the other one-third of patients).

Although the CML stem cell is multipotent, production of B cells from the neoplastic

clone occurs only at low levels, and only rare T-cell precursors can be detected. This

indicates that lymphopoiesis, particularly the development of T cells, is compromised by

BCRABL expression. (21)Erythrocytes from patients with chronic myeloid leukemia

(CML) are fragile. Their osmotic fragility and thermal sensitivity has been attributed to

membrane skeletal defects. (2) Basu et al equate these skeletal defects and deficiencies

to those observed in anemias which have been associated with decreased red cell

deformability, leading to splenic sequestration of erythrocytes and consequent anemia in

patients. However, the mechanisms underlying anaemia in CML are not entirely

elucidated. Identification of erythrocyte membrane abnormalities in

myeloproliferative disorders will help in identifying mechanisms responsible for

altered mechanical properties and biological recognition of these erythrocytes by

macrophages and reticuloendothelial cells (16)

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Review of Literature

.

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Until the end of its life span of 120 4 days with 120 miles of travel and 1.7x10 5

circulatory cycles, the human RBC successfully copes with a number of dangers, such as

passage across narrow capillaries and splenic slits, periodic high turbulence and high

shear stress, and extremely hypertonic conditions. The external RBC surface is non-

immunogenic and non-adhesive, to avoid adhesion to endothelia and phagocytosis by

spleen, liver and bone marrow macrophages which are ready to phagocytose any cell

showing even subtle membrane alterations. (1) The integrity of the erythrocyte membrane

is dictated by several structural proteins of the membrane-skeletal complex and processes

which modulate these interaction.

Molecular architecture of erythrocyte membrane skeleton

The molecular architecture of the erythrocyte membrane displays an asymmetric

lipid bilayer. The outer monolayer contains neutral phospholipids, phosphatidyl choline

(PC), and spingomyelin.wheras phosphatidyl ethanolamine (PE) and phosphatidyl serine

are mainly restricted to the inner leaflet of the bilayer. Underlying and within the bilayer,

there is a protein network called the membrane skeleton. In general, the RBC membrane

proteins are classified as either 'integral proteins' or 'peripheral proteins'. The forms

anchored in the lipid bilayer are glycoproteins and include glycophorins, band 3 and band

4.5 proteins. The peripheral proteins constitute the 'red cell cytoskeleton' and consist of

spectrin, actin and ankyrin Fig 3).(5)

Fig 3

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Structural and molecular alterations in erythrocyte senescence

Loss of integrity of the components of membrane-skeleton is reported in

senescent erythrocytes. They appear to play a causal role in this process as evident from

the major contending hypotheses as enumerated below.

i) Specific modifications of RBC membrane components, such as loss of

carbohydrates by enzymatic desialylation, release ofvesicles, or endopeptidase action .

ii) Time-dependent modifications of natural components of RBC surface, such as

band 3, glycophorin, thus rendered 'foreign antigens', which in turn elicit the

production of autoimmune antibodies:

iii) Loss of RBC membrane phospholipid asymmetry resulting in the progressive

appearance of phosphatidylserine at the cell outer leaflet.iv) Irreversible oxidative damage of SH groups resulting in recognition of oxidized

membrane glyco proteins.

(5)

I. Alterations in the membrane and senescence

a. Micro-vesicle formation from erythrocyte membrane during ageing

Greenwall and coworkers reported that micro vesicles (50 to 200 in diameter) can be

found in small numbers by ultracentrifugation at 70000 g of sufficiently large volumes of

fresh human plasma. These vesicles have a varying amount of hemoglobin mid spectrin

depending on the nature of inducing factors of vesiculation. They contain half the

amount of membrane proteins bound in native membrane when referred to the content of

phospholipids.

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This is primarily due to the reduction in spectrin content. One hypothesis

postulates that the impaired binding between spectrin/ ankyrins and band3 results in

vesicles containing lipid bilayer and band 3. Another possibility might be the formation

of band 3- free areas by an increased lateral mobility of band 3. Due to the reduced

stability of the lipid bilayer in these areas, blebs develop and are subsequently released.

However, the major integral membrane proteins, such as glycophorin as well as the

phospholipids and cholesterol of vesicle membranes are present in vesicles in similar

quantities per surface area as in RBC membrane. In conclusion, it is now accepted

generally that the so-called fragmentation of RBC membrane explains why old RBCs are

denser and smaller than the young ones. Elimination of the resulting spherocytes from the

circulation is primarily a function of the spleen and its phagocytic system. However the

mechanism of phagocytosis has not been elucidated in details.(20,5)

b.Band 3 :center of RBC removal

Band 3 is the major integral membrane protein with a molecular weight of 95kDa

possessing two distinct structural domains. The transmembrane domain with the

carboxy-terminus bear the anion transport function and the amino-terminal cytoplasmic

domain serves an anchor for the membrane cytoskeleton and the glycolytic enzymes.

Spectrin tetramer interacts with actin, protein 4.1 and adducin to form the cytoskeleton

network which is linked to the membrane by interaction of protein 4.1 with glycophorin

and ankyrin with band 3.(14). Various models have been proposed which explain the

role of band 3 in erythrocyte senescence.

Kays model

Marguerite Kay was first to show that dense (presumably old) human RBC bound

increased amounts of autologous IgGs. Those eluted auto-antibodies induced

phagocytosis in an in vitro assay, re-bound only to band 3 and to a 62-kDa protein

presumably derived from band 3 and including most of the 35-kDa carboxyl terminal

fragment and the 17-kDa anion-transport region with the glycosylated side chain ( ).

