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UPTEC-K12018
Examensarbete 30 hp
September 2012
An investigation
of protective
formulations containing enzyme
inhibitors Model experiments of trypsin
Erika Billinger
Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 AN INVESTIGATION OF PROTECTIVE FORMULATIONS CONTAINING ENZYME INHIBITORS. MODEL EXPERIMENTS ON TRYPSIN. ERIKA BILLINGER MASTER THESIS SPRING 2012 SVENSKA CELLULOSA AKTIEBOLAGET UPPSALA UNIVERSITY 2 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 ABSTRACT This master thesis considers an investigation of protective formulations (ointment, cream) containing enzyme inhibitors. Model experiments have been made on the enzyme trypsin. It is well accepted that feces and urine are an important causing factor for skin irritation (dermatitis) while using diaper. A protective formulation is a physical barrier that separates the harmful substances from the skin. It can also be an active barrier containing active substances, which can be active both towards the skin, and the substances from feces and urine. By preventing contact from these substances the skin will not be harmed, at least for a period of time. A number of different inhibitors were tested towards trypsin and they all showed good inhibition, two of the inhibitors were selected to be immobilized with the help of NHS-­‐
activated Sepharose. Immobilization of these two inhibitors leads to a lesser extent of the risk of developing allergy and also that the possible toxic effect can be minimized. 3 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 POPULÄRVETENSKAPLIG SAMMANFATTNING Det är väl känt att avföring och urin är en viktig faktor som ger upphov till irriterad hud (dermatitis) för barn som använder blöjor. Tillsammans med avföringen följer enzymer från mag-­‐tarmkanalen med och det är dessa enzymer som ”äter” på huden och ger upphov till dermatitis. Då urin avges i blöjan stiger pH vilket leder till en mer trivsam miljö för enzymerna som i sin tur leder till en ökad aktivitet och resulterar i en större area irriterad hud. Ytterligare anledningar till dermatitis är även den fuktiga miljön som skapas av stängd miljö och även friktionen som uppkommer då rörelser sker vid användning av blöjan. En skyddande formulering, i form av exempelvis en salva eller kräm, kan separera de skadliga ämnena från huden. Man kan även tänka sig använda en aktiv barriär innehållande aktiva substanser, exempelvis inhibitorer, som kan vara skyddande mot huden och samtidigt aktiv mot de skadliga ämnena från avföring och urin. Inhibitorerna i en salva eller kräm hämmar då enzymernas aktivitet och huden skyddas och blöjeksem undviks. Genom att undvika hudkontakt med de skadliga ämnena kan huden skyddas, åtminstone under en viss tidsperiod. Då avföringen innehåller ett matspjälkningsenzym trypsin har olika inhibitorer mot enzymet undersökts. För att även minimera risken för både toxiska och allergena effekter har två av dessa inhibitorer immobiliserats på NHS-­‐aktiverad Sepharose. 4 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 TABLE OF CONTENTS An investigation of protective formulations containing enzyme inhibitors. Model experiments on trypsin. Abstract ............................................................................................................................................................. 3 Populärvetenskaplig sammanfattning ................................................................................................... 4 Introduction ..................................................................................................................................................... 6 What is an enzyme ............................................................................................................................................................... 7 Enzymes in feces .................................................................................................................................................................... 7 Trypsin ....................................................................................................................................................................................... 8 Substrate for trypsin ................................................................................................................................................. 9 Inhibitors ....................................................................................................................................................... 10 Aprotinin ................................................................................................................................................................................ 10 Soybean trypsin inhibitor ............................................................................................................................................... 11 α-­‐1-­‐antitrypsin .................................................................................................................................................................... 12 Leupeptin ............................................................................................................................................................................... 12 Antipain .................................................................................................................................................................................. 14 Immobilization ............................................................................................................................................. 15 Schiff bases ............................................................................................................................................................................ 15 NHS-­‐activated Sepharose ............................................................................................................................................... 17 Materials & methods .................................................................................................................................. 18 Aprotinin ................................................................................................................................................................................ 18 Soybean trypsin inhibitor ............................................................................................................................................... 18 α-­‐1-­‐antitrypsin .................................................................................................................................................................... 18 Leupeptin ............................................................................................................................................................................... 18 Immobilization of α-­‐1-­‐antitrypsin ............................................................................................................................. 19 Coupling between NHS-­‐activated Sepharose 4 Fast Flow and α-­‐1-­‐antitrypsin. .......................... 19 Test of immobilization .......................................................................................................................................... 19 Immobilization of leupeptin .......................................................................................................................................... 20 Coupling between NHS-­‐activated Sepharose 4 Fast Flow and Leupeptin ...................................... 20 Test of immobilization of leupeptin ................................................................................................................ 20 Results ............................................................................................................................................................. 20 With each inhibitor ........................................................................................................................................................... 21 Experiments of immobilized inhibitor ...................................................................................................................... 