Studies of the interaction between heme oxygenase - 1 and human HBP

Universidade de Aveiro 2014 Departamento de Quimica

Iga Karolina

Jodłowska

Studies of the interaction between heme

oxygenase-1 and human HBP

Universidade de Aveiro 2014 Departamento de Quimica

Iga Karolina

Jodłowska

Studies of the interaction between heme

oxygenase-1 and human HBP.

Thesis is submitted to the University of Aveiro to fulfill the necessary requirements for the degree of Master of Biotechnology, made under the scientific supervision of Prof. Doutor Brian James Goodfellow Professor auxiliar do Departamento de Química da Universidade de Aveiro,

Jury

 Prof. Bruno Miguel Rodrigues das Neves

Professor Auxiliar Convidado do Departamento de Química da Universidade de Aveiro

 Prof. Doutor Brian James Goodfellow

Key words heme oxygenase-1, heme, human HBP, interaction, NMR spectroscopy, purification.

abstract The presented work aimed to examine for the first time the interaction between heme oxygenase -1 (HO-1) and heme bound human HBP (hHBP). The protein hHBP is thought to be a heme binding protein involved in heme transport during heme metabolism in cells, although the exact function is unknown. Heme binds with a mM Kd. Therefore if hHBP is being used to transport heme, a partner needs to be present to accept the heme ring. Unpublished results have shown that HO-1 is present when hHBP is knocked down in hepatic cell moodels, therefore HO-1 could be possible partner for hHBP. Both proteins were overexpressed in a bacterial host system. HO-1 was grown in non isotopically labeled media while hHBP was grown in M9 media with 15N, labeling. hHBP was purified using affinity chromatography while anion exchange chromatography was used for HO-1. To initially study the binding of heme to HO-1 UV-vis spectroscopy was used with the heme absorbance at 405nm being followed. Binding was observed as expected. Heme tritration with HO-1, hHBP and mixtures of hHBP/HO-1 were carried out to follow the fate of the heme molecule. NMR spectroscopy was also used to see if HO-1 could remove heme from heme-bound hHBP. The results indicate that HO-1 cannot in fact remove heme from hHBP.

Thanks I would like to thank my supervisor, Prof. Doutor Brian James Goodfellow Professor auxiliar do Departamento de Química da Universidade de Aveiro, for the patient guidance, encouragement and advice he has provided.

I have been extremely lucky to have a supervisor who cared so much about my work, and who responded to my questions so promptly.

Contents

1.3. Heme binding proteins 9

1.3.1. Extracellular heme binding proteins 10

1.3.2. Cytoplasmic heme binding proteins 10

1.4. Heme oxygenase-1 12

1.4.1. Heme oxygenase-1 structure 13

1.5. Studies of heme-protein interaction 15

1.5.1. NMR spectroscopy 15 1.5.2. NMR spectroscopy of proteins. 16 1.5.2.1. Sample preparation. 18 1.5.3. UV-vis spectroscopy. 18 1.5.3.1. Sample preparation. 19 2. AIM. 20

3. MATERIALS AND METHODS. 21

3.1. Overexpression of HO-1. 21 3.1.1. Materials 21 3.1.1.1. Plasmid. 21 3.1.1.2. Bacterial stain. 21 3.1.1.3. Culture medium. 21 3.1.2. Methods 22 3.1.2.1. Transformation of DH5α. 22

3.1.2.2. Cell culture/ expression of HO-1 in cells. 22

3.2. Purification of heme oxygenase-1. 23

3.2.1. Materials. 23

3.2.1.1. Buffers. 23

3.2.1.2. Dialysis membrane. 24

3.2.1.3. QA52 anion exchange column. 24

2

3.2.2.1. Cell lysis. 24

3.2.2.2. Salting out. 24

3.2.2.3. Dialysis. 25

3.2.2.4. Purification on QA52 anion exchange column. 25

3.2.2.5. SDS-PAGE analysis. 25

3.2.2.6. Concentration of purified protein. 26

3.3. Overexpression of hHBP 26 3.3.1. Materials. 26 3.3.1.1. Bacterial stain. 26 3.3.1.2. Culture media. 26 3.3.2. Methods. 27 3.3.2.1. Expression of hHBP in cells. 27 3.4. Purification of hHBP. 28 3.4.1. Materials 28 3.4.1.1. Buffers. 28 3.4.2. Methods 28 3.4.2.1. Cell lysis. 28 3.4.2.2. Purification of hHBP. 29 3.4.2.3. SDS-PAGE analysis. 29 3.4.2.4. Concentration of protein. 29 3.4.3. Quantitative analysis. 30

3.5. Studies of the interaction. 30

3.5.1. Material. 30

3.5.1.1. Hemin solution. 30

3.5.2. Methods 30

3.5.2.1. UV-vis spectroscopy. 30

3.5.2.2. Nuclear magnetic resonance. 31

3.5.2.3. Preparation of sample for NMR. 31

4. RESULTS AND DISCUSSION. 33

4.1. Overexpresion and purification of HO-1. 33

4.2. Overexpression and purification of hHBP 35

4.3. Heme – protein interaction 36

4.3.1. UV-vis spectroscopy 36

4.3.2. NMR spectroscopy. 48

5. CONCLUSION. 51

3

1. Introduction.

1.1.Heme

Heme is a chemical compound of a type known as a prosthetic group containing a Fe2+ (ferrous) ion. The organic part of heme is a large heterocyclic organic ring called a porphyrin, consisting of four pyrrole rings joined together by methane bridges. Ferrous (Fe2+) heme has a neutral chemical charge, whereas ferric (Fe3+) heme is positively charged, and can bind anions [11]. These states are the most common within the heme structure, although oxidative states of the ferrous ion can vary from Fe2+ to Fe5+ [12]. The porphyrin in the heme molecule is planar. The ferrous ion can be situated in the plane of the porphyrin ring or above it. The structure of heme B is shown in Fig 1.

Fig 1. Heme B structure [31].

Over the years, a wide variety of heme types have been identified in living organisms. They include types A, B, C, and D. The most common type found in mammals is heme B (present in hemoglobin and myoglobin), other important types are heme A and heme C, which are found in cytochrome c oxidase and cytochromes c, respectively. The main difference lies in the structure of the molecules; more exactly in the substituents of the porphyrin ring. In heme A at position 8 a formyl group is present, and in position 2 a hydroxyethylfarnesyl group, and isoprenoid chains are attached to the vinyl group [26]. In heme C two vinyl groups in position 2 and 4 of porphyrin ring are replaced by covalent, thioether linkages to the apoprotein [25]. The structures of heme A and heme C are shown in Fig 2.

4

Fig 2. Structure of Heme A and heme C [7].

The heme molecule is able to bind to proteins via covalent or non-covalent interactions. In hemoglobin the prosthetic group is bound non-covalently that allows easy release of the heme, but in cytochromes it is bound covalently to the sulfhydryl groups of cystein by the thioether bonds [1].

The heme prosthetic group has two main functions in living organisms:

 The iron atom in heme is capable of binding two ligands ( ex. O2, CO2,

and CN- anion ) via a coordination bond orthogonal to the heme plane. For example an oxygen molecule is bound by heme in hemoglobin and is responsible for transportation of oxygen with blood to the tissues, it is reversible reaction. In the case of an irreversible reaction, when a CN -anion or CO binds, the oxidation state of ferrous ion does not change.

 Electron binding – the ferrous cation is able to change its oxidation state, this reaction is used in cytochromes for the transport of electrons.

Heme is a common component in a range of hemoproteins that play a fundamental role in many biological process. This includes gas transport and storage, drug metabolism, signal transduction and regulation of gene expression, and in the mitochondrial electron transport chain [1;3]. Furthermore, under oxidative stress, heme can be released from hemoproteins as free heme. These molecules can act as a potent cytotoxic pro-oxidant.