Lows model

According to Low and his group aggregation of band 3 induces clustering of potential

antibody-binding sites and promotes deposition of autologous IgGs. This mechanism

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does not imply covalent modifications of band 3 but simply assumes that antibodies with

affinities too weak to bind to band 3 monovalently would react avidly with band 3

aggregates due to enhanced affinity of the bivalent interaction typically. Band 3

aggregation and ensuing deposition of autologous IgG was considered to be primarily

elicited by hemichrome deposition. In studies performed in phenylhydrazine-treated

RBCs and confirmed with hemoglobinopathic RBCs, it was shown that clustered band 3

and surface-bound IgG had superimposable localization over Heinz bodies, extremely

large clumps of irreversible hemichromes. Co-localization of Heinz bodies, band 3

cluster and anti-band 3 deposition in phenylhydrazine-treated RBCs might have been

overshadowed by the artefactual liberation of spectrin from partially lysed RBCs and

membrane deposition of spectrin-anti-spectrin immune complexes ( ).

Lutzs model

This model shares important elements with Lows model ( ). Naturally occurring auto-

antibodies (Nabs) are low affinity and well below saturating concentration. Therefore

they cannot operate as efficient opsonins. Their efficiency is increased by complement

components that lower by about 100-fold the number of antibody molecules required for

phagocytosis induction. As pointed out earlier, Nab and specifically antiband 3

antibodies activate the classical complement pathway and stimulate complement

amplification and overstoichiometric deposition of C3b component. complement

amplification compensates low affinity of NAbs and generates very efficient opsonins.

Autologous anti-band 3 antibodies appear to possess the unique ability to stimulate

alternative pathway C3b deposition. It has been shown that naturally occurring anti-band

3 antibodies stimulated C3b deposition on oxidatively stressed RBCs in presence of 500-

to 1000-fold excess of autologous or allogeneic IgG molecules. They have shown that

naturally occurring anti-band 3antibodies have high affinity to C3 and contain a binding

site for C3 probably within the Fd region of IgG. The affinity for C3 is assumed to

potentiate the effect of antiband 3 by stimulating alternative pathway C3 deposition.

Oxidative damage to Hb and formation of hemichromes leads to the association between

hemichromes and the cytoplasmic domain of band 3 and to band 3 oligomerization and

subsequent clustering to large aggregates. These clusters show enhanced affinity for

normally circulating anti-band 3 antibodies and these in turn activate the complement

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system. It has been shown that less than 1% oligomerized band 3 was sufficient to elicit

deposition of autologous anti-band 3 IgG.(1)

Model which suggests a connection between band 3 and vesiculation

One of the most intensively studied post-translational modifications of

erythrocyte proteins is the phosphorylation of tyrosine residues of band 3, which is

strictly regulated in vivo by PTKs (protein-tyrosine kinases) and PTPs (protein-

phosphotyrosine phosphatases). Band 3 undergoes tyrosine phosphorylation upon a

decrease in cell volume, as occurs when erythrocytes treated with Ca2+/Ca2+ ionophore

(A23187) lose KCl and release micro vesicles. Analysis of erythrocytes of different cell

ages revealed that PTP1B, unlike the other enzymes examined, was quantitatively

conserved during erythrocyte aging. This suggests important roles for the down-

regulation of tyrosine phosphorylation of band 3 in erythrocyte physiology, and for

vesiculation as a mechanism of human erythrocyte senescence.(19)

c. Exposure of phosphatidyl serine in the outer leaflet of human RBCs.

Although a variety of chemical and physical methods are available for measuring

membrane phospholipid asymmetry, none is sensitive enough to detect small alterations

in asymmetry. Perhaps that could explain the paucity of data regarding the physiological

and pathological alterations in RBC membrane phospholipid asymmetry until the advent

of annexin V, a calcium dependent protein of the annexin family . In 1990, Thiagarajan et

al demonstrated the ability of annexin V (placental anticoagulant protein I) to bind to

activated platelets. This was attributed to the exposure of phosphatidylserine (PS)

resulting from the activation of platelets. Indeed Tait et al, in 1994, documented such

changes in RBC from normal as well as from sickle cell patients. Phosphatidylserine is

present truly in small amounts (about 300 sites per cell) in the outer leaflet of fresh

unseparated human erythrocytes, it increases to a level of 300,000 per cell when treated

with the ionophore A23187 . In RBC from patients with sickle-cell anemia, it may be

even as high as 12000- 13000 sites per cell. They postulated that the presence of external

phosphatidylserine in the outer leaflet of RBCs might serve as a signal for triggering their

recognition by macrophages. Such a mechanism was proposed to explain the increased

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binding to macrophages by erythrocytes whose surface has been altered by the

incorporation of lipid vesicles containing phosphatidylserine or a phosphatidylserine

analogue, the 1-acyl-2[(N-4-nitro benzo-2-oxa-1, 3 diazole) aminocaproyl]

phosphatidylserine . Later, Schroit and collaborators and Allen et al demonstrated

unambiguously that the presence of phosphatidyiserine on the cell surface directly

correlated with the propensity of the RBCs to be bound in vitroby autologous inonocytes

and to be rapidly cleared in vivoby the spleen. (5)

II. Changes in the cytosolic components of senescent erythrocytes

a. Damaged hemoglobin

During the binding of oxygen to form oxy-Hb, one electron is transferred from iron to

the bound oxygen forming a ferric-super oxide anion complex. The shared electron is

normally returned to the iron when oxygen is released during deoxygenation. However, a

fraction of the electrons remains and transforms oxygen into a super oxide anion radical

(super oxide or O2-). In this process, iron is left in the ferric state and Hb is transformed

into met-Hb.

Both super oxide and met-Hb are potentially dangerous to the RBC membrane. Met-

Hb is unable to bind oxygen and is the first step in the formation of harmful

hemichromes. Super oxide is easily transformed into the potent oxidant H2O2 by superoxide dismutase, abundantly present in the RBC. Under normal conditions, the RBC is

able to reduce met-Hb back to ferrous-deoxyHb by the NADH-dependent met-Hb

reductase. H2O2 is detoxified by catalase and by GSH peroxidase (GPOx). In case of

insufficient NADPH generation, catalase is inactivated and thus the anti-oxidant defence

of the RBC impaired. The GPOx-dependent peroxide detoxication route is also

dependent of efficient supply of NADPH, the reducing cofactor in the GSH-regenerating

reaction catalyzed by glutathione reductase.