25 α-­‐1-­‐antitrypsin ......................................................................................................................................................... 25 Leupeptin .................................................................................................................................................................... 26 Discussion and conclusions ..................................................................................................................... 29 References ..................................................................................................................................................... 30 Acknowledgements .................................................................................................................................... 32 Appendix ........................................................................................................................................................ 33 5 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 INTRODUCTION It is well known that feces and urine are two factors that affect the skin in a negative way and together they can cause skin irritation, dermatitis. The biggest difference between an adult skin and a baby’s skin lies primarily in the anatomical differences in the surface of the skin. The development of the skin layers, distribution and size of certain glands, the nerve vessels development and also the hair growth differs between a baby and an adult. The outer layer of the skin protects the baby’s skin from mechanical stress and temperature changes that makes the skin less resilient. The baby’s skin is 20-­‐30% thinner and therefore less resistant than adult skin and the baby’s skin both absorbs and loses moisture more quickly than an adult skin. This results in that when urine and feces mix together in the diaper the external environment penetrates the infant’s skin more easily as the skin barrier is not equally developed as an adult. Also the friction between the skin and the diaper makes it easier for the skin to become irritated. The closed environment caused by the diaper results in that very little air reaches the skin under the diaper. This makes the baby’s skin warmer and moister, which leads to a higher pH. Excessive moisture in the skin makes it more easily to be penetrated by irritants and also vulnerable to swelling. When friction also applies to this area the moist skin can become more exposed to irritants and constant wiping can facilitate the penetration of irritating substances into the skin. (Williams, 2011). What is the biggest reason for skin irritations? Well, the feces contain enzymes that may irritate the skin of a baby such as proteases and lipases. In addition to this, feces can contain organisms that may cause skin infection, as for example different bacteria. The high urea content in urine may be degraded by urease and release ammonia. This results in a pH rise and the enzymes from the feces become more active due to a friendlier environment for them. This may lead to skin irritation and further on to diaper rash. The purpose of this master thesis is to study the enzyme trypsin and the possibilities of inhibiting trypsin both with an inhibitor alone but also with an immobilized inhibitor. The aim is to create a model that can be applicable to the other enzymes in the human feces. 6 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 WHAT IS AN ENZYME An enzyme is a protein that catalyzes a chemical reaction. By catalyze it means that the rate of the reaction will increase or decrease by the help of an enzyme. In an enzymatic reaction the molecules that goes in to the reaction is called substrate and the enzyme turns them into products. Almost all process in the human body needs enzymes so the reaction occurs at a speed that is sufficient for the body. Every enzyme binds the substrate to a specific site that is called the active site. That is the part of the enzyme that catalyzes the chemical reaction. The enzymes lowers the activation energy for reactions, which leads to that products can form faster, and that reactions equilibrium also can be reached faster. Most of the enzymatic reactions occur millions of times faster than without the enzymes. Just like any other catalytic molecule the enzymes are not consumed during the reaction not either do they change the equilibrium of the reaction, but what differs the enzymes from other catalysts is that they are much more specific. The activity of an enzyme can be affected of other molecules, for example inhibitors. Fig. 1 – The variation in the energy for the Many pharmaceuticals are enzyme stabilisation of the transition state by an enzyme is inhibitors, as for example ibuprofen (COX-­‐inhibitor). shown as a function of reaction coordinate The activity can also be affected by temperature, chemical environment as for example pH and the concentration of the substrate. (Hermanson, 2008) ENZYMES IN FECES Enzymes may have difference sizes, ranging from ≈60 amino acids to more than thousand amino acids. As proteins, enzymes are long, linear chains of amino acids that has folded itself to a 3D-­‐structure. There are a number of different enzymes in the human feces that come from the gastro intestinal tract and they are called the digestive enzymes. The most common enzymes in feces are lipase and proteases. Lipase is an enzyme that breaks down fat in our body so it can be absorbed in the intestines. Lipase is primarily produced in pancreas and is released in duodenum, but also in our stomach (gastric lipase) and mouth (lingual lipase, that is released from the von Ebner glands on the upper side of the tongue). Lipase cleaves lipids, as for example triglycerides. There are also hepatic lipase, lysosomal lipase and endothelial lipase. These enzymes cleaves the body’s own lipids. More generally, lipase hydrolyses the ester linkage coupling of fatty acids. (http://www.umm.edu/altmed/articles/lipase-­‐000311.htm, 20120412) Proteases are a group of enzymes that catalyze degradation of linkages between amino acids in proteins. The reaction that takes place is a hydrolysis. At this moment proteases are divided into six groups and they fall into different number of classes due to the way they cleave the molecule: 1.
2.
3.
4.
5.
6.
Serine proteases Metallo proteases Cysteine proteases Aspartic proteases Treonin proteases Glutamic acid proteases 7 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 The proteases fall into different number of classes due to that several mechanisms can be used for fragmenting the polypeptide chain. Some proteases cleave only the peptide bond adjacent to particular amino acid residues and thus fragment a polypeptide chain in a predictable and reproducible way. (Laskowski (1950)). Digestive proteases are mostly excreted in zymogen form i. e. as inactive proenzymes, in order to prevent the proteases degrading the pancreas or spread to abdomen and other organs. Trypsin, a serine protesase, is one of the most important and also most studied digestive proteases. In pancreas, trypsin is produced as an inactive precursor but well in duodenum it is activated by enteropeptidase and also by already activated trypsin molecules and activates itself the other proenzymes prochymotrypsinogen, proelastase and procarboxypeptidase to the active forms chymotrypsin, elastase and carboxypeptidase TRYPSIN Trypsin can be found in the gastro-­‐intestinal tract where it catalyzes the hydrolysis of proteins. The peptides are further hydrolyzed into single amino acids by other proteases before the amino acids can enter the blood stream. The first step of the hydrolysis, where trypsin acts, is very important because proteins are generally to big to be absorbed through the intestinal wall. Trypsin belongs to the pancreatic secretions and it passes into the small intestine through the pancreatic duct. Arrival of amino acids in the upper part of the intestine (duodenum) causes release into the blood of the hormone cholecystokinin, which stimulates secretion of several pancreatic enzymes with activity optima at pH 7-­‐8. The zymogen form (inactive enzyme precursor) of trypsin are synthesized and secreted by the exocrine cells of pancreas. Trypsinogen is then converted to its active form, trypsin, by enteropeptidase. The mechanism of the trypsin reaction can be compared to similar serine protease