5 It has been shown that free heme may participate in the pathogenesis of immune-mediated inflammatory disease [1;2].

1.1.1. Heme metabolism

Porphyrin synthesis, is the enzymatic process that produces heme. The process is highly conserved across the species. In humans this pathway is only used to synthesize heme, but in other species, it produces substances such as vitamin B12. The organs mainly

involved in heme synthesis are the liver and the bone marrow, although it is noteworthy that every cell requires heme to function properly. Heme synthesis occurs through a sequence of eight reactions, four of which take place in the cytoplasm and four in the mitochondria [35] (Fig 4). The pathway is initiated by the synthesis of D-Aminolevulinic acid from the amino acid glycine and succinyl-CoA (formed from the citric acid cycle). ALA synthase is responsible for rate-limitation of this reaction, and the enzyme is negatively regulated by glucose and heme. Inhibition by heme induces mRNA instability and also decreases uptake of mRNA in the mitochondria [1;6;14]. Degradation of heme begins inside macrophages in the spleen, where old and damaged erythrocytes are removed from circulation. In the first step, heme is converted to biliverdin by enzymatic activity of the heme oxygenases (encoded by the HMOX gene). There are two isoforms; heme oxygenase-1 (1) and heme oxygenase-2 (2). HO-2 is constitutively expressed by most cells [HO-2HO-2]. On the other hand, when cells are exposed to free radicals, it induces the expression of the stress-responsive HO-1, increasing the rate of heme catabolism and preventing its (putative) cytotoxic effects [13]. HO activity catalyzes the oxidative splitting of the porphyrin ring, in presence of NADPH, molecular oxygen enters the reaction which results in biliverdin (BV), and the release of Fe ions and carbon monoxide (CO). In second reaction biliverdin is converted to bilirubin by biliverdin reductase (BVR), [1;2] the reaction is shown in Fig 3.

6

Fig 3. Degradation of heme B to bilirubin [8]. 1.1.2. Heme transport

The hydrophobic character of heme, allows it to diffuse passively across cellular membranes. However, heme cellular trafficking appears to be controlled by a range of evolutionary conserved heme transporters [14]. These transporters, have different functions: some of them control the translocation of heme as well as that of heme precursors between the cytoplasm and mitochondria [16], others control extracellular heme import [17]. Also, ATB-binding cassette and the feline leukemic virus receptor are able to export heme from cells [15].(Fig4)

7

Fig 4. Heme metabolism and transport in cell [1]. 1.2. Hemoproteins

As it was mentioned in section 1.1., heme is a prosthetic group found in a wide variety of proteins, which are called hemoproteins. These proteins have different spectra of biological functions. They include transport and storage of diatomic gaseous molecules, electron transfer reactions, modulation of gene transcription, they are also involved in regulation of signal transduction pathways, ion-channel processing and metabolism of xenobiotics and drugs [2]. The biological function of hemoproteins are shown in Table 1.

8

Table 1. Examples of hemoproteins [2].

Function Hemoprotein Specific Biological function Heme acts as a stable

prosthetic group

Gas carriers Hemoglobin O2 carrier in red blood cells

Myoglobin O2 storage and delivery in muscle

Neuroglobin Uncertain function in neuronal cells

Cytoglobin Uncertain function in fibroblasts and related cells

Nitrophorins NO delivery in salive Electron

transporters

Cytochrome c Electron transfer between complex III and IV in the

mitochondria electron transport chain Cytochrome c

oxidase

Converts molecular O2 to H2O2 using

electrons from cytochromes c Cytochrome c

reductase

Catalyzes the reduction of cytochrome c by oxidation of coenzyme Q

Cytochrome b5 Electron donor

Cytochrome b558 Catalytic subunit of NADPH oxidase Catalysts of

biodegradation or biosynthesis

Cytochrome P450 superfamily

External monooxygenases that catalyze the incorporation of one atom of O2 into a substrate

Indoleamine 2,3 – dioxygenase

Catalyzes of oxidative metabolism of tryptophan

Tryptophan 2,3 dioxygenase

Initiates oxidative metabolism of tryptophan Catalases Decompreses H2O2 to H2O and O2

Myeloperoxidase Produces HOCl from H2O2 and Cl-

Cytochrome c peroxidase

Uses H2O2 to oxidize Fe2+ cytochromes c

Eosinophil peroxidase

Produces ROS

Lactoperoxidase Uses H2O2 to oxidize thiocyanate

NO synthase 1 NOS1-3 catalyze the oxidation of L-arginine to l-citrulline with a concomitant production of NO

NO synthase 2 NO synthase 3 Cystathione b-synthase

Catalyzes the condensation of serine and L-homocysteine to form cystathionine

Heme-based gas sensors

FixL Regulates microaerobic adaptation (bacteria) DOS Oxygen sensor ( bacteria)

AxPDEA1 Regulates cellulose extraction (bacteria) Neuronal PAS2 Mammalian CO-regulated transcription factor

implicated in circadian rhythm regulation hemATs Aerotaxis transducers ( bacteria) GRegs Gene regulator ( bacteria)

CooA protein CO-sensing transcription factor (bacteria) Soluble guanylyl

cyclase

Synthesis of cGMP from GTP Heme acts as a cellular

messenger Heme-sensing Heme-regulated proteins Heme activator protein (HAP1)

Transcription factor (yeast) Bach1 Transcription repressor

δ –

aminolevulinate synthase 1

Rate-limiting enzyme of heme synthesis

Iron regulatory protein 2 ( IRP2 )

Regulates Fe metabolism; heme binding induces oxidation

DiGeorge critical region-8

RNA-binding protein involved in microRNA processing; heme binding required for function

Eucaryotic initiation factor 2a kinase

Control protein synthesis in response to heme availability; inhibited by heme

Slo1 BK channel Transmembrane movement of K+; inhibited by heme

9 Heme is found mainly in the “heme pockets” of hemoproteins, that frequently include aromatic amino acids, including phenylalanine, tyrosine or tryptophan and few uncharged amino acids, which gives the hydrophobicity required for stable heme binding [18]. Five conserved amino acids, such as histidine, methionine, cysteine, tyrosine and lysine are able to act as a axial heme ligands in heme pockets [18]. Most often histidine is found in hemoproteins that bind heme B or C. What is more, cystein promotes binding of heme B, whereas methionine binds heme C [1]. Other hydrophobic amino acids (leucine, isoleucine, valine) can create interactions with porphyrins, and arginine and positively charged amino acids can interact with the heme propionate group, that is negatively charged.

Several binding motifs have been found and described, commonly the heme-binding motifs are formed by two amino acids. However, there are some exceptions, the GX..HR..XC..PLAV..G motif is incorporated with heme B, the motifs that bind primarily heme C are CXXCH and CXXCK [18], another motif found in mammalian cytochrome c and in plants, FXXGXXCXG also binds heme B (in each motif the “X” refers to any amino acids) [1].

In some hemoproteins heme can undergo chemical and electronic modifications. That can lead to changes in the structure of the protoporphyrin ring, like in the case of reaction with hydrogen peroxide (H2O2). In other molecules heme does not undergo any

modifications and that is the state in which hemoproteins binds gaseous molecules. Under oxidative stress hemoproteins can also release heme from the structure, an effect that produces cytotoxic free heme [1].

1.3.Heme binding proteins

Free heme due to the presence of iron, has ability to generate reactive oxygen species through Fenton reactions. The cytotoxic effect of free heme can be prevented by heme-binding proteins or heme chaperones. The aim of these proteins is to isolate or transport heme.

10

1.3.1. Extracellular heme binding proteins

Free hemoglobin (Hb) is released from cells during hemolysis and is one of the main sources of extracellular free heme. Cell-free Hb can be scavenged by hepatoglobin (Hp), a plasma glycoprotein with hemoglobin-binding capacity. Therefore, Hp inhibits the pro-oxidative effect of Hb and also free heme. The creation of complex lead to its endocytosis and heme catabolism by HO-1 [2]. The appearance of polymorphism in human Hp, effects the amount of protein in plasma, decreasing its concentration. This suggests that neutralization of free heme by Hp exerts a protective effect against immune-mediated inflammatory diseases.

Hemopexin (Hpx) is an acute-phase protein that binds free heme in plasma, with the highest known affinity (Kd ~10-13 M) [2]. The heme – Hpx complex is not noxious, and

it delivers heme to HO-1for degradation.