Any pathological situation, which increases the turnover of this cycle whether

increased oxidative stress or impaired antioxidant defenses will enhance production of

met-Hb and generation of active oxygen species. Enhanced formation of met-Hb leads to

formation of hemichromes. Hemichromes are ferric Hb derivatives with characteristic

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absorption and electron spin resonance spectra in which the sixth heme ligand is either

the distalhistidine, in which case the process is reversible (reversible hemichromes) or

another aminoacid residue from the globin (irreversible hemichromes). Hemichrome

formation depends on the amount of met-Hb formed and is accelerated by oxidants such

as super oxide or H2O2 that enhance the formation of met-Hb. The damaging activity of

hemichromes derives from their potential role as generators of powerful oxidant hydroxyl

radical via a so-called iron-catalyzed Haber-Weiss reaction. A second mechanism by

which hemichromes exert a damaging effect is due to their liberation of free oxidized

heme (hemin). Oxidized Hb or hemichromes easily lose hydrophobic heme that readily

associates with membrane lipids.

RBCs of all pathologic conditions characterized by increased susceptibility to

oxidation such as sickle cell anemia, thalassemia and G6PD-deficiency contain increased

amounts of membrane-bound hemin. Reports indicate that micromolar hemin can induce

potassium leak, decrease osmotic fragility, cause RBC swelling, interact with spectrin,

actin and protein 4.1, mediate dissociation of membrane skeletal proteins, and destabilize

the RBC membrane. Lipid derivatives of oxidant attack, most notably malondialdehyde,

exert a number of detrimental effects on RBCs: they can damage membrane structure

with formation of membrane pores, increase potassium leak and alter water permeability;

polymerize membrane components and decrease cell deformability; cross-link membrane

proteins; enhance IgG binding and complement activation; finally, they may enhance

exposure of phosphatidylserine (PS) on the outer cell surface.(1)

b. Apoptotic machinery of erythrocyte

The groups headed by Klaus Schulze-Osthoff and the tandem formed by Jean-

Claude Ameisen and Jean Montreuil report the intriguing finding that Ca2+ ionophores

(A23187 or ionomycin) induce an in vitro erythrocyte senescence process characterized

by cell shrinkage, membrane microvesiculation, and phosphatidylserine externalization.

This process culminates in erythrocyte disintegration, phagocytosis in the presence of

macrophages in vitro, and in clearance from the circulation in vivo. Surprisingly, in vitro

senescence is not just due to a disruption of ion homeostasis and rather involves the

activation of proteolytic enzymes, as indicated by the fact that cysteine protease

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inhibitors such as Ac-DEVD-CHO or leupeptin prevent all the features of Ca2+

ionophore-induced eythrocyte senescence. Erythrocytes were found to contain pro-

caspase-3 and -8 as well as -calpain. Upon Ca2+ exposure, -calpain is cleaved to its

active fragments which in turn are likely responsible for the degradation of spectrin

(erythrocyte fodrin). In contrast, neither pro-caspase-3 nor pro-caspase-8 become

activated in erythrocytes, which can be explained by the absence of two essential

apoptosome components, Apaf-1 and cytochrome c. Thus, calpain but not caspases

participate in erythrocyte senescence, and the effect of Ac-DEVD-CHO must be

attributed to the inhibition of another protease (presumably -calpain) than its classical

target, caspase-3.(8) Apoptosis and erythrocyte senescence share the common feature of

exposure of phosphatidylserine (PS) in the outer leaflet of the cells. Western analysis

showed that mature red cells contain Fas, FasL, Fas-associated death domain (FADD),

caspase 8, and caspase 3. Circulating, aged cells showed colocalization of Fas with the

raft marker proteins G_s and CD59; the existence of Fas-associated FasL, FADD and

caspase 8; and caspase 8 and caspase 3 activity. Aged red cells had significantly lower

aminophospholipid translocase activity and higher levels of PS externalization in

comparison with young cells. In support of our contention that caspases play a functional

role in the mature red cell, the oxidatively stressed red cell recapitulated apoptotic events,

including translocation of Fas into rafts, formation of a Fas-associated complex, andactivation of caspases 8 and 3. These events were independent of calpain but dependent

on reactive oxygen species (ROS) as evident from the effects of the ROS scavengerN-

acetylcysteine. Caspase activation was associated with loss of aminophospholipid

translocase activity and with PS externalization. Fas-dependent signaling processes play a

role in regulating PS externalization, one of the signals for red cell clearance from the

circulation, by down regulating manner. They play a distinct role, at least under certain

conditions, in regulating red cell survival.(17)

b.Alterations in activities of various enzymes

The activities of cytosolic protein kinase C (PKC),cAMP-dependent kinase

(PKA) , and casein kinase C type I and II (CK I and II) were all found to undergo an age

dependent decrease of twofold to fourfold over the week lifespan of the cells.Membrane

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associated tyrosine kinase showed little or no decrease , but membrane-associated CKI

showed a dramatic eightfold decrease over the 8-week period. By contrast, various

cytosolic enzymes, including Lactate dehydrogenase , Phosphoglycerate kinase

Pyruvate kinase, and Acid phosphatase, showed no change in activity over the same time

period .Density separated human erythrocytes showed qualitatively similar decreases in

cytosolic protein kinase activities in the densest fractions,which contain the oldest cells.

The aging erythrocytes undergo progressive loss of protein kinases that may adversely

affect various cellular processes. (11)

Erythrocyte senescence in anemia and leukaemia

In leukaemias and anemias, abnormalities of the erythrocyte membrane-skeletal

components have been reported which could explain the premature senescence oferythrocytes. They are compiled in Table 1.