actions. The enzyme contains a catalytic triad consisting of histidine-­‐57, aspartate-­‐102 and serine-­‐195. The three residues form a charge relay, which makes the active site nucleophilic. Trypsin cleaves at the carbonyl side of the positively charged lysine and arginine (if the next residue is not proline). As mentioned, trypsin catalyzes the hydrolysis of only those peptide bonds where the carbonyl group is contributed by either lysine or arginine residues, regardless of the length or amino acids sequence of the chain. Trypsin is commonly used as a biochemical tool since, it’s easy to predict the number of smaller peptides produced by trypsin due to the total number of arginine or lysine. (Voet, 2008) Fig. 2 – The amino acid Arginine Fig. 3 – The amino acid Lysine The optimal operating parameters for trypsin are a temperature of 37 °C and a pH about 7,5-­‐8,5. Trypsin has to be stored at -­‐20 °C to -­‐80 °C to prevent autolysis. Trypsin can also be stored at lower pH, around 3, to prevent autolysis. When the pH is adjusted back to optimal parameter the activity of trypsin returns. (Campbell, 2008) 8 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 Trypsin can be inhibited by trypsin inhibitors, which also are known as serine protease inhibitor (serpins). They are the largest and most diverse family of protease inhibitors. Serpins control the activation and catabolism of proteins by the inhibition of serine protease in vivo. (Lehninger, 2005) SUBSTRATE FOR TRYPSIN During each reaction BAPA has been used as a substrate for trypsin. BAPA stands for N-­‐α-­‐Benzoyl-­‐L-­‐arginine-­‐4-­‐nitroanilide, which is a chromogenic substrate for trypsin and also for other proteolytic enzymes. CAS Number: 21653-­‐40-­‐7 Molecular Weight: 434.88 Da The reaction takes place at the carboxyl side of arginine and the detection is at 410 nm where the formed 4-­‐nitroaniline product absorbs light. (Hiroyasu Nakata, Shin-­‐Ichi Ishii (1970)). Fig. 4 – 4-­‐nitroaniline is shown to the left and BAPA to the right. 9 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 INHIBITORS The activity of an enzyme can be prevented by the use of inhibitors, which prevents the enzyme to work in a normal manner. The inhibitors can be divided into different groups due to the way the inhibition works on the enzyme. The variety of types of inhibitors includes nonspecific, irreversible and reversible that in turn can be divided in different groups, such as competitive and noncompetitive. As mentioned earlier are drugs and poisons example of inhibitors. Nonspecific inhibitors affect all the enzymes in the same way that includes any physical or chemical changes that denatures the enzyme and are therefore irreversible. For example, the activity of the enzyme can be controlled by pH. As the pH increases or decreases, the protonation state of the various acid and amine groups on side chains is altered with resulting changes in the overall shape structure of the enzyme. In this case it can be hard to control the pH in as for example diapers when body fluids has a certain pH when it leaves the body. Specific inhibitors act upon a single enzyme. Most of the poisons work by specific inhibition of enzymes and there are also many drugs that exert their effect on a single enzyme. A competitive inhibition is any molecule that is similar in the chemical structure and molecular geometry of the substrate. As the word “competitive” indicates are the inhibitors competing for the same active site as the substrate molecule. When the inhibitor and the enzyme binds, the enzyme is prevented to function as normal and the inhibition also prevents the substrate from reacting with the enzyme. The competitive reaction can be reversible if there is enough amount of substrate so the inhibitor can be displaced. This results in that the amount of enzyme inhibition depends on the inhibitor concentration, substrate concentration and the affinities of the inhibitor and substrate for the active site. A non-­‐competitive inhibitor is a substance that interacts with an enzyme but usually not at the active site. The non-­‐competitive inhibitor reacts on another part of the enzyme then the active site and the net effect of the inhibition is to impair the catalytic mechanism itself, possibly by a small change in the shape of the enzyme. Non-­‐competitive inhibition is not affected by the substrate concentration as the reversible competitive inhibitors are. Irreversible inhibitor reacts with the enzyme and forms a covalent bond to permanently inhibit the enzyme. The inhibitor may act near or at the active site of the enzyme but it can also react on other part of the enzyme than at the active site. Excess substrate will not displace the inhibitor. There are many examples of natural trypsin inhibitors, such as bovine pancreas trypsin inhibitor (aprotinin), ovomucoid, soybean trypsin inhibitors and lima bean trypsin inhibitor. They all act as a competitive substrate analog and an inactive complex are formed. Trypsin inhibitors can offer unique results depending on the mission. Below there are a few inhibitors described that were used in this master thesis. There are a numerous amount of other inhibitors available for trypsin but the ones below are the ones that fell into interest due to the possibilities of immobilization. (Fersht, 1984) 10 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 APROTININ Molecular weight: ~6 511 Da CAS-­‐number: 9087-­‐70-­‐1 While aprotinin and bovine pancreatic trypsin inhibitor (BPTI) are the same protein sequence, the term aprotinin is typically used when describing the protein derived from bovine lung. Aprotinin is a protein consisting of 58 amino acids arranged in single peptide chain with three disulfide bonds. A disulfide bond is a chemical bond between two cysteine’s thiol groups. The two cysteines lose their hydrogen atom and together forms a S-­‐S-­‐bond. These bonds are important for the protein three-­‐dimensional structure. Aprotinin is a competitive serine protease inhibitor that inhibits chymotrypsin, kallikrein, plasmin and trypsin. Aprotinin forms a reversible but strong complex with the enzyme Fig 5 -­‐ Aprotinin and blocks the enzymes active site. The binding is reversible with most of the protease-­‐aprotinin complexes with a dissociation at pH <3 or >10. One trypsin inhibitor unit will decrease the activity of two trypsin units by 50 %. The effective concentration of Aprotinin is equimolar with protease. (Cited http://www.sigmaaldrich.com/catalog/product/sigma/a1153?lang=en&region=SE, 120312) Fig. 6 – Aprotinin 11 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 SOYBEAN TRYPSIN INHIBITOR Molecular weight: ~ 20,1 kDa CAS-­‐number: 9035-­‐81-­‐8 Soybean trypsin inhibitors (SBTI) are found in the seeds of soybean. Several trypsin inhibitors have been identified and isolated from soybean with a molecular weight ranging from 8,000 to 24,000 and the biological and physicochemical properties have been studied. (Kuntiz (1946); Scung-­‐Ho (1985)) Kunitz crystallized a soybean trypsin inhibitor with a molecular weight at 21,500 with optimal pH at 7.0 in a complex with trypsin from porcine in 1945. (Kunitz (1946); Scung-­‐Ho (1985)) The Kunitz soybean inhibitor consists of a single polypeptide chain crosslinked by two disulfide bridges. SBTI inhibits trypsin mole for mole and also to a lesser extent chymotrypsin. One mg will approximately inhibit 1.0-­‐3.0 mg of trypsin. SBTI is extremely stable; in a 1 % sterile filtered solution the inhibitor maintained the activity for over three years Fig 7 – Soybean trypsin inhibitor stored at a temperature at 4-­‐8°C. SBTI forms a 1:1 stoichiometric complex with trypsin and upon the complex formation, trypsin may cleave a single arginine-­‐isoleucine bond in the inhibitor. When the complex dissociate it may lead to formation of the native inhibitor. At pH 8.0, which is the optimal pH, the association constant is ≥ 10×108. It acts as an inhibitor only when it is in its native state, denaturation of the soy protein by heat, acid or alkali is accompanied by a loss in its inhibiting power. (Scung-­‐Ho, (1985); (Cited: http://www.sigmaaldrich.com/catalog/product/sigma/t9003?lang=en&region=SE, 120423) There are three different types of Kunitz-­‐type trypsin inhibitors, Tia, Tib and Tic. All the three inhibitors consist of 181 amino acids residues and what differs them are the sequences of the amino acids. Tia has the sequence of Pro(60)-­‐Ser(61) and Asp(154)-­‐Asp(155)-­‐Gly(156)-­‐His(157) where Tib and Tic had the sequence Ser(60)-­‐Pro(61) and His(154)-­‐Asp-­‐Asp-­‐Gly(157) respectively. (Kunitz (1946)) The mechanism of the reaction is the action of SBTI to form an irreversible stoichiometric compound with trypsin. The combination is instantaneous and independent, within a wide range, of the pH of the solution. SBTI inhibits the proteolytic action of an equal weight of crystalline trypsin by combining with trypsin to form a stable compound. (Scung-­‐Ho, (1985)) 12 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 α-­‐1-­‐ANTITRYPSIN Molecular weight: 44,3 kDa CAS-­‐number: 9041-­‐92-­‐3 α-­‐1-­‐antitrypsin is the most abundant serine protease inhibitor in the human plasma and it is mainly produced in the liver parenchymal cells. Normally an amount of 34 mg/kg in 24 hours is produced, which will give a normal plasma concentration between 0,9-­‐1,75 g/l with a half-­‐life on 3-­‐5 days. 5 to 10 mg of α-­‐