Human serum albumin binds different types of proteins and it can also bind heme. Albumin has a high concentration in plasma (30-50 g/L), that compensates for the low affinity (Kd ~10-8 M) towards free heme [3].

Other proteins present in plasma can also bind heme such as α1 –microglobulin which

belongs to the lipocalin protein superfamily. It is able to neutralize the pro-oxidative effect of free heme, however, the role of this protein under pathophysiologic condition is still unknown [3].

Lipoprotein such as low-density lipoproteins (LDL) and high-density lipoproteins (HDL) can also bind free heme from plasma, with faster kinetics than those of Hpx or albumin. The heme complex with LDL or HDL can be removed by Hpx or albumin. Also, the oxidative modification of lipoproteins can be cause by the heme binding, which then produce a cytotoxic form of HDL and LDL [3].

1.3.2. Cytoplasmic heme binding proteins

Heme has high chemical reactivity and is poorly soluble in aqueous solution. Therefore, the amount of free heme in cells must be under tight control. These two reasons, suggest the existence of intracellular heme binding proteins. One such possibility is the soul/p22HBP protein family which has a wide variety of possible biological functions. The gene is expressed in eye, pineal gland, liver and kidney, which suggests possible

11 functions in circadian rhythms, anti-inflammatory response in cell differentiation and necrosis[5].

The SOUL and p22HBP proteins are evolutionary conserved proteins. Genes encoded for heme binding proteins have been originally identify in mouse and human (p22HBP) and in chicken (SOUL) [5].

This protein family share some common characteristics including:

 amino acids sequence similarity (~ 48%);

 size (22-28 kDa);

 tertiary structure;

 cytosolic distribution.

The value of tetrapyrrol mM affinity and cellular localization ( cytoplazma ) of soul/p22HBP proteins suggest that they play a role in part of porphyrin metabolism where they operate as an intercellular buffer.

The structure of mHBP was determined in 2006 by NMR [26]. Molecular modeling studies of HBP from murine and human shows high sequence identity 86% and the binding pocket of the human homolog is almost identical to the murine protein. It is thought that the highly conserved lysine 177 ( 176 human ) and arginine 56 stabilize the tetrapyrrol propionate side chains in both complexes [4]. Studies of mHBP allowed a model of the heme hHBP complex to be proposed.

12

Fig 5. The heme binding pocket in hHBP. As a sticks are shown Lys 176, Met 59 and Met 63.

The heme is located in an hydrophobic protein-binding pocket, and it is surrounded by the αA helix and β8 – β9 loop. Interestingly the binding of heme to mHBP does not

involve iron coordination [4]. Heme binding is stabilized by lysine and arginine by strong electrostatic interactions with one of the propionate groups in both the murine and human complexes. These strong interactions and hydrophobic interactions can explain the strong 0,5nM affinity [33] of mHBP to heme.

1.4.Heme oxygenase-1

Oxidative stress can lead to release of free heme from hemoproteins. The pro-oxidative effect of free heme can be avoided through different mechanisms. One of these relies on the induction of HO-1, which increases the catabolism of free heme.

Heme oxygenases (HO) encoded by the HMOX gene are evolutionarily conserved enzymes that catabolize heme, whose structure was discussed in section 1.1. Humans and rodents have two different isoforms of heme oxygenase, namely HO-1 (EC 1.14.99.3) and HO-2 (EC 1.14.99.39) encoded by the HMOX1 and HMOX2 genes respectively. These genes are expressed by most living organisms such as bacteria, alga, plants, insects and mammals suggesting that the need for these enzymes appeared early

13 in evolution. The simplest explanation of free heme catabolism is, that it involves the extraction of Fe from the protoporphyrin ring, (section 1.2.). It is worth noting that free heme catabolism by HO-1 is very important for cytoprotection in organisms [2].

Expression of HO-1 is regulated at the transcriptional level, and it can be induced by variety of signal transduction pathways that activate different transcription factors [2]. Regulation of the HMOX1 gene is in response to oxidative stress. Normally Bach1, a transcriptional repressor, inhibits the transcription of the HMOX1 gene by binding to stress-responsive elements in the HOMX1 promoter. In the presence of free heme in cells, Bach1 binds heme which modifies the protein conformation. Bach1 cannot now bind to the gene promoter which allows the transcription factor Nrf2 to bind and induce HMOX1 expression. Reactive oxygen species can also target directly the sulfhydryl groups of Bach1 to inhibit binding to the HMOX1 promoter, and also promote nuclear export and degradation [2].

HO-1 has a general healing effect, as demonstrated by several pieces of evidence: firstly, HMOX1 deletion in mice, increases the intensity of many experimental diseases like malaria; secondly, microsatellite polymorphism in the HMOX1 promoter contributes to many diseases in humans; thirdly, pharmacological induction of HO-1 or administration of the end products of heme catabolism can be used therapeutically in many diseases and fourthly, several molecules known to have a protective effect against a wide variety of diseases appear to do so through a mechanism that relies on the activation of HO-1, called the HO-1 therapeutic amplification funnel [2].

The main function of HO-1 is to protect the cell against the cytotoxic effect of free heme, that would otherwise enforce cells to undergo apoptosis. HO-1 is expressed at higher protein level in renal cancer tissues, than in normal cell tissues [30]. Although, the HO system may have a protective role in several conditions including diabetes, inflamation, heart disease, hypertension, neurological disorders, organ transplantation and endotoxemia. What is more HO-1 has potential therapeutic applications in cancer.

1.4.1. Heme oxygenase-1 structure

Sequence alignments have show that all known HO-1 proteins are homologues and share the same fold and therefore mechanism of action [23]. Mammalian HO-1 is a membrane-bound protein, (through the C-terminal hydrophobic tail), that catalyzes the

14 breakdown of heme to biliverdin. The human HO-1 is 288 AA protein, the UniProt access P09601 [30] (encoded by HMOX1 13,148 kpb). The weight of protein is 32,819 kDa [30]. The protein sequence is shown below [30].

MERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKLVMASLYHIYV ALEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHE VGRTEPELLVAHAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIASATKFKQLYR SRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTHDTKDQSPSRAPGLRQRASNKV QDSAPVETPRGKPPLNTRSQAPLLRWVLTLSFLVATVAVGLYAM

The X-ray structure of the protein (PDB access 1N3U) is mostly helical 69% [30].

Fig 6. Structure of HO-1. Is built with 14 helices [29].

The first studies of the structure of HO-1 was in 1999, published in paper by Schuller at al. The structure was determined by X-ray crystallography and provided information on how the enzyme controls regioselectivity. Furthermore, it was assumed that the closed form of enzyme represented the active form.

The heme is situated between two helices defined as the proximal and distal helices. Both helices form the heme pocket in which the proximal helix provides His25 as a heme ligand, along with Ala28, Glu29 and Thr21. The distal helix provides Gly139 in highly conserved sequence Asp-Leu-Gly-Gly. Furthermore the distal helix does not have polar residues, to directly stabilize iron-bound ligands. That correct orientation of

15 the heme within the heme pocket is provided the interaction of propionate charges with Lys179, Arg183, Lys18 and Lys22 [23;24]. The heme pocket is shown in Fig7.

Fig 7. Heme pocket structure. The blue helix is the proximal helix with His25 which bind heme. The orange helix represent the distal helix, in which are situated Gly139 residues.

1.5.Studies of heme-protein interaction

Studies of ligand-protein interactions and protein-protein interactions have great importance in functional studies. The interactions between enzymes and substrates and between receptors and large molecules can be studied. It is also very important from the point of creating new drugs. Nowadays the main methods used for interaction studies, are NMR spectroscopy, UV-vis spectroscopy and Fluorescence Quenching.