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DISEASE MECHANISM OF RBC SNESCENCE

1. Warmautoimmune

hemolytic

anemia

(WAIHA)

It is characterized by an accelerated clearance of red bloodcells (RBCs) associatedwith the presence of anti-RBC

immunoglobulin (Ig)G autoantibodies. Anti-RBC IgG

autoantibodies of patients with WAIHA shareextensive

similarity with natural antiRBC autoantibodies of healthydonors and suggest that defective control of IgG autoreactivity

by autologous IgM is an underlying mechanism for

autoimmune hemolysisin WAIHA(24)

2. Sickle cellanemia

(i)It is reported that where Heinz bodies are found associatedwith the cytoplasmic surface of the membrane, clusters of band

3 are usually colocalized within the membrane. In contrast,

normal erythrocyte membranes and regions of sickle cell

membranes devoid of Heinz bodies display an uninterrupted

staining of band 3. Similarly, ankyrin and glycophorin areperiodically seen to aggregate at Heinz body sites, but the

degree of colocalization is lower than for band 3. Thesedatademonstrate that the binding of denatured hemoglobin to the

membrane forces a redistribution of several major membrane

components.(27)

(ii)copolymerisation of degraded hemoglobin binds to the

cytoplasmic domain of Band3 leads to its clustering and IgG(auto antibodies) bind to it and results in removal of

erythrocytes in sickle cell anemia.(22)

(iii)Exposure of phosphatidyl serine in the outer leaflet.InRBC from patients with sickle cell anemia it may be even as

high as 12000-13000 sites per cell.(5)

(iv)Accelerated transbilayer movement of phospholipids has

been observed in SCA.(2)

3. Hemoglobin

Koln disease

Copolymerisation of degraded hemoglobin binds to the

cytoplasmic domain of Band3 leads to its clustering and IgG

(auto antibodies) bind to it and results in removal of

erythrocytes (22)

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5. Primary acquired

sideroblasticanemia

(i)Accumulation of iron and Higher permeability of erythrocyte

membrane for iron and defect in heme synthesis.(9)

(ii) the mechanism of elevated ADA activity in this acquired

defect was similar to that found in hereditary hemolytic anemia

associated with ADA production.(12)

6. Chronic largegranularlymphocytic

leukaemia

Lytic activity of natural killer cells to autologous red bloodcells.activity against autologous red cells not against allogenicred cells.(10)

7. Chronic

lymphocytic

leukaemia(CLL)

CD5+B lymphocytes produces auto-antibodies against RBC

leading to autoimmune hemolytic anemia.(26)

8. Acute lymphoid

leukaemia(ALL)

Abnormalities in theskeletal protein organization as well as

alternations in transbilayer distribution of phospholipids in the

erythrocyte membrane,which may,in part,account for anaemiaassociated with this disease.(15)

9. Heriditaryspherocytosis

Increased band 3 density and aggregation representingaccelerated red cell ageing in hereditary sphrocytosis(20)

Membrane-skeletal alterations in CML erythrocytes

In CML eythrocytes spectrin becomes abnormal due to cross-linking of its two

subunits via disulphide bonds. Sulphydryl oxidizing agents in controlled condition

,exclusively cross-linked spectrin via disulphide bonds associated with a decrease in

erythrocyte deformability, inhibition of discocyte-echinocytes shape changes and reduced

lateral diffusion of band 3 polypeptides, resulted in extensive cross linking of membrane

4. Thalassemia (i)Aggregated Band3 ,deposition of complement and IgG andwere intensively phagocytosed by human monocyte,where

membrane protein aggregates also contained large amounts of

IgG and C3 complement fragments and where deposition ofIgG and complement C3c and phagocytic susceptibility were

correlated to the amount of membrane bound hemichromes.(1)

(ii)Ps exposing red cell population associated with thalassemia(17)

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proteins and consequently aggregation of intramembrane particles and enhanced rate of

transbilayer movement of phosphatidyl choline(PC). Abnormalities in erythrocyte

spectrin are invariably associated with abnormal membrane phospholipid organization

and therefore support the view that the aminophospholipid-spectrin interaction are the

major determinants of transmembrane lipid asymmetry in red cells. And it is suggested

that externalization of PS in CML erythrocytes would hyperactive the blood coagulation

system and hence may cause thrombosis.(13). In another report the treatment of

erythrocyte ghost with low ionic strength buffer for 40c for a prolonged period (24-48hr)

and subsequent fractionation on sepharose 4B leads to three major peaks attributable to

the spectrin-actin-band4.1 complex, tetrameric spectrin, and dimeric spectrin. Normal

ghost samples have spectrin tetramers predominantly. CML samples have reduced

number of tetramer when compared to dimers. (4)

SO42- self exchange rates were measured in both normal and CML samples to

know their anion-transport capabilities and it suggested no significant alternation in the

anion-transport site of band3. Ankyrin depleted vesicle analysis with 125I-labeled

ankyrin in normal and CML revealed the significant reduction in the number of

ankyrin binding sites in CML erythrocytes and a marked alternation in the

cytoplasmic domain of band 3. (4)

A possible explanation for the anemia in CML is provided by Kundu et al ( ).

They have described increased binding of CML erythrocytes by autologous antibodies,

probably through the aggregated band 3 on their cell surface, as the probable mechanism

for their premature removal from circulation leading to anemia. This is similar to the

autologous IgG-mediated removal of the senescent normal erythrocytes. Subsequently

the same group reported increased activity of protein kinase C (PKC) and associated

hyperphosphorylation of protein 4.1 in CML erythrocytes, which provided explanation

for aggregation of band 3. Hyperphosphorylation of protein 4.1 weakens its binding to

band 3, thereby increasing its lateral mobility and aggregation (Fig 4).

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Fig 4

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Objectives

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The present study was undertaken with an objective to

delineate the alterations in the membrane-skeletal

components of the CML erythrocytes with a view to better

understand the phenomenon of premature senescence.