1-­‐antitrypsin will inhibit 1.0 mg of trypsin. (Cited; http://www.sigmaaldrich.com/catalog/product/sigma/a6150?lang=en&region=SE, 20120510) α-­‐1-­‐antitrypsin protects tissues from degrading enzymes, protease, and the most important assignment α-­‐
1-­‐antitrypsin has is to protect lung tissues. When the lung are infected of or in the presence of foreign substance (as for example smoking), white blood cells are released in the lungs to “clean” the lungs from undesirable substances. The release of white blood cells will lead to the amount of a certain enzyme, neutrophil elastase, will increase and the enzyme kills bacteria’s and cleans up dead lung tissue. When the foreign substances are gone α-­‐1-­‐antitrypsin are released to decrease the effect of the destructive enzyme, otherwise healthy lung tissue will be attacked. Some people suffer from α-­‐1-­‐antitrypsin deficiency, which can lead to that healthy lung tissue breaks down which in turn leads to emphysema. The lungs will lose their elasticity and it will be harder and harder to breath. LEUPEPTIN Molecular weight: 463,01 Da CAS number: 24125-­‐16-­‐4 Leupeptin, also known as N-­‐acetyl-­‐L-­‐leucyl-­‐L-­‐leucyl-­‐L-­‐
argininal, is a small peptide that is composed of three linked amino acids and it has some unusual attributes. The amino group of the compound has an acetyl group on it; also an aldehyde group replaces the terminal carboxy group. Fig. 8 -­‐ Leupeptin Leupeptin is a protease inhibitor that inhibits the proteases with endopeptidase activity (plasmin, trypsin, papain, calpain, and cathepsin B) and the soil bacteria Streptomyces produces it naturally. It has an effective concentration of 10-­‐100 µM (of course depending on the concentration of the enzyme) and it inhibits the serine protease trypsin with a Ki = 3,5 nM. Leupeptin is soluble in water (stable for about one month at -­‐20 °C), ethanol, acetic acid and DMF. (Cited: http://www.sigmaaldrich.com/catalog/produ
ct/sigma/l9783?lang=en&region=SE, 20120518) One positive effect with Leupeptin is that it has very low toxicity towards humans. It has been shown in research on animal models that leupeptin protects hair cells in the ear from being killed by loud noises. It’s also being widely used during the first part of protein purifications to keep the protease in the tissue from breaking down the protein of interest. The wide inhibition potential leupeptin has makes it also very useful in Figure 9. Crystal structure of Leupeptin (silver) in the Trypsin (green) binding pocket. Hydrogen b
onds a
re shown as yellow dotted lines. 13 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University biochemical research and in large-­‐scale production. It is common that leupeptin is used in so called protease cocktail due to its potency, low toxicity and as mentioned, activity on a broad range of proteases. (Drapalova (2008); Stys (2008); Lukesova (2008); Kopecky (2008)) 120612 ANTIPAIN Molecular weight: 604.7 Da CAS number: 37691-­‐11-­‐5 Ki = 2×10!! 𝑀 with serine protease as substrate. Synonym: [(S)-­‐1-­‐Carboxy-­‐2-­‐
phenylethyl]carbamoyl-­‐L-­‐arginyl-­‐L-­‐valyl-­‐
argininal
Fig. 10 -­‐ Antipain Antipain is an oligopeptide that has been isolated from actinomycetes and it is common to use antipain as protease inhibitor, specifically for trypsin and papain. Antipain is a serine and cysteine reversible protease inhibitor and besides trypsin and papain it also inhibits a small amount of plasmin to some extent. Antipain is mostly used to evaluate the role of proteases in cell transformation and to help identify new proteases IC50 for trypsin is approximately 0.25 µg/mL and a solution of 10 mM in water or buffer is usually stable for one week at 4 °C and one month at -­‐20 °C. The activity of antipain can be compared to the activity of leupeptin due to that antipains action resembles leupeptins, but antipain inhibits plasmin less and cathepsin A more. (Cited: http://www.sigmaaldrich.com/catalog/product/sigma/10791?lang=en&region=SE, 20120520) There are a number of other inhibitors that has been proven to have a good inhibiting effect towards trypsin. For example ovomucoids, fetal bovine serum (FBS) and trypsin inhibitors from lima beans also show a good inhibiting effect. The focus has been on the five inhibitors above but the choice doesn’t stop there. 14 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 IMMOBILIZATION Immobilization of molecules to a carrier phase is frequently used for e.g. affinity chromatography and enzyme reactors. There are many activated gel matrices available at market and what differ between them are reactive groups, the extent of activation, introduced spacer length and type, particle size and also porosity. Most of the gel matrices are based on beaded agarose. In one article (Sommeren et. al. (1993)) three different activated agaroses were compared in matters of coupling performance and ligand leakage. One of these three was N-­‐hydroxysuccinimide agarose and the result showed a very fast and complete binding to the ligand. SCHIFF BASES Aldehydes and ketones can react with primary and secondary amines to form Schiff bases. A Schiff base is a relatively labile bond that is readily reversed by hydrolysis in aqueous solution. The formation of Schiff bases is enhanced at alkaline pH values, but not completely stable unless it is reduced to a secondary or tertiary amine linkages. There are a number of reducing agents that can be used to specifically convert the Schiff base into an alkylamine linkage. Once the linkage is reduced, the bond is highly stable. The use of reductive amination to conjugate an aldehyde containing molecule to an amine containing molecule results in a zero-­‐length crosslink where no additional spacer atoms are introduced between the molecules. The reduction reaction is best facilitated by the use of reduction agent such as sodium cyanoborohydride, because the specificity of this reagent is for the Schiff base structure and it will not affect the original aldehyde groups to the same extent. Sodium borohydride can also be used in this reaction but its strong reducing power will and can rapidly convert aldehydes that has not reacted into non-­‐reactive hydroxyls, effectively eliminating them from further participation into the conjugation process. Borohydride may also affect the activity of some sensitive proteins. Cyanoborohydride is gentler, successfully preserving the activity of some labile monoclonal antibodies. Cyanoborohydride has been shown to be five times milder than borohydride in reductive amination process with antibodies (Peng et al., 1987) There have been an investigation of other reductive agents that includes various amine boranes and ascorbic acid. (Cabacungan et al., 1982; Hornsey et al., 1986) The end result of the reaction is a compound where the C=O double bond is replaced by a C=N double bond (see figure 11 below). This type of compound is known as an imine or Schiff base. Fig. 11 – Overall reaction Mechanistically, the formation of the Schiff base involves two steps. At the first step the amine nitrogen acts as a nucleophile and attacks the carbonyl carbon (figure 12). This can be compared to hemiacetal and hemiketal formation.