1.5.1. NMR spectroscopy

Over last years, NMR ( Nuclear Magnetic resonance ) spectroscopy has become the preeminent technique for determining the structure of molecules [35]. The technique is based on NMR a physical phenomenon in which nuclei in a magnetic field absorb and re-emite electromagnetic radiation. This energy is at a specific resonance frequency which depends on the strength of the magnetic field and the magnetic properties of the nucleus. NMR allows the observation of specific quantum mechanical magnetic properties of a nucleus. All isotopes that contains an odd number of protons and/or of neutrons have a magnetic moment and angular momentum, in other words a non-zero

16 spin, while all nuclei with even numbers of both have a total spin of zero. The most commonly studied nuclei are 1H and 13C, however nuclei from isotopes of many others elements like 2H, 6Li, 10B, 11B, 14N, 15N, 17O, 19F, 23Na, 29Si, 31P, 35Cl, 129Xe, 195Pt can also be studied.

In the case of small proteins, when we are able to get signals well separated, to determine a structure, 2D-NMR of homonuclear 1H-1H experiments are sufficient. The resonance assignment for the protein (assigning a chemical shift to each nucleus) is the first step. The procedure is based on carrying out NMR experiments such as correlation spectroscopy (COSY) and total correlation spectroscopy (TOSCY). Those two experiments allow spin systems to be identified. To correlate a spin system to the amino acid sequence, we need to perform a nuclear Overhause effect spectroscopy (NOESY) experiment.

When overlap of signals is present, we need to use heteronuclear NMR and 3-D and/or 4-D spectroscopy. Heteronuclear techniques are used to correlate the chemical shift of heteronuclei 13C and/or 15N with 1H. One of the most useful heteronuclear experiment is Heteronuclear Single Quantum Coherence (HSQC), which correlates the chemical shift of a heteronucleus and its directly bond proton.

NMR spectroscopy can give detailed information about the dynamics, structure, reaction state, and chemical environment of molecules. Most frequently, NMR spectroscopy is used by chemists and biochemists to investigate the properties of organic molecules, although is applicable to any kind of samples that contains nuclei possessing spin. Many scientific techniques are based on the NMR phenomena, to study molecular physics, crystal and non-crystalline materials, furthermore is also used in advanced medical imaging techniques like MRI.

1.5.2. NMR spectroscopy of proteins.

NMR spectroscopy plays a important role in the field of structural biology where it is used to obtain information about the structure and dynamics of proteins, and also nucleic acids, and their complexes. The main goal of this method in experiment is to obtain the high resolution 3-D structure of the protein. The first proton spectrum of a protein was determined in 1957, for ribonuclease. Currently there are 101207 protein structures in the PDB database out of which 10 431 structures (10%) were determined

17 by NMR [?]. In comparison to X-ray crystallography the number of determined structures is much lower but NMR has advantage in that the protein dynamics in solutions, can be studied. A disadvantage is that, this method is limited to small proteins (~ 35kDa), and the solutions have to be highly concentrated to 0,1 -0,3 mM [25].

For protein NMR 15N, 13C, 1H, 31P are the nuclei that are normally studied. Each nuclei in the molecule has different electronic environment and thus has distinct chemical shift. Furthermore, in large molecules the number of resonance can be counted in thousands which can cause the overlap of signals. To prevent this occurrence, we need to performed multidimensional NMR such as, 2D-NMR and 3D-NMR that correlate the frequencies of distinct nuclei. There exist two main categories in NMR experiment for proteins: first one, the magnetization transformed through the chemical bonds, to assign the different spin signals (COSY, TOSCY). Second one, is when magnetization is transferred through space, used to generate the distance restraints, which are used to calculate the structure (NOESY).

As it was describe in section 1.5.1. heteronuclear techniques correlate the chemical shift of heteronuclei 15N-1H. As 15N is only present at 0.37% naturally. Labeled proteins have been produced by overexpression in bacterial host system in 15N labeled media. This method is not too expensive, and the experiment is highly sensitive and can be performed very quickly 20 minutes. HSQC is often used to screen candidates for determination of structure, also for optimization of the sample it can also be used in relaxation analysis and dynamics.

If an HSQC spectrum is assigned it can be used to study protein interactions as well as ligand – protein interaction by technique known as Chemical Shift Mapping (CSM). The spectra of a free protein is compared with the protein – ligand spectrum. Binding of a ligand can cause perturbation in the chemical shifts of some peaks, which lie at the binding site. In this thesis CSM was used to examine the interaction of between heme and hHBP as well as with HO-1. The assignments for hHBP were obtained by comparing hHBP and mHBP spectra. Fig 8 shows what happens to threonine43 (Thr43), when heme binds to the protein.

18

Fig 8. Chemical shift map ping for mHBP. The green spectra represent shift after binding of hemin and the blue spectra represent the PPIX binding.

We were able to use mHBP shifts map, due to similarity in the sequence between mHBP and hHBP ( 89% homology).

1.5.2.1. Sample preparation.

Samples are prepared in aquous solution with the pH, ionic strength and temperature being close to physiological conditions. NMR studies of a protein have to be performed on samples of highly purified protein. The protein can be obtained from a natural host or can be produced by using an overexpression system. The use of recombinant DNA techniques also allows isotopic labeling. The sample consist of 300 – 600 microliters in which protein has a concentration of between 0,1 – 3 mM.

1.5.3. UV-vis spectroscopy.

Ultraviolet – visible spectroscopy (UV-vis) have been used for the last 35 years, and over this period of time become one of most important technique. It is used for many a wide variety of analysis, due to its simplicity, versality, speed, accuracy and cost-effectivness.

19 UV-vis spectroscopy refers to absorption spectroscopy, in the ultraviolet-visible spectral region (length of wave from 200nm to 1100nm). The absorption or reflectance in the visible range directly affect the perceived color of the chemicals involved. The method is based on that many molecules absorb ultraviolet or visible light. The absorbance is increasing with the increase of attenuation of the beam. The absorbance is directly proportional to the path length (d), and the concentration (c) of the absorbing solution, it is stated by the Beer – Lambert law.

Different molecules absorb radiation of different wavelengths. Molecules containing n numbers of electrons or non-bonding electrons can absorb the energy in a reason of that they excite to higher anti-bonding molecular orbitals. Method is mostly use to determine the concentration of absorbent in the solution, also it can be used as a detector for HPLC. Spectroscopic analysis is commonly carried out in solutions but it can be also used to study gases and solid.

In a case of colored solution, it absorb visible light, that the bases for analysis of heme – protein interactions. It was stated that, according to changes of heme concentration the absorbance is changing at 405nm. It can be used to examine the interactions between ligand and protein, by observation of shifts at the maximum absorbance in 405nm.

1.5.3.1. Sample preparation.

Samples are usually prepared in aquous solution with the pH, ionic strength and temperature being close to physiological conditions. Samples must be clear solutions, to do not give the erroneous results. The sample is placed in the glass cell in which a solution path leangth is exactly 1cm. The volume of glass cuvette is 1ml.

20

2. Aim.

Heme is one of the most common prosthetic group in a wide variety of proteins named hemoproteins. Under oxidative condition or during biosynthesis of heme, heme can be released from the proteins into the cytoplasm. Heme has high chemical reactivity and its poorly soluble in aqueous solution, that is why it needs to be under tight control in the organisms. Heme buffers or transporters must be present during heme proaction.

Human heme binding protein (hHBP) belongs to the SOUL/p22HBP protein family. Although, the function of hHBP is still unknown the protein binds heme with a 0,5nM Kd. If hHBP is a heme transporter there may need a binding partner.

Unpublished results have shown that HO-1 is present when hHBP is knocked down in hepatic cell moodels, therefore HO-1 could be possible partner for hHBP. The aim of the work was therefore to examine the ability of HO-1( a possible binding partner) to accept heme from heme bound to human heme binding protein. UV-vis and NMR spectroscopy were used to study possible heme transfer between hHBP and HO-1.

21

3. Materials and methods.

3.1.Overexpression of HO-1. 3.1.1. Materials

3.1.1.1.Plasmid.

The vector pBAce contain HO-1 gene which sequence is shown below, stop and start codons are marked in capitals letter. The gene was placed between Nde1 and Sal1 restriction sites. The plasmid vector was kindly provided to us by Professor Montellano (UCSF, USA). The sequence is shown bellow. The native sequence has 13,148kbp [30] and the sequence provided is truncated from rat and has 795bp.