Separation of the erythrocyte ghost fraction from young and

aged erythrocytes on SDS-PAGE followed by silver staining

of the separated proteins and mass spectrometric

identification of the differentially stained bands.

Using inhibitors and activators of PKC, the role of this

kinase on the protein profile of CML and normal erythrocyte

ghosts was assessed.

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Methodology

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1.Separation of erythrocytes from blood sample

Reagents

A TBS (Tris buffered saline) wash buffer:

1M Tris pH 7.6 10ml

Sodium chloride 9 g

Make up the volume to1000ml with distilled water.

B. 10Mm PBS (phosphate buffered saline),PH 7.4

(i)10mM Na2H PO4 +150mM NaCl

(ii)10mMNaH2PO4+150mM NaCl

Add dropwise the (ii) to (i) to make the PH 7.4

Method

1. Blood sample was collected in an Ethylene diamine tetraacetic acid (EDTA)-

containing tube and allowed to stand on ice till separation of plasma .

2. Plasma was separated and the settled erythrocyte layer was further processed.

3. RBCs were washed 3 times each with 40ml of TBS wash buffer followed by

centrifugation in sorvall RC5C high speed centrifuge at 1000rpm, 15min at 4C .

4 RBC count was taken by counting the number of erythrocytes in a haemocytometer

using a 1:500 dilution of the cells in chilled PBS.

5. The RBC layer was tightly pelleted by centrifugation and the pellet was

reconstituted in wash buffer to give a cell dilution of 1:40

2.Treatment of erythrocytes for stimulation / inhibition of PKC

Reagents

A. TBS(Tris buffered saline)10mM

B.DMSO (Dimethyl sulphoxide) 99.9% A.C.S Reagent, Sigma- Aldrich Cat.

No.472301

C. PMA (Phorbol myristate acetate)-1 M Sigma- Aldrich Cat no.P8139--------

D. 4 PDD(4 Phorbol 12,13 Didecanoate) Sigma Aldrich Cat no. P8014

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E.Rottlerin- 50 M,Cal Biochem, Cat No.557370

Method

1.The cells were separated in to seven equal aliquots containing 1X108cells

2.The cells were treated with either of the following agents

A. TBS(Tris buffered saline) 10 l

B. DMSO(Dimethyl sulphoxide) 10 l

C. PMA (Phorbol myristate acetate) 5 l

D. 4 PDD(4 Phorbol 12,13 Didecanoate) 6.8 l

E. Rottlerin 3 l, 30 l

F. Rottlerin+PMA 30 l+5 l

3.the tubes were kept in water bath for 20 min at 37C and lysed thereafter.

3.Lysis of erythrocytes

Reagents

A. Lysis Buffer stock

1M Tris pH 7.6-10ml

0.5M EDTA-2ml

Make upto 1000ml with distilled water.

B. Working solution of lysis buffer

Lysis buffer stock 10ml

PMSF (Phenyl methyl sulphonyl fluoride) 2mg in 200 l of

DMSO(freshly prepared)

Sodium orthovanadate(1mM) Sigma,S6508-50 l ,

Method

1.One ml RBC lysis buffer was added to all tubes and kept in ice for 1hr

2.After the incubation, the tubes were kept for centrifugation at 14,000rpm for

20 min at 4C in sorvall RC5C high speed centrifuge

3.Cytosol (supernatant) and ghost(pellet) were separated.

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4.Ghosts were washed with lysis buffer at 14,000 rpm,4C,20 min in Plastocrafts Rota

4R-table top centrifuge.

4.Estimation of proteins

Reagents

1. Standard BSA (bovine serum albumin)-1mg/ml

2. Solution A: copper tartarate carbonate (CTC)

(i) 20% Sodium carbonate( Na2CO3)

(ii) 0.2% Copper Sulphate (CuSO4)

(iii) 0.04% Potassium tartarate

Mix (ii) &(iii) and adjust the final volume to 100ml with

Distilled water. Then add (i) by stirring

3. Solution B: 10% SDS

4. Solution C: 0.8N NaOH

5. Reagent A (prepared just before use)

Mix solutions A,B,C and distilled water in 1:1:1:1 ratio.

6. Reagent B: Folin & Ciocalteus Phenol Reagent , sigma,Cat No.F-9252

(diluted 1:5)

Method

1. Stock BSA was aliquoted into5,10,15,20,25,30 l as standard and distilled water was

used as a blank.

2. Volume was made to one ml in all tubes.

3. Protein estimation was done for cytosol (2ul) and ghost (5ul) of the lysed erythrocytes

stimulated with None, DMSO, PMA, 4 PDD, R3, R30, R+P respectively

4. The volume of solution in all the tubes was made to 1ml with distilled water

5. 1ml of Reagent A was added to all the tubes and incubated for 10min at room

temperature.

6. After the incubation, 0.5ml of Reagent B was added in to all the tubes and vortexed.

7.All the tubes were kept at room temperature for 30min.

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8. The colour developed was read at 750 nm in a Kontron uv spectrophotometer.

5. Separation of proteins by SDS- PAGE

Reagents

1. 30% acrylamide: 29.2g acrylamide and 0.8g bisacrylamide were dissolved

in 80ml distilled water and the final volume made up to 100ml with distilled water.

2. 1M Tris HCl pH 8.8-buffer for preparation of separating gel

3. 1M Tris HCl pH 6.8-buffer for preparation of stacking gel

4. 20% SDS

5. 20% APS (Ammonium per Sulphate)

6. TEMED (Tetra methyl ethylene diamine)

7. 10% polyacrylamide gel solution for separating gel(30ml): 10ml 30% Acrylamide,1M

Tris Hcl (pH 8.8), 200 l 20%SDS, 200 l APS and 10 l TEMED

4.5% stacking for stalking gel(10ml): 1.67ml 30% Acrylamide, 1M Tris

Hcl (pH 6.8), 100 l 20%SDS, 100 l APS and 5 l TEMED

8. Electrode buffer:

Glycine 72g

Tris 15g

SDS 10g

The volume was made upto 5l with distilled water.