15 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 Fig. 12 – Nitrogen acts as a nucleophile Based on the mechanism of acetal and ketal formation the next step would be the attack be a second amine to form a compound where a carbon binds to two amines, but this is not the case here. The next thing that happens is that the nitrogen is deprotonated and the electrons from the N-­‐H bond “push” the oxygen off the carbon, leaving a C=N double bond, an imine, and a water molecule. Fig. 13 – Nitrogen deprotonates, a C=N bond is left. The conversion of an imine back to an aldehyde or ketone is a hydrolysis and the reverse formation of the imine. Fig. 14 – The reverse formation of the imine Immobilization by reductive amination of amine-­‐containing biological molecules onto aldehyde-­‐
containing solid supports has been used for quite some time and the reaction proceeds with excellent efficiency (Sanderson and Wilson, 1971; Domen et al., 1990). The optimum pH for the reaction is alkaline although a good yield can be given by the pH range from 7 to 10. At the higher end of pH is the formation of the Schiff base reaction more efficient and the yield of the immobilization can be increased. (Hornsey et al., 1986). 16 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 The introduction of aldehyde functional groups into proteins and other molecules can be accomplished by a number of methods. Glycoproteins may be oxidized at their carbohydrate residues using sodium periodate or specific sugar oxidase. Amine groups may be modified to produce a formyl group by reacting with NHS-­‐aldehydes or p-­‐nitrophenyl diazopyruvate. (Hermanson, 2008) NHS-­‐ACTIVATED SEPHAROSE Pre-­‐activated agarose matrix increases the choice of coupling chemistries. NHS-­‐
(N-­‐hydroxysuccinimide)-­‐esters of introduced carboxylate groups allow the formation of chemically stable amide bonds with ligands containing primary amino groups. NHS-­‐activated Sepharose provides a spacer arm and is therefore particularly suitable for immobilizing small protein and peptide ligands. The NHS-­‐
activated Sepharose is a bead-­‐formed, highly crosslinked pre-­‐activated matrix prepared by coupling Sepharose 4 fast flow with 6-­‐aminohexanoic acid via a spacer arm. The terminal carboxyl group is activated by esterification with N-­‐
hydroxysuccinimide. Ligands containing primary amino groups couple directly to Fig. 15 -­‐ NHS this active ester to form a chemically very stable amide linkage. A pH between 6 and 9 are normally used due to higher pH can lead to premature hydrolysis of the NHS-­‐ester groups. (Hermanson, 2008, GE Life Science 20120611). Medium characteristics Mean particle size Particle size range Bead structure Linear flow velocity Ligand density pH stability, long term1 pH stability, short term1 90 µm 45 to 165 µm Highly crosslinked 4 % agarose, spherical 150 cm/h at 100 kPa 16 to 23µmol NHS/ml drained medium 3 to 13 2 to 13 Table 1 – Characteristics of the medium 1Depends on the ligand. Tested with lysine as ligand with single-­‐point attachment. 17 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 MATERIALS & METHODS Enzyme and inhibitors The enzyme trypsin and the four inhibitors aprotinin, soybean inhibitor, leupeptin and alfa-­‐1-­‐antitrypsin were obtained from Sigma Aldrich. NHS-­‐activated Sepharose was obtained from GE Healthcare and ethylenediamine (99%) was obtained from Kebo lab (EC no 203-­‐468-­‐6). All the inhibitors were dissolved in to a solution consisting of following: -­‐
-­‐
-­‐
-­‐
50 µL BAPA (substrate for trypsin) 100 µL NH4HCO3 x µL inhibitor distillated water To each inhibitor solution a certain volume of trypsin solution was added. Different amount of inhibitor was added to each solution, depending on the molar ratio. Following that the resulting activity of trypsin was measured. All the reactions were measured with UV-­‐visible spectrophotometer, Shimadzu, UV-­‐1601 at 410 nm for 180 and 700 seconds. APROTININ A1153 SIGMA, Aprotinin from bovine lung, lyophilized powder A aprotinin solution of 66,6 µM and a trypsin solution of 2,69 µM were used. In the first experiment the molar ration 2:1 (aprotinin:trypsin) and in the second experiment a ratio of 3:2 (aprotinin:trypsin) were used. SOYBEAN TRYPSIN INHIBITOR T9003 SIGMA, Trypsin inhibitor from Glycine max (soybean), Type I-­‐S, lyophilized powder. A soybean trypsin inhibitor solution of 35,1 µM and a trypsin solution of 2,25 µM were used. Different experiments with different mole ratios were tested. The mole ratio that were tested were 1-­‐6:1 (soybean trypsin inhibitor:trypsin). α-­‐1-­‐ANTITRYPSIN A6150 SIGMA α1-­‐Antitrypsin from human plasma, salt-­‐free, lyophilized powder A α-­‐1-­‐antitrypsin inhibitor solution of 35,5 µM and a trypsin solution of 2,25 µM were used. Different molar ratio (0,79-­‐8,94) between α-­‐1-­‐antitrypsin:trypsin were tested. LEUPEPTIN L9783 SIGMA, Leupeptin hydrochloride, microbial, ≥90% (HPLC) A leupeptin inhibitor solution of 100 µM and a trypsin solution of 54,53 µM were used. The molar ratio 1:1, 2:1 and 3:1 (leupeptin:trypsin) were tested. 18 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 IMMOBILIZATION OF α-­‐1-­‐ANTITRYPSIN COUPLING BETWEEN NHS-­‐ACTIVATED SEPHAROSE 4 FAST FLOW AND α-­‐1-­‐ANTITRYPSIN. NHS-­‐activated Sepharose is supplied as a suspension in 100% isopropanol with ligand density 16-­‐23 µmol NHS/ml drained medium. In order to retain maximum binding capacity of the pre-­‐activated medium prior to the coupling step, cold (0°C to 4°C) solutions was used. Also the time interval between all the steps was minimized, because of that, all of the solutions were prepared before the coupling started. (GE Life Science) 1.