CATatggagcgtccgcaacccgacagcatgccccaggatttgtcagaggccctgaaggaggccaccaag gaggtgcacacccaggcagagaatgctgagttcatgaggaactttcagaagggccaggtgacccgagacg gcttcaagctggtgatggcctccctgtaccacatctatgtggccctggaggaggagattgagcgcaacaagg agagcccagtcttcgcccctgtctacttcccagaagagctgcaccgcaaggctgccctggagcaggacctgg ccttctggtacgggccccgctggcaggaggtcatcccctacacaccagccatgcagcgctatgtgaagcggc tccacgaggtggggcgcacagagcccgagctgctggtggcccacgcctacacccgctacctgggtgacctgt ctgggggccaggtgctcaaaaagattgcccagaaagccctggacctgcccagctctggcgagggcctggcct tcttcaccttccccaacattgccagtgccaccaagttcaagcagctctaccgctcccgcatgaactccctggag atgactcccgcagtcaggcagagggtgatagaagaggccaagactgcgttcctgctcaacatccagctctttg aggagttgcaggagctgctgacccatgacaccaaggaccagagcccctcacgggcaccagggcttcgccagc gggccagcaacaaagtgcaagattctgcccccgtggagactcccagagggaagcccccactcaacacccgct cccaggctTAA 3.1.1.2.Bacterial stain.

During this experiment the DH5α strain was used [25].

The DH5α strain genotype is: F- 80dlacZ M15 (lacZYA-argF) U169 recA1 endA1hsdR17(rk-, mk+) phoAsupE44 -thi-1 gyrA96 relA1.

3.1.1.3.Culture medium.

The bacterial culture media, LB was used to grow DH5α cells. And SOC was used medium for the transformation of the DH5α cells with HO-1.

22 LB medium – yeast extract, tryptone, NaCl, 1,5% agar ( for solid media ). Sterilization by autoclaving. The pH was adjusted to 7,4.

SOC media – tryptone, yeast extract, NaCl, KCl. Sterilization by autoclaving. MgCl2,

MgSO4, glucose added after sterilization. The pH was measured as 6-8.

The antibiotic used for selection was ampicillin. The stock was prepared in distillated water and sterilized by filtration 0,22nm membrane filter. Stored in 4˚C. The concentration of stock was 100 μg/ml.

3.1.2. Methods

3.1.2.1.Transformation of DH5α.

For the transformation of the DH5α cells, the heat-shock method was used. Tubes containing 100μl of commpetent cells were thawed on ice. Then, 5μl of plasmid DNA was added ( 0,4ng ). The tube was gently agitated, and the mixture was incubated on ice for 30 minutes. Heat shock 42˚C for 45 sec was applied. Followed by 2min on ice. Then 0,9 ml of SOC media was added, and the mixture was incubated at 37˚C, 180rpm, for 1h.

The resulting cells were spread on the LB+agar plates and grown overnight at 37˚C, the properly transformed cells were white.

For long term storage of transformed DH5α cells, glycerol stocks were prepared. After growth in LB media, 375μl was transferred to a sterilized eppendorf tube to which 175μl of 80% glycerol was added. The mixture was gently mixed and store at -80˚C.

3.1.2.2.Cell culture/ expression of HO-1 in cells.

Overexpression of HO-1 was carried out on a 2L scale. A single colony was selected from overnight growth on LB plates with ampicylin (100 μg/ml ). This was used to inoculate 5ml of LB media with ampicilin ( 100 μg/ml ) and grown overnight at 37˚C, 180rpm. 1ml of innoluated cells were transferred to 1L of LB/amp media and incubated at 37˚C, 180rpm, for 16-18h.

The cells were collected after 16-18h in 50ml tubes, by centrifuging them for 15min, at 5000 RPM ( 4˚C ).

23

3.2.Purification of heme oxygenase-1. 3.2.1. Materials.

3.2.1.1.Buffers.

During the purification, the following buffers were used, the recipes are shown in Table 2 .

Table 2. Buffers used during purification of HO-1.

Name of process Characteristic of buffers

Cell Lysis 100mM Tris pH 8,0; 2mM EDTA pH 8,0; 2mM PMSF ( dissolved in EtOH ) Lysozyme ( 0,5mg/ml ) Volume: 20ml Dialysis 10 – 20mM potassium phosphate pH7,4; 1mM EDTA

Buffer for resuspending the pellet after precipitation by ammonium sulfate. Volume: 30ml 10mM potassium phosphate pH7,4; 1mM EDTA Dialyze buffer. Volume: 4L.

QAE A-25 mobile phase

10mM potassium phosphate pH7,4; 1mM EDTA Wash/binding buffer Volume:25mL 10mM potassium phosphate pH7,4; 1mM EDTA 0-100mM NaCl gradient Elution buffer. Volume of a single fraction: 15ml SDS-PAGE buffers 2,175ml of H2O; 1,25ml Tris-HCl 1,5M pH 8,8; 1,5ml of 40% acrylamide; 50μl of 10% SDS; 2,5μl TEMED and 25μl of 10% APS Running gel 795μl of H2O; 315μl Tris-HCl 0,5M pH 6.8; 125μl of 40% acrylamide; 12,5μl of 10% SDS; 1,25μl TEMED; 6,25μl of 10% APS Stacking gel 200μl of 10% SDS; 125μl Tris-HCl 0,5M pH 6,8; 100μl mercaptoethanol; 75μl of glycerol and bromophenol

blue

Sample loading buffer

1g of Coomassie Billiant Blue resuspend in 1 liter of the following solution: Methanol (

50%[v/v]), glacial acetic acid (10%[v/v]) and H2O (40%).

4h stirring.

24 3.2.1.2. Dialysis membrane.

For purification purposes a dialysis membrane was used, with cut off at 10K. Pre-soaking of dialyze tube was carried out by incubation in boiling water for 1h, before use.

3.2.1.3.QA52 anion exchange column.

For purification of HO-1 after dialysis, a QAE Sephadex A-25 column was used. It is strong basic, quaternary amine-bearing anion exchange medium, with high protein capacity. When fully ionized, it bears constant change under all pH conditions. QAE Sephadex A-25 60 ml was loaded onto a 100 ml column and washed with 700 – 1000ml of 10mM potassium phosphate pH 7.4.

3.2.2. Methods.

3.2.2.1. Cell lysis.

Sonication was used for cell lysis. As one of the best tool for fragmentation of cell membrane and release of cell contents.

Pellets were resuspended, in 20ml of lysis buffer 100mM Tris pH 8,0, 2mM EDTA pH 8,0 and 2mM PMSF (dissolved in EtOH) (table 2) then lysozyme was added to final concentration of 0,5mg/ml and stirred intermittently for 30min.

Cell lysates were sonicated for 10min, with 59sec of 70% power impulse and 59sec of intervals, while the process tubes were placed on ice to prevent denaturation of protein. Centrifugation for 45min at 35000 rpm (4˚C), was used to separate the cells, from the supernatant.

3.2.2.2.Salting out.

In the first stage of protein purification, as no purification tag was used in the HO-1 plasmid (such as a his-tag) salting out was used. Under high concentration of a salt (NH4SO4), proteins can precipitate from solution.

To precipitate protein from the supernatant after cell lysis, ammonium sulfate was added to final concentration of 35% saturation and stirred intermittently for 40min. The

25 solution was then centrifuged for 20min at 15000 rpm (4˚C). Then ammonium sulfate was added to final concentration of 60% saturation and stirred intermittently for 1h. After centrifuging for 20min at 15000 rpm (4˚C), the pellet was resuspended in 20ml of 10mM potassium phosphate, 1mM EDTA pH 7,4 buffer.

3.2.2.3.Dialysis.

Dialysis is a process of separating molecules in solution based on molecular size. Dialysis was used to separate small molecules like salts from the supernatant. Dialysis was performed using pre-soaked tube, with 20ml of resuspended pellet. Dialysis against 4L of dialysis buffer: 10mM potassium phosphate pH7,4 and 1mM EDTA overnight at 4˚C was carried out.