9. 3x sample buffer

Glycerol 1.0ml

1M Tris Cl pH 6.8 0.625ml

- mercaptoethanol 0.5ml

The volume was made to 3.33ml with distilled wate and 20-40 l ofBromophenol Blue dye was added.

Method

1. Gel plates were set along with the spacer and the sides were sealed with 2%

agar.

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2. eparating gel solution (10%) was poured in the space between the plates, so as to

occupy three quarters of the volume and allowed to set and overlayered with distilled

water

3. After polymerization, the water was drained and the stacking gel (4.5%) was poured

above it to completely fill the remaining one third space and comb was inserted before

the gel polymerized.

4. After proper polymerization, the combs were removed.

5. :Ghost sample equivalent to 60 g protein was dissolved in half the

volume of 3X sample buffer and boiled for 5min.

6. The samples were loaded in to the wells along with marker

7. The plates were secured in electrophoresis chamber and the chamber was filled with

electrophoresis buffer . The separation of proteins was achieved by running the gel at 200

volts for one hour.

6. SILVER STAINING

Reagents:

1. 50% Methanol

2. Solution 1: 0.02% Sodium thiosulphate.

3. Solution 2: 0.2% silver nitrate +0.75% Formaldehyde.4. Solution 3: 2%Sodium carbonate(chilled) +0.5%Formaldehyde.

5. Stop solution: 10% acetic acid

Method

1. The gel was fixed in in 50% methanol.

and was subsequently washed in distilled water for 2hr or more, changing the water

2-3 times. Decanted the water.

2. The gel was kept in in solution 1 and the solution was decanted after one minute.

3. A quick rinse of distilled water was given.

4. The gel was kept in solution 2 for 20 min

5. The gel was rinsed with distilled water thrice (quick washes, just rinse for few seconds

not in shaker)

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6. The gel was kept in the developer (solution3) and shaken till solution turns brown

7. The stop solution was added.

6. Mass spectrometry- Identification of proteins

Reagents

1. 50mM Aammonium Bicarbonate (NH4HCO3)

2.30mM Potassium Ferricyanide (K3Fe(CN)6)

3.100mM Sodium Thiosulphate (Na2S2O3.5H2O)

4.10mM Dithiotreitol (DTT) (HSCH2(CHOH)2 CH2SH

5.55mM Iodacetamide (light sensitive) in 50mM Ammonium

Bicarbonate.

6. 25mM Ammonium Bicarbonate

7. 1:1 mix solutions

a. Potassium Ferricyanide and Sodium Thiosulphate

b. Ammonium Bicarbonate and neat Acetanitrile

c. Dithiotriotol and Ammonium Bicarbonate

8. Soution 1: 500 l of 50% CACN(Acetanitrile) +450 l Distilled

water + 50 l TFA(Trifluro acetic acid)

9. 0.1% TFA

10. Trypsin (10ng/ l), sigma, proteomic grade,Cat No.T6567

Method

WASHING OF GEL PIECES

1. The specific bands were cut in to small pieces and taken into an fresh eppendorf

tube.

2. Washed the gel pieces with 500 l distilled water 3-4 times for 5 min each with

the help of vortex till the acetic acid smell goes.

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3. Added 100 l of freshly prepared destaining solution- 1:1 mix of potassium

ferricyanide and sodium thiosulphate.

4. Put on vortex for 10min or till silver colour gone from band

5. Removed destaining solution and washed with 500 l of distilled water 3 times

for 5 min of each with the help of vortex.

6. Now added 100 l of 1:1 mix of 50mM ammonium bicarbonate and acetanitrile

(100% neat acetanitrile)

7. kept on vortex for 15 min and thrown the supernatant.

8. Added 100 l of neat acetanitrile and kept for 5 min

9. Removed acetanitrile and vaccum dried in speed vac for 5-15 min.

WASHING STEP OF COOMASSIE STAINED GEL

The gel was washed 2 times with distilled water each of 10 min.

Excised the spots of interest from the gel and separated in to eppendorf tubes.

Washed the gel pieces with 500 l of distilled water, 3 times,5 min of each with the help

of vortex.

Washed the gel pieces with 200 l of 1:1 mix of 50mM ammonium bicarbonate and

acetanitrile for 5min, 2times

Removed the remaining liquid and added enough acetanitrile to cover the gel particles

for 5 min.

Removed the acetanitile after the gel plugs shrink and stick together.

Rehydrated the gel pieces in 100 l 50mM ammonium bicarbonate for 5 min and added

an equal volume of acetanitrile.

Removed all liquid after 15min of incubation

Added enough acetanitrile to cover the gel pieces.

After the gel pieces shrunk, removed the acetanitrile.Dried down the gel particles in a vaccum centrifuge.

II REDUCTION AND ALKYLATION

1. Swelled the gel particles in 200 l of 1:1 mix of 10mM Dithiotrietol (DTT)

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and 50mM ammonium bicarbonate(freshly prepared).

2. Incubated for 45min at 56C .

3. Chilled the tubes to room temperature.

4. Removed excess liquid and replaced it quickly by roughly the same

volume of freshly prepared 55mM iodoacetamide(light sensitive) in

50mM ammonium bicarbonate

5 .Incubated for 30min at room temperature in dark

6. Removed iodoacetamide solution

7. Washed the gel particles with 200 l of 1:1 mix of 50mM ammonium bicarbonate

and acetonitrile.

8. Given 15min vortex and removed the solution .repeated for twice.

9. Added enough 100% neat actonitrile to cover the gel particles

10. Removed the acetonitrile and dried the gel particles in speed vac for 5-10min

11. Now added 10-15 l of trypsin. 25mM ammonium bicarbonate was used to soak

the gel pieces.And kept in 37C for over night.

PEPTIDE EXTRACTION

1. Next day removed the trypsin and added 100 l of solution 1 to each tube

2. Kept in ultasonicator for 20min

3. Taken out the supernatant in to an another ependorf tube

4. Added again 50 l of solution 1 to the gel pieces and ultrasonicated for 20min.

5. Once again collected the supernatant in to the same ependorf tube

6. Vaccum dried the supernatant for 2 hr

7. Add 10 l of 0.1%TFA vortex and centrifuge for 2-3 min

8. 2 l of the sample was taken to spot along with matrix solution ( 1:1)l

9. Acquisition and analysis done.

10. The peaklist is taken and given for the search in protein databases.

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Results and discussion

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As part of the on-going experiments in the laboratory, ghost fractions of normal and CML

erythrocytes were separated on SDS-PAGE and the gels were silver stained. These gels were used

for the study reported in this thesis. The gels were compared manually to detect differentially

expressed bands. The gel images are given below:

Fig 5 N#5, C#16 silver stained gel

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Fig 6 N#6 ,C#18 silver stained gel

Fig 7 N#18.C#13 silver stained gel

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Fig 8 N#19,C#14 silver stained

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Fig 9 N,C#11,15.N#16,C#6 silver stained

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N#14,C#10,N#17,C#12 Coomassie stained

41

N14 C10 N17 C12

170

135

95

72

55

43

34

26

17

M N D P R30 M N D P R30 M N D P R30 M N D P R30

N14 C10 N17 C12

170

135

95

72

55

43

34

26

17

M N D P R30 M N D P R30 M N D P R30 M N D P R30

N14 C10 N17 C12

170

135

95

72

55

43

34

26

17

M N D P R30 M N D P R30 M N D P R30 M N D P R30

N14 C10 N17 C12

170

135

95

72

55

43

34

26

17

M N D P R30 M N D P R30 M N D P R30 M N D P R30

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The bands in the silver-stained gels which were different in normal and CML ghosts were

compiled as given below:

Molecular

Weight.

NORMAL SAMPLE (CONTROLS)

N5 N6 N18 N19 N11 N15 N16 N14 N17

Above

170kDa

presentpresent presentpresentpresent present present present present

72kDa present present presentpresentpresent present present present present

55kDa Light

bands

Light

bands

Light

bands

Light

bands

absent Light

band

Light

band

absent absent

43kDa Two

bands

Two

bands

One

band

Below

43kDa

One

light

band

One

band

Two

bands

Not

visible

Not

visible

34kDa present present presentabsent present Two

bands

Two

bands

absent absent

26kDa present present presentpresentabsent absent absent absent absent

17kDa present present Not

visible

Not

visible

present present present Not

visible

Not

visible

11kDa present N S N S N S N S N S N S N S N S

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Molecular

Weight. CML PATIENTS SAMPLE

C16 C18 C13 C14 C11 C15 C6 C10 C12170kDa absent absent absent absent absent absent absent absent absent

72kDa absent absent absent absent absent absent present absent absent

55kDa above

55kDa

above

55kDa

above

55kDa

absent Above

55kDa

Above

55kDa

Light

band

present present

43kDa One

band

One

band

One

band

Below

43kDa

Below

43kDa

One

band

One

band

One

band

One

band

34kDa absent absent absent absent absent absent present

26kDa present present present present present present Absent present present

17kDa Above

17kDa

absent Not

visible

Not

visible

present present present present present

11kDa present N S N S N S N S N S N S N S N S

*NS- Not separated.

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Bands at molecular masses 170, 72, 55, 43,26 , 20, 17,11kDawere differentially

present in normal and CML ghosts. They were identified by mass spectrometry. The

results are given in the following tables.

N O R M A L S AM P LE S

k D a S am p leD a ta ba a c e s s io n nopI m o l.wt s co re s e que np e p t i d e sp e p t i d e sp pm m is se d Pro te in

c o v e r a g em a t c h e ds ub m itte d c le va g e s

1 7 0 k D a

N # 1 4 sw i ssp ro tZ N 2 2 4 _ H U M A N9 .0 1 8 4 8 7 4 5 8 1 2 % 7 1 4 1 3 0 1 Z inc finge r prote in 224

N # 1 9 sw i ssp ro tK 2 C 1 _ H U M A N 8 .1 6 6 6 1 4 9 5 6 2 5 % 8 4 4 1 0 0 1 K e r a t i n t y p e I I c y t o s k e l e t a l

N # 5 sw i ssp ro tS P T B 1 _ H U M A N 5 .1 3 2 4 7 0 2 5 6 3 1 1 % 1 9 5 2 1 0 0 1 Spe c tr in be ta c ha in , e r y thr oc

N # 6 N C B I n r g i | 1 1 9 6 0 1 2 8 7 4 .9 3 1 7 3 8 8 0 6 8 1 3 % 1 5 4 1 1 0 0 1 s pe c tr in be ta , e r y thr oc yt i c

7 2 k D a

N # 1 4 b a nds a not visible

N # 1 9 sw i ssp ro tS P T B 1 _ H U M A N 5 .1 3 2 4 7 0 2 5 6 3 1 2 % 2 0 5 6 1 0 0 1 Spe c tr in be ta c ha in , e r y thr oc

N # 5 sw i ssp ro tT R F L _ H U M A N 8 .5 8 0 0 1 4 6 1 2 3 % 8 3 5 1 0 0 1 L ac to tr ans fe r r in pr e c ur s or

N # 6 not g iven signif icant sco res in bo th N C BInr & sw issp rot

5 5 k D a

N # 1 4 N C B I n r g i | 7766942 9 .4 8 5 3 7 5 7 1 0 7 2 6 % 1 2 3 0 1 0 0 1 C h a i n C , c r y o g e n i c c ry s t a l s

o f H u m a n M y e lo p e r o x id a s e

N # 1 9 sw i ssp ro tg i | 7766942 9 .4 8 5 3 7 5 7 1 0 6 2 9 % 1 2 3 2 1 1 0 1 C h a i n C , c r y o g e n i c c ry s t a l s

o f H u m a n M y e lo p e r o x id a s e

N # 5 b a nds a not visible

N # 6 b a nds a not visible

N O R M A L S A M P L E S

k D a S a m p l eD a t a a c e s s i o n n op I m o l . w ts c o r e s e q u p e p t i d e sp e p t i d e sp p m m i s s e dP r o t e i n

c o v e r a g em a t c h e ds u b m i t t e d c l e v a g e s

4 N # 1 9 N C B I n rg i|6 2 0 8 8 4 1 0 5 . 2 32 6 9 0 4 2 1 0 0 1 2 % 2 1 4 2 1 0 0 1 S p e c t r i n b e t a e r y t h r o

s p h e r o c y t o s i s , c

v a r i a n t [ H o m o s a p i e n4 N # 1 9 s w is s p r o tP E R M _ H U M A N9 . 4 8 5 3 7 5 7 6 6 2 1 % 1 1 3 0 7 5 1 M y e l o p e r o x i d a s e p r

H o m o s a p ie n s ( H u m a

2 N # 1 4 s w is s p r o tL R C 5 3 _ H U M A N7 . 5 7 5 7 5 9 6 7 1 2 1 % 8 4 1 1 5 0 1 L e u c i n e - r i c h r e p e a t -

p r o t e i n 5 3 - H o m o s a p

2 N # 1 4 n o t g iv e n s ig n ific a n t s c o r e s in b o th N C B I n r & s w is s p r o t

2 6N # 1 9 s w is s p r o tE L N E _ H U M A N9 . 7 1 2 9 1 2 7 6 4 2 8 % 7 3 8 1 0 0 1 L e u k o c y t e e l a s t a s e

H o m o s a p ie n s ( H u m a

2 N # 1 9 n o t g iv e n s ig n ific a n t s c o r e s in b o th N C B I n r & s w is s p r o t

2 0 ( 2 )N # 1 9 s w is s p r o tP R D X 2 _ H U M A N5 . 6 6 2 2 0 4 9 6 1 2 9 % 6 3 7 1 0 0 1 P e r o x i r e d o x i n - 2 - H o1 7N # 1 9 n o t g iv e n s ig n ific a n t s c o r e s in b o th N C B I n r & s w is s p r o t

1 1N # 5 N C B I n rg i|6 3 0 8 0 9 8 8 9 . 0 1 1 0 7 8 9 7 0 8 3 % 6 2 3 1 0 0 1 H e m o g l o b i n a l p h a 2 -

[ H o m o s a p i e n s ]

N # 5 N C B I n rg i|6 1 6 7 9 6 0 4 6 . 7 5 1 6 1 0 2 6 9 5 5 % 5 2 3 1 0 0 1 C h a i n B , T - T o - T ( H i g

T r a n s i t i o n s I n H u m a

D e s h i s 1 4 6 D e o x y L

1 4 5 M i x t u r e 1 g i | 6 3 0 8 0 9

1 1N # 6 s w is s p r o tH B A _ H U M A N8 . 7 2 1 5 3 0 5 5 7 5 9 % 7 3 1 2 0 0 2 H e m o g l o b i n s u b u n i t

H o m o s a p ie n s ( H u m a

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C M L S A M P L E S

k D a S a m p l eD a t a a c e s s i o n n op I m o l . w ts c o r es e q u p e p t i d e sp e p t i d e sp p m m i s s e dP r o t e i n

c o v e r a g em a t c h e ds u b m i t t e dc l e v a g e s

1 7 0 k D a

C # 1 0n o t g i v e n s i g n i f i c a n t s c o r e s i n b o t h N C B I n r & s w i s s p r o t

C # 1 6n o t g i v e n s i g n i f i c a n t s c o r e s i n b o t h N C B I n r & s w i s s p r o t

C # 1 8n o t g i v e n s i g n i f i c a n t s c o r e s i n b o t h N C B I n r & s w i s s p r o t

5 5C # 1 0N C B I n rg i | 7 7 6 6 9 4 2 9 . 4 85 3 7 5 72 0 3 4 1 % 2 3 4 2 1 0 0 1 C h a i n C , c r y o g e n i

o f H u m a n M y e l o pC # 1 0n o t g i v e n s i g n i f i c a n t s c o r e s i n b o t h N C B I n r & s w i s s p r o t

C # 1 0N C B I n rg i | 2 3 9 2 2 3 0 1 1 . 5 12 5 7 6 5 9 2 3 7 % 1 1 3 9 1 5 0 1 C h a i n A , H u m a n

1 7C # 1 6n o t g i v e n s i g n i f i c a n t s c o r e s i n b o t h N C B I n r & s w i s s p r o t

1 1C # 1 6N C B I n rg i | 2 2 8 7 9 7 8 . 6 8 3 7 8 8 7 4 8 0 % 4 1 4 1 0 0 2 n e u t r o p h i l g r a n u l e

C # 1 8N C B I n rg i | 2 3 9 2 2 3 0 1 1 . 5 12 5 7 6 5 7 1 4 0 % 8 3 7 2 0 0 1 C h a i n A , H u m a n

N C B I n rg i | 2 0 6 6 4 2 2 11 1 . 3 62 7 0 8 3 6 8 3 8 % 8 3 7 2 0 0 1 C h a i n B , C a t h e p s

C # 1 8s w is s p r o tC A T G _ H U M A N1 1 . 1 92 9 1 6 1 6 1 3 5 % 1 0 3 8 2 5 0 3 C a t h e p s i n G p r e c

( H u m a n )

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Summary and Conclusion

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CONCLUSION

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References

49

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