2.
3.
Coupling solution was prepared, a standard buffer of 0.2M NaHCO3, 0.5M NaCl at pH 8.3 was used. Normally pH between 6 and 9 are used, higher pH can lead to premature hydrolysis of the NHS-­‐
ester groups. The NHS-­‐activated Sepharose 4 Fast Flow was washed with 10 medium volumes of cold 1 mM HCl immediately before use. α-­‐1-­‐antitrypsin (A1AT) was dissolved the appropriate buffer (0.2M NaHCO3, 0.5M NaCl, pH 8.3). A ligand concentration of 20 µM was made. The washed medium and coupling solution was mixed and allowed to react selected times. A volume ratio of 0.5:1 with coupling solution and the medium was used. Time of mixing (min) Amount A1AT (ml) Amount NHS (ml) 15 0.25 0.5 30 0.25 0.5 60 0.25 0.5 120 0.25 0.5 Table 2 – Amount and time of mixing 4.
After the coupling was completed the medium/mixture was kept in 0.1M Tris-­‐HCl, pH 8.5 for about one hour in order to block any non-­‐reacted groups. The medium was after that washed with the same buffer three times and to prevent microbial contamination, the four mixtures were left in 20% ethanol in 4°C. TEST OF IMMOBILIZATION For each immobilized α-­‐1-­‐antitrypsin according to the mixing times, except for trypsin, immobilized α-­‐1-­‐
antitrypsin was added and then 3 µL of 50 µM trypsin solution was added and during the experiment 15 µL more 50 µM trypsin solution was added. So to explain the experiment, 3 µL of 50 µM trypsin solution was added at time 0 sec. At about 300 sec additional 15 µL 50 µM trypsin solution was added. A total of 18 µL 50 µM trypsin solution was added. IMMOBILIZATION OF LEUPEPTIN COUPLING BETWEEN NHS-­‐ACTIVATED SEPHAROSE 4 FAST FLOW AND LEUPEPTIN NHS-­‐activated Sepharose is supplied as a suspension in 100% isopropanol. In order to retain maximum binding capacity of the pre-­‐activated medium prior to the coupling step, cold (0°C to 4°C) solutions should be used. Also the time interval for all the steps must be minimized, because of that, all of the solutions were prepared before the coupling started. (GE Life Science) 19 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 1. Coupling solution was prepared, a standard buffer of 0.2M NaHCO3, 0.5M NaCl at pH 8.3 was used. 2. The NHS-­‐activated Sepharose 4 Fast Flow was washed 10 medium volumes of cold 1 mM HCl immediate before use. A 1:1 solution of the 1M ethylenediamine is added and left for 60 minutes. Different volumes of 1M ethylenediamine were tested: 10 µL 20 µL 30 µL 40 µL Before Leupeptin was added, 10 µL of concentrated NaBH3CN (99%) was added to each test tube. Leupeptin was dissolved in the appropriate buffer (0.2M NaHCO3, 0.5M NaCl, pH 8.3). The washed medium and coupling solution was mixed and left for 60 minutes. A volume ratio of 1:0,5 with coupling solution and the medium was used. −
−
−
−
3.
4.
5.
Time of mixing (min) 60 60 60 60 Amount Leupeptin (ml) 1 1 1 1 Amount NHS (ml) 0.5 0.5 0.5 0.5 Amount NaBH3CN (µl) 10 10 10 10 Amount 1M Ethylendiamine (µl) 10 20 30 40 6.
TEST After that the coupling time, the medium was kept in 0.1M Tris-­‐HCl, pH 8.5 for about one hour to block any non-­‐reacted groups. The medium was after that washed with the same buffer three times and to prevent microbial contamination, the four mixtures were left in 20% ethanol in 4°C. Table 3 – Amount and time of mixing OF I MMOBILIZATION O F L EUPEPTIN Trypsin was incubated in the presence of different molar ratios of immobilized leupeptin and with four different amounts of ethylenediamine. The first experiment was with the presence of immobilized leupeptin 1:1 (leupeptin:trypsin) and the second experiment was 2:1 (leupeptin:trypsin) for each volume of ethylenediamine. Calculations for each experiment can be found in the appendix. A solution of substrate, buffer and immobilized leupeptin was mixed and just before the measurement started trypsin was added. 20 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 RESULTS The effect of each inhibitor on trypsin is shown below in diagrams. The blue curve always represents trypsin that’s not inhibited. Different mole ratios between the inhibitor and trypsin are shown in each diagram. The absorbance was measured at λ = 410 nm. WITH EACH INHIBITOR Trypsin inhibited by Aprotinin 2,5 Absorbans, 410 nm 2 y = 0,0095x + 0,2571 R² = 0,99164 1,5 y = 0,0056x + 0,0963 R² = 0,99761 1 Trypsin 1:1 1:2 0,5 y = 0,002x + 0,0315 R² = 0,99867 0 0 20 40 60 80 100 Time (sec) 120 140 160 180 200 Aprotinin was tested at different molar ratios. The blue line represents the degradation of BAPA without the inhibitor. The red line represents the experiment at 1:1 molar ratio inhibitor to trypsin. The green line represents the experiment at 2:1 molar ratio inhibitor to trypsin. Inhibition ratio 1:1 → 41 % of trypsin is inhibited Inhibition ratio 1:2 → 79 % of trypsin is inhibited 21 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 Trypsin inhibited by STI 0,9 y = 0,0043x + 0,0692 R² = 0,99546 0,8 Absorbans, 410 nm 0,7 0,6 y = 0,0025x + 0,0291 R² = 0,99874 0,5 Trypsin 1 till 1 2 till 1 0,4 3 till 1 0,3 4 till 1 5 till 1 0,2 y = 0,0014x + 0,0256 R² = 0,99937 0,1 6 till 1 0 0 20 40 60 80 100 120 140 160 180 200 Time (sec) Soybean trypsin inhibitor (STI) was tested at different molar ratios. The blue line represents the degradation of BAPA in absence of the inhibitor. The other lines show the degradation of the present of STI at different molar ratios. (All