3.2.2.4. Purification on QA52 anion exchange column.

For the final purification of HO-1, anion exchange chromatography was carried out using a QAE Sephadex A-25 column. The cellulose had quaternary amine-bearing anion exchange media. The purification was based on changing the charge inside the column, by using NaCl salts. The pI of the protein is [].

The supernatant after dialysis was loaded on the column, and washed by elution buffer 10mM potassium phosphate pH7,4, 1mM EDTA with a step NaCl gradient (0mM, 20mM, 40mM, 60mM, 80mM and 100mM ). Fraction were collected into 50ml tube. The presence of protein was determined by SDS-PAGE.

3.2.2.5.SDS-PAGE analysis.

SDS-PAGE electrophoresis was used to examine the purity of fractions after purification on the QA52 anion exchange column. A Vertical Mini-Protean 3 unit (BioRad) was assembled for SDS-PAGE gel running. A 12% running gel and 4% stacking gel was prepared prior to electrophoresis.

The 12% running gel was prepared by adding 2,175ml of H2O, 1,25ml Tris-HCl 1,5M pH 8,8, 1,5ml of 40% acrylamide, 50μl of 10% SDS, 2,5μl TEMED and 25μl of 10% APS. The 4% stacking gel was prepared by adding 795μl of H2O, 315μl Tris-HCl 0,5M pH 6.8, 125μl of 40% acrylamide, 12,5μl of 10% SDS, 1,25μl TEMED, 6,25μl of 10% APS. Each sample was mixed with 50μl of reducing buffer, which was compound 200μl

26 of 10% SDS, 125μl Tris-HCl 0,5M pH 6,8, 100μl mercaptoethanol, 75μl of glycerol and bromophenol blue. Followed by denaturing step at 90˚C for 5 minutes. Then, the samples with the SDS marker were loaded into appropriate lanes of the SDS-PAGE gel. The gel was run at 190V for 45 minutes. After that, the gel was put in to the water and boiled in a microwave for 1min, followed by cooling for 3min (repeated three times). Finally, the gel was immersed in Coomassie Blue and boiled in the microwave for 1min. After removing Coomassie, distilled water was added, 2 min of shaking was sufficient to visualize the bonds.

3.2.2.6.Concentration of purified protein.

SDS-PAGE analysis allowed the purity of the fraction to be seen. The fraction containing the biggest and at the same time the purest protein (fraction containing 20mM of NaCl) was concentrated using 10kDa centricons (Milipore). The centricon was filled by the purest fraction, and centrifuge for 10min at 5000 (4˚C), it was concentrated to the required final volume. During concentration the fraction was washed three times with potassium phosphate buffer to wash from it EDTA and NaCl.

3.3.Overexpression of hHBP 3.3.1. Materials.

3.3.1.1. Bacterial stain.

E.coli stain was transformed before. During the experiment, were used cell from glycerol stock. Bacterial stain was stored at -80˚C in 80% of glycerol.

3.3.1.2. Culture media.

Here are showed media used during the experiment, to overexpressed cells with hHBP gene.

LB medium yeast extract, tryptone, NaCl, 1,5% agar ( for solid media ). Sterilization by autoclaving. The pH 7,4.

M9 medium ( minimal )

950 ml of distilled water with 50 ml M9 salts. Sterilization by autoclaving.

27 In the cell culture the antibiotic kanamycin was used as a selective agent. The stock was prepared in distillated water and sterilized by filtration 0,22nm membrane filter. Stored in 4˚C. The concentration of stock was 50μg/ml.

3.3.2. Methods.

3.3.2.1. Expression of hHBP in cells.

Cell expression was carried out in 2L scale. For the starter culture 50ml of LB media with 50μl of kanamycin (50μg/ml). Media was inoculated by cells from glycerol stock and growth overnight at 37˚C, 150 rpm.

After overnight growth, 2L of LB with 1ml kanamycin (50μg/ml) were inoculated by 10ml of starter culture each. Cells were growth for 5h, at 37˚C 150rmp. After, that cell were centrifuge for 3min, at 8000 rmp (4˚C) in 50ml sterile tubes. The pallet was resuspented in 2L of M9 media to which was added 0,5ml MgSO4 1M, 0,5ml CaCl

0,1M, 4g glucose, 0,25ml of 0,2% tiamin-HCl, 0,5ml FeSO4 0,1M, 1ml kanamycin

50μg/ml, 1g NH4Cl (N15), all of the operations were under sterile conditions. The

incubation with M9 media was for 2h at 30˚C, 150rpm. To induced growth after 2h, 1ml of 0,5M IPTG was added to the M9 media and then growth overnight at 30˚C, 150rpm.

The cells were collected after night in 50ml tubes, by centrifuging them for 3min, at 8000 rmp (4˚C).

28

3.4.Purification of hHBP. 3.4.1. Materials

3.4.1.1.Buffers.

During the purification, few buffers were used, the recipes are shown in the table below (Table 3).

Table 3. Buffers used for purification of hHBP

Phosphate buffers

Phosphate buffer+ NaCl 50mM KH2PO4; 300mM NaCl pH 8.0 Phosphate buffer 50mM KH2PO4 pH 8,0

Phosphate buffer+ NaCl + Imidazol

50mM KH2PO4; 300mM NaCl; Imidazol gradient ( 10mM, 20mM, 50mM, 75mM, 175mM, 500mM )

SDS-PAGE buffers The running gel

2,175ml of H2O; 1,25ml Tris-HCl 1,5M pH 8,8; 1,5ml of 40% acrylamide; 50μl of 10% SDS; 2,5μl TEMED and 25μl of 10% APS

The stacking gel

795μl of H2O; 315μl Tris-HCl 0,5M pH 6.8; 125μl of 40% acrylamide; 12,5μl of 10% SDS; 1,25μl TEMED; 6,25μl of 10% APS

Sample loading buffer

200μl of 10% SDS; 125μl Tris-HCl 0,5M pH 6,8; 100μl mercaptoethanol; 75μl of glycerol and bromophenol blue

Coomassie Buffer

1g of Coomassie Billiant Blue resuspend in 1 liter of the following solution: Methanol ( 50%[v/v]), glacial acetic acid (10%[v/v]) and H2O (40%). 4h stirring.

3.4.2. Methods

3.4.2.1.Cell lysis.

For extraction of hHBP from cells, as a disruption method Sonication was used. After overnight growth, cells were collected by centrifugation for 3min, at 8000 rmp (4˚C). Pallet was resuspended in 50mM KH2PO4 with 300mM NaCl pH 8.0 buffer and

incubated for 1h at 37˚C with lysozyme (0,5mg/ml).

Cell lysates were sonicated 10min, with 59sec of impulse and 59sec of intervals power 70%, while the suspensions were placed on ice to prevent denaturation of protein.

29 Centrifugation for 60min at 20000 rpm (4˚C), was used to separate the cells from cytoplasmic fraction. For the further purpose, subernatant was kept.

3.4.2.2.Purification of hHBP.

The purification of hHBP was performed by affinity chromatography, is a method for separation of biochemical mixtures based on highly specific interactions. The column used during the process was His-Trap IMAC HP. The separation of proteins was based on IMAC system. It involves the natural tendency of histidine to form a complex with metal at neutral pH. The changes of pH using chelators lead to elution of his-tagged proteins. hHBP was expressed as a histidine-tagged protein. To release the protein from the column, the bound between histidine and Ni need to be broken. It is possible by reducing pH or increasing the ionic strength of the buffer (imidazol).

The supernatant after cell lysis was loaded on the His-Trap IMAC HP column, which firstly was equilibrated with 50mM phosphate buffer with 300mM NaCl pH 8. The elution of protein was performed using 50mM phosphate, 300mM NaCl buffer containing imidazol gradient (10mM, 20mM, 50mM, 75mM, 175mM, 500mM). The obtained fractions were analyzed by SDS-PAGE.

3.4.2.3.SDS-PAGE analysis.

The separation of each fraction was carried out by SDS-PAGE, in the same condition as described in section 3.2.2.5.

3.4.2.4.Concentration of protein.

Fraction contained the highest purity of protein, was concentrated in 10kDa Centricon (Milipore) to volume 3,5 ml by centrifugation at 5000rpm for 10min. The concentrated fraction was putted on the PD-10 column, to change the buffer form the one containing NaCl and imidazole to 50mM phosphate buffer. The volume of washed fraction was 2,5ml. If it was required the fraction was concentrated.

30

3.4.3. Quantitative analysis.

The quantification of pure sample was carried out by UV-vis spectroscopy method. According to the Beer-Lambert law, the absorbance was measured at 280nm. The equation from which concentration of protein was counted, is showed below.

A- Absorbance, measured at 280nm.

ε – extinction coefficient M-1cm-1 for HO-1 it was 24410 M-1cm-1 [34] and for hHBP it was 36800 M-1cm-1[33].

l – the thickness of the cell (cm) c – concentration (M)

3.5. Studies of the interaction. 3.5.1. Material.

3.5.1.1. Hemin solution.

Hemin solution was prepared using recipes presented below.

To prepared hemin in 10ml scale, 1mg of hemin was used and 50μl of 25% NH3 was

added, 137μl of 1,5% m/v tween was added, and the solution was prepared by dissolving 150mg of tween in 1ml of H2O. After 8ml of H2O was added. The pH of the

solution should be 8, it was obtained by adding 1M NaH2PO4. 3.5.2. Methods

3.5.2.1.UV-vis spectroscopy.

To examine the binding of heme to HO-1 and to hHBP, a series of reactions with different concentration of proteins and heme were carried out. The spectra were measured from 900nm to 190nm. The experiment was performed based on the changes of the solution staining during addition of hemin. The absorbance was measured by Spectrophotometer UV/VIS JASCO V-580, from 900nm to 190nm.

31 The measurement was performed using different HO-1 and hHBP concentration for HO-1 it was: 61 μM, 30 μM and 10 μM. For hHBP 29,3 μM and 9,77 μM. The added hemin has different concentrations for different samples, the initial concentration was 270 μM which was respectively diluted . To the sample containing 10 μM HO-1 in volume 600 μl, was added 0,5 μl. The samples were placed in 1ml volume glass cuvette with d=1cm. The analysis was based on the paper Wilks at al.[25].

Table 4. reaction analysed by UV-vis spectroscopy.

Number HO-1 [μM] Hemin [μM] hHBP [μM] peroxide [μM]

Experiment 1 30 30 - -

Experiment 2 10 Final concentration 0,4

initial concentration 10 - -

Experiment 3 10 0,4 - 10

Experiment 4 10 0,4 - 10

Experiment 5 - Final concentration 0,56;

initial concentration 10 9,8 -

Experiment 6 - 0,56 9,8 10

Experiment 7 - PPIX was used in

concentration of 270 10

Experiment 8 36,2 170 29.3 -

Experiment 9 36,2 170 29,3 -

3.5.2.2.Nuclear magnetic resonance.

The NMR of hHBP samples were recorded on a 500MHz Bruker spectrometer. All the NMR experiments were performed at 303K. All the obtained spectra were processed by NMRPipe/NMRDraw software and all the spectra analysis were performed by CARA software. For chemical shift mapping 15N HSQC had been performed.

3.5.2.3. Preparation of sample for NMR.

For the NMR studies of the interaction between heme and hHBP and HO-1, sample was prepared to final volume of 250 μl in a 50mM phosphate buffer pH 8 and 10% D2O.

32 To the samples containing labeled hHBP first hemin was added in different concentrations: 28 μl of 1,5 mM of hemin and 28μl of 0,15mM of hemin. The sample with the higher concentration of hemin was mixed in 1:1 ratio with HO-1 solution. The reactions that were carried out are shown in table 5.

Table 5. Concentration of proteins and hemin that were used in NMR studies.

Sample number [HO-1] mM [hHBP] mM [hemin] mM

Sample 1 - 0,17 0,15

Sample 2 - 0,17 1,5

33

4. Results and discussion.

4.1. Overexpresion and purification of HO-1.

Gene of HO-1 was overexpressed in DH5α stains, in 2L scale in LB media using as a selective agent actibiotic, which was ampicilin ( 100 mg/ml ). To extract protein from cells, as a disruption tool Sonication was used. Furthermore to obtain more protein after cell lysis, ammonium sulfate was used to precipitate the protein. As a first step of purification dialysis was carried out against the 10mM potassium phosphate with 1 EDTA buffer pH 7.4. The supernatant after dialysis, was purified on anion exchange column as a elution buffer 10mM potassium phosphate, 1mM EDTA pH 7,4 with 0-100mM NaCl gradient. All the fractions and samples collected at each step of purification protocol were analyzed by SDS-PAGE. In a reason of many collected samples the analysis needed to be performed on two gels. Results are shown in Fig 8 and Fig 9.

Fig 9. SDS-PAGE analysis of purified HO-1. Lanes 1 – leader nzytech Low Molecular Weight Protein Marker; 2 - supernatant after lysis; 3 - pellet after cell lysis; 4- resuspended pellet after precipitation; 5 – supernatant after dialysis; 6 – purification on the column, flow of supernatant after dialysis; 7- purification on the column, elution with phosphate buffer 0mM NaCl; 8- purification on the column, elution with phosphate buffer +20mM NaCl; 9 - supernatant after lysis.

The band (marked by arrows) presented in lanes 7 and 8 between 32,0 kDa and 26,0 kDa are representing HO-1 which molecular weight is around 30,0 kDa [30]. The

34 analysis showed that HO-1 is eluted from the column by 10mM potassium phosphate with 1mM EDTA pH 7,4 buffer and by the same buffer with 20mM of NaCl.

Fig 10. SDS-PAGE analysis of purified HO-1. Lanes 1 – leader nzytech Low Molecular Weight Protein Marker; 2 – wash of the column by 10mM potassium phosphate buffer with 1mM EDTA ( elution buffer ), 0mM NaCl; 3- elution by elution buffer with 100mM NaCl; 4- elution by elution buffer with 80mM NaCl; 5- elution by elution buffer with 60mM NaCl; 6-elution by elution buffer with 40mM NaCl; 7 – elution by elution buffer with 20mM NaCl; 8 – elution by elution buffer with 0mM NaCl; 9 – purification on the column, flow of supernatant after dialysis.

The result of purification on the QA52 anion exchange column is shown in Fig 9. The band (marked by the arrows) presented in lanes 7 and 8 between 32,0kDa and 26kDa are representing the molecular weight (around 30,0 kDa) of HO-1. It was also observed band (marked by orange arrows) in lanes 2 and 3 which are situated between 66,0 kDa and 48,0 kDa, what can suggest that it is dimer of HO-1, which molecular weight is around 55,1 kDa.

35

4.2.Overexpression and purification of hHBP

The human p22HBP gene was expressed in 2L scale fist in LB media, then in M9 media to label the protein with 15N. As a selective agent kanamycin was used in a concentration of 50mg/ml. Sonication was used to extract protein from the cells. For purification the affinity chromatography was used, on His-Trap IMAC HP column. The expressed protein had 6xHis to be able to bound to Ni in the column. Protein was eluted using 50mM phosphate and 300mM NaCl buffer pH 8 by imidazol gradient (0mM, 10mM, 20mM, 50mM, 75mM, 175mM, 500mM). The results are shown in Fig 11.

Fig 11. SDS-PAGE analysis of purified hHBP. Lanes 1 – leader nzytech Low Molecular Weight Protein Marker; 2- supernatant after lysis; 3- flow of supernatant after lysis through column; 4 - elution with 0mM imidazole; 5- elution with 10mM imidazole; 6 – elution with 20mM imidazole; 7- elution with 50mM imidazole; 8- elution with 75mM imidazole; 9 – elution with 175mM imidazole; 10- elution with 500mM imidazole.

A distinct band was observed on the gel after the SDS-PAGE. The band marked by red arrows is situated between 26,0 kDa and 18,5 kDa, and it is from hHBP (molecular weight 22,0 kDa).

36

4.3.Heme – protein interaction 4.3.1. UV-vis spectroscopy

The aim of the experiment was to check the ability of heme to bind to HO-1 and hHBP, by increasing the concentration of heme in the sample and measuring the changes in absorbance at 405nm, by examining the increase in absorbance at 405nm, for both proteins and by comparing the position of the band maximum the interaction of heme with proteins can be followed. The experiment with HO-1 was based on the protocol from paper by Wilks at al. [25].

No specific information about and concentration of HO-1 was given in the previous paper [25], therefore we needed to optimize the experiment.

Experiment 1

Initially the experiment was performed at a HO-1 concentration of 0,03mM and a hemin concentration of 0,03mM. The tritration used additions 2μl and 20μl of hemin. The experiment was performed in 1ml volume.

Fig 12. HO-1 reaction with hemin. Blue line - HO-1; green line - HO-1 with 2μl hemin: violet line - HO-1 with 20μl hemin. The concentrations are shown in Table 4.

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 800 782 764 746 728 710 692 674 656 638 620 602 584 566 548 530 512 494 476 458 440 422 404 386 368 350 332 314 A b sor b an ce Wavelength [nm]

37 The absorption at 405nm increased slightly, during addition of 2μl heme, but when 20μl hemin was added value of the absorbance at 405nm increased significantly. This resulted in a non-linear increase in absorbance.

Experiment 2

The experiment was therefore repeated using 10μM concentration of HO-1 and a 30μM concentration of hemin, it was performed in 600μl. The results are shown in Fig 13.

Fig 13. The reaction of hemin and HO-1. Hemin was added in 0,5μl volume for each reaction. The orange curve represent hemin in concentration of 0,51μM.

It can be seen that the absorption at 405nm is increasing, due to the higher concentration of hemin and the creation of a heme-HO-1 complex. The absorption increases linearly with the increase of hemin concentration (Fig 14). The changes in the absorbance at 405nm are a result of the formation of heme-HO-1 complex. [24].

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 800 782 764 746 728 710 692 674 656 638 620 602 584 566 548 530 512 494 476 458 440 422 404 386 368 350 332 314 A b sor b an ce Wavelength [nm]

38

Fig 14. Graph of absorbance versus the heme concentration, while adding heme to HO-1.

Experiment 3

To confirm the creation of heme-HO-1 complex, we performed reaction with peroxide (10μM), which aimed to destroy heme. It cause the decrease of absorption at 405nm and increase it at ~ 680nm [25]. The course of reaction was monitored by measuring absorption before addition of peroxide and in 0, 5, 10, 20min time after addition. The results are shown in Fig 15.

0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0 0.1 0.2 0.3 0.4 0.5 A b sor b an ce Heme [μM]

39

Fig 15. Reaction with heme-heme oxygenase complex with H2O2. Blue line – immediately after; red line – 5min

after; green line – 10min after; violet line – 20min after; turquoise line – HO-1 with heme.

As it was mentioned before the reactions were based on the protocol from paper by Wilks at al. [25]. The course of reaction with peroxide is different than that described in the paper. It was stated that after addition of peroxide the absorbance at 405nm should decrease over time. In Fig 14 the absorption (405nm) is lower after addition of peroxide in 0min time, but after incubation it increase. This is due to the fact, that HO-1did not create complex with hemin. What can be seen by the look of the pick of HO-1 curve in Fig 15.

Experiment 4

To confirm obtained result, another reaction with peroxide was carried out. During the reaction 10μM of peroxide was added firstly in 1μl volume then in 2 μl, to the solution of heme-HO-1 complex, the reaction was performed in 600μl volume. Whole amount of added peroxide was 3μl. Then the absorbance was measured at 0, 10 and 15min time.

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 800 782 764 746 728 710 692 674 656 638 620 602 584 566 548 530 512 494 476 458 440 422 404 386 368 350 332 314 A b sor b an ce Wavelength [nm]

40

Fig 16. Reaction of peroxide with hemin-HO-1 complex. Addition of hemin 1μl each step. Time of incubation with enzyme 0, 10 and 15minutes.

Fig 16 shows the influence of peroxide on the HO-1 complex with hemin. The absorbance is decreasing at 405nm, due to transformation of hemin to biliverdin in presence of peroxide. This is in agreement with Wilks at al. [25]. Also the absorbance at 680nm is increasing, over time. Both of these facts suggest that the complex of hemin and HO-1 was formed. The concentration of peroxide changes are shown in Fig 17.

Fig 17. Graph of absorbance versus concentration of peroxide.

0 0.02 0.04 0.06 0.08 0.1 0.12 750 736 722 708 694 680 666 652 638 624 610 596 582 568 554 540 526 512 498 484 470 456 442 428 414 400 386 372 358 A b sor b an ce Wavelength [nm] 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 0 0.01 0.02 0.03 0.04 0.05 A b sor b an ce concentration [μM]

41 Experiment 5

The some reaction was carried out for hHBP to examine the possibilities of removing heme from hHBP. For this reaction 9,8 μM hHBP was used and 30 μM of hemin was added in 0,4 μl. Initially the binding of hemin to hHBP was followed, the reaction was performed in 400μl volume. The results for addition of hemin to hHBP are shown in Fig 18.

Fig 18. Reaction of hemin binding to hHBP. Addition of 0,4μl hemin each step.

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 800 782 764 746 728 710 692 674 656 638 620 602 584 566 548 530 512 494 476 458 440 422 404 386 368 350 332 314 A b sor b an ce Wavelength [nm]

42 The increase in absorbance at 405nm confirms that hemin binds to hHBP. It can be seen at the graph of absorbance versus concentration of hemin (Fig 19).

Fig 19. Graph of absorbance versus heme concentration. While reaction of heme with hHBP.

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0 0.1 0.2 0.3 0.4 0.5 0.6 A b sor b an ce Concentration [μM]

43 Experiment 6

As stated previously if hemin can be broken down by peroxide and therefore could have the some influence on the UV-vis spectrum as for the hemin-HO-1 complex. To the solution with the highest concentration of heme, peroxide (10μM) was added in 0,8μl steps, to reach the 8,8 μl final volume. Results are shown in Fig 20.

Fig 20. Reaction of peroxide with hemin-hHBP complex. Addition of 0,4 μl peroxide each step. Absorbance at 405nm decrease and at 680nm increase.

As it was noticed for hemin hHBP complex during reaction with peroxide the absorbance at 405nm was decreasing in order to higher concentration of peroxide. Furthermore, the absorbance at 680nm was increasing with the higher concentration of peroxide. It can be stated that the graph in Fig 20 represent the some changes that appeared during reaction of peroxide with hemin - HO-1 complex. It can be seen that as peroxide is added the absorbance at 405nm is decreasing. It appears therefore that hemin is being break down by peroxide. The changes of peroxide concentration are shown on Fig 21. 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 800 782 764 746 728 710 692 674 656 638 620 602 584 566 548 530 512 494 476 458 440 422 404 386 368 350 332 314 A b sor b ac e Wavelength [nm]

44

Fig 21. Graph of concentration versus absorbance 9405nm), for reaction of peroxide with heme-hHBP complex. 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0 0.05 0.1 0.15 0.2 0.25 A b sor b an ce concentration [μM]

45 Experiment 7

A final reaction was carried out on the hHBP complex with protoporphyrin ( PPIX ). Here 10μM peroxide was added to the hHBP – PPIX complex. The results are shown in Fig 22.

Fig 22. Graph shows reaction of PPIX - hHBP complex with peroxide. Green line – after addition of 5μl of peroxide; violet line – after addition of 5μl of peroxide ( 10μl whole volume ); blue line – after addition of 5μl of peroxide ( 15μl whole volume ).

Here it appears that, the PPIX cannot be broken down when complexed to hHB. Experiment 8 and 9

An experiment with HO-1 and hHBP was carried out next. A concentration of HO-1 of 36,2μM and a concentration of 29,3μM for hHBP was used. The initial concentration of hemin was 0,17mM. 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 700 687 674 661 648 635 622 609 596 583 570 557 544 531 518 505 492 479 466 453 440 427 414 401 388 375 362 A b sor b an ce Wavelength [nm]