of the equations are not shown in the diagram due to crowding, see appendix for all the linear fits). Inhibition ratio 1:2 → 42 % of trypsin is inhibited Inhibition ratio 1:6 → 67 % of trypsin is inhibited 22 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 Trypsin vs α-­‐1-­‐antitrypsin 0,6 Absorbans, 410 nm 0,5 y = 0,0025x + 0,0271 R² = 0,99861 0,4 0,3 0,2 0,1 0 0 20 40 60 80 100 120 y = 0,0023x + 0,0304 R² = 0,99867 y = 0,002x + 0,0271 R² = 0,99792 y = 0,0018x + 0,0278 R² = 0,99649 Trypsin y = 0,0012x + 0,0384 1:0,8 R² = 0,98811 1:1,6 1:3,2 1:6,3 1:7,9 1:8,9 140 160 180 200 Time (sec) α-­‐1-­‐antitrypsin was tested with different molar ratios. The blue line represents the degradation of BAPA without the inhibitor. The red line represents the experiment at 0,8:1 molar ratio inhibitor to trypsin. The green line represents the experiment at 1,6:1 molar ratio inhibitor to trypsin, and the purple 3,2:1 molar ratio inhibitor to trypsin. The light blue, orange and grey all shows the same inhibition even though there are different molar ratio for the three. Inhibition ratio 1:0,8 → 8 % of trypsin is inhibited Inhibition ratio 1:1,6 → 20 % of trypsin is inhibited Inhibition ratio 1:3,2 → 28 % of trypsin is inhibited Inhibition ratio 1:6,3 → 52 % of trypsin is inhibited Inhibition ratio 1:7,9 → 52 % of trypsin is inhibited Inhibition ratio 1:8,9 → 52 % of trypsin is inhibited 23 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 Trypsin vs Leupeptin 1,2 y = 0,0013x + 0,064 R² = 0,9932 Absorbans, 410 nm 1 0,8 Trypsin 0,6 Leupeptin 1:1 0,4 y = 0,0005x + 0,039 R² = 0,99469 0,2 0 0 100 200 300 400 500 Leupeptin 2:1 y = 0,0002x + 0,0336 R² = 0,97158 600 700 800 Time (sec) The blue line represents the degradation of BAPA in absence of the inhibitor. The red line represents the experiment at 1:1 molar ratio inhibitor to trypsin. The green line represents the experiment at 2:1 molar ratio inhibitor to trypsin. Inhibition ratio 1:1 → 60 % of trypsin is inhibited Inhibition ratio 1:2 → 98 % of trypsin is inhibited 24 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 EXPERIMENTS OF IMMOBILIZED INHIBITOR α-­‐1-­‐ANTITRYPSIN Trypsin vs immobilized α-­‐1-­‐antitrypsin 1,2 Absorbance, 410 nm 1 0,8 15 min 30 min 0,6 60 min 0,4 120 min No inhibitor 0,2 0 0 100 200 300 400 500 600 700 Time (sec) Yellow curve, No inhibitor: 50 µL 20 mM BAPA + 100 µL 250 mM NH4HCO3 + dest H2O + 18 µL 50 µM Trypsin. Blue curve (15 min): 50 µL 20 mM BAPA + 100 µL 250 mM NH4HCO3 + dest H2O + 100 µL A1AT & NHS + 3 µL 50 µM Trypsin at t=0s + 15 µL 50 µM Trypsin at t=300s Black curve (30 min): 50 µL 20 mM BAPA + 100 µL 250 mM NH4HCO3 + dest H2O + 100 µL A1AT & NHS + 3 µL 50 µM Trypsin at t=0s + 15 µL 50 µM Trypsin at t=300s Green curve (60 min): 50 µL 20 mM BAPA + 100 µL 250 mM NH4HCO3 + dest H2O + 100 µL A1AT & NHS + 3 µL 50 µM Trypsin at t=0s + 15 µL 50 µM Trypsin at t=300s Purple curve (120 min): 50 µL 20 mM BAPA + 100 µL 250 mM NH4HCO3 + dest H2O + 100 µL A1AT & NHS + 3 µL 50 µM Trypsin at t=0s + 15 µL 50 µM Trypsin at t=300s 15 min, 30 min, 60 min and 120 min represents the coupling times for the inhibitor. For each curve, except for trypsin alone, I first added the immobilized α-­‐1-­‐antitrypsin and then added a small amount of trypsin and during the experiment more trypsin was added. So to explain the diagram, 3 µL of 50 µM trypsin was added at time 0 s. At about 300 s additional 15 µL 50 µM trypsin was added. A total of 18 µL 50 µM trypsin was added. The diagram shows that the immobilization works and that α-­‐1-­‐antitrypsin coupled to NHS inhibits trypsin. The diagram also show that 30 min of immobilization is enough for the bond between the inhibitor and the agarose to be formed. It differs some between the curves when additional trypsin was added at t=300s, this can be due to the different immobilization times and that different amount of α-­‐1-­‐
antitrypsin coupled to NHS. The “jump” in all four curves in the beginning of the experiment can be due to 25 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 that trypsin “found” the substrate BAPA pretty quick compared to the time “finding” the immobilized α-­‐1-­‐
antitrypsin. LEUPEPTIN Trypsin vs immobilized leupeptin Absorbance, 410 nm 2 1,8 Trypsin 50 µl 1,6 10 µl EA 31,25 1,4 10 µl EA 62,5 1,2 20 µl EA 31,25 1 20 µl EA 62,5 0,8 30 µl EA 31,25 0,6 30 µl EA 62,5 0,4 40 µl EA 31,25 0,2 40 µl EA 62,5 0 0 100 200 300 400 500 600 700 Trypsin 25 µl Time (sec) In this first diagram all the runs are shown. The blue line represents trypsin alone. For each volume of ethylenediamine two runs has been made, the first with a volume of 31,25 µl immobilized leupeptin (1:1) and the second with a volume of 62,5 µl (1:2). Also, one run with 25 µl trypsin solution without inhibitor is shown in the diagram. 26 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 Trypsin vs immobilized leupeptin (1:1) 2 1,8 Absorbans, 410 nm 1,6 1,4 Trypsin 50 µl 1,2 10 µl EA 31,25 1 20 µl EA 31,25 0,8 30 µl EA 31,25 0,6 40 µl EA 31,25 0,4 Trypsin 25 µl 0,2 0 0 100 200 300 400 500 600 700 Time (sec) For each volume of ethylenediamine the ratio one to one are shown in the diagram above. The most effective immobilizations are with the volumes 30 µl and 40 µl ethylenediamine. Trypsin vs immobilized leupeptin (1:2) 2 Absorbance, 410 nm 1,8 1,6 1,4 Trypsin 50 µl 1,2 10 µl EA 62,5 1 20 µl EA 62,5 0,8 30 µl EA 62,5 0,6 40 µl EA 62,5 0,4 Trypsin 25 µl 0,2 0 0 100 200 300 400 500 600 700 Time (sec) For each volume of ethylenediamine the ratio one to two are shown in the diagram above. The most effective immobilization is with the volume 40 µl ethylenediamine. 27 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 Trypsin vs immobilized leupeptin (40 mikroliter EA) 2 Absorbance, 410 nm 1,8 1,6 1,4 1,2 Trypsin 50 µl 1 0,8 40 µl EA 31,25 0,6 40 µl EA 62,5 0,4 0,2 0 0 100 200 300 400 500 600 700 Time (sec) With 40 µl of ethylenediamine the diagram shows that the immobilization work. With the ratio 1:2 (trypsin:immobilized leupeptin) a larger inhibition can be seen. The blue line represents the degradation of BAPA without the inhibitor. The red line represents a solution with BAPA, trypsin and 31,25 µl of the immobilized leupeptin. The immobilization had been left for incubation for 60 minutes with 40 µl ethylenediamine. This amount immobilized leupeptin gave a molar ratio of 1:1. The green line represents a solution with BAPA, trypsin and 62,5 µl of the immobilized leupeptin. The immobilization had been left for incubation for 60 minutes with 40 µl ethylenediamine. This amount immobilized leupeptin gave a molar ratio of 1:2 (trypsin:leupeptin). 28 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 DISCUSSION AND CONCLUSIONS The evaluation of the different inhibitors shows that they all work efficiently, but with quantitative differences. A lot more of the soybean trypsin inhibitor (STI) has to be used to be able to come up in the same level as aprotinin. A ratio of 1:7 (trypsin:STI) is at least needed compared to aprotinin 1:2 (trypsin:aprotinin). α-­‐1-­‐antitrypsin is a molecule that is really interesting since it is a natural inhibitor from the human body and with using endogenous substance the risk of develop sensitivity lowers. It’s a better option for allergic people too. The inhibition works quite well and with a molar ratio of 1:6 (trypsin: α-­‐1-­‐antitrypsin) gives an inhibition towards 50 %. Leupeptin has shown to be the most efficient where a molar ratio of 1:2 (trypsin:leupeptin) was enough to inhibit 98 % of the enzymes activity. Due to the efficiency of leupeptin and the advantage with an endogenous substance as α-­‐1-­‐antitrypsin, these two substances were immobilized. The immobilization of α-­‐1-­‐antitrypsin was successful and with good inhibition effect on trypsin and the bond between the inhibitor and the NHS-­‐agarose was formed. Inhibition with immobilization leupeptin using ethylenediamine and NHS-­‐agarose was also successful but with a weaker inhibition than immobilized α-­‐1-­‐antitrypsin. The weaker inhibition from leupeptin so far can be due to that the amounts of leupeptin, ethylenediamine and the reducing agent was mostly calculated with the consideration to molar ratio one to one, with a possible low coupling yield. A modified immobilization protocol can be expected to give more efficient inhibition. It is possible that the inhibition effect of α-­‐1-­‐antitrypsin can be further improved as well. The time of incubation also matters and since α-­‐1-­‐antitrypsin was immobilized during a longer time, this can be one reason that a lower amount leupeptin was immobilized. The conclusion is that both α-­‐1-­‐antitrypsin and leupeptin serve well as immobilized with room for quantitative improvement. In the end immobilized inhibitors can possible work in an ointment or cream and as a result of this diaper rash can be prevented on the babies skin. The approach may lead us to working solutions and possibilities to apply this idea and model on to other digestive enzymes and inhibitors of interest. 29 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 REFERENCES Cabacungan, J.C. Ahmed, A.I., Feeney R.E. (1982), Amine boranes as alternative reducing agents for reductive alkylation of proteins. Anal. Biochem 124, 272-­‐278. Campbell, Mary K., Farrell, Shawn O., (2008), Biochemistry, 6th edition, p. 133. Domen, P.L., Nevens, J.R., Mallia, A.K., Hermanson, G.T., Klenk, D.C. (1990) Site-­‐directed immobilization of proteins. J. Chromatogr. 510, 293-­‐302. Drapalova, Petra; Stys, Dalibor; Lukesova, Alena; Kopecky, Jiri (2008), Genus Nostoc – A source of novel trypsin inhibitors, Algological studies, Vol 127, 61-­‐82. Fresht, A., (1984) Enzyme structure and mechanism, 2nd edition. 98-­‐118. Hermanson, Greg T. (2008). Bioconjugate Techniques, 2nd edition, Pierce biotechnology, Thermo Fisher Scientific, Rockford, Illinois, Usa. p. 170-­‐173, 230-­‐233. Hiroyasu Nakata, Shin-­‐Ichi Ishii (1970) Substrate activation in the trypsin-­‐catalyzed hydrolysis of Benzoyl-­‐L-­‐Arginine p-­‐Nitroanilide, Biochem. Biophys. Research communications, Vol 41. No 2. Hornsey, V.S., Pnowse C.V., Pepper D.S. (1986) Reductive animation for solid-­‐phase coupling of protein. A practical alternative to cyanogen bromide. J. Immunol. Meth. 93, 83-­‐88. Hwang David. L., Foard Donald E., Chin Hsuan Wei (1977). “A soybean trypsin inhibitor”. The J. Bio. Chem., Vol 252, No. 3, Feb 10th: 1099-­‐1101 Kunitz, M. (1946) ”Crystalline soybean trypsin inhibitor.” J. Gen. Physiol. 29, 149-­‐154
Laskowski, M., (1950), “Proteolytic enzymes.” Dep. Biochemistry, Marquette Uni. School of Medicine, Milwaukee, WI, USA. Nelson, David L., Cox, Michael M. (2005), Lehninger, Principles of biochemistry, 4th edition, p. 231-­‐232, 650, 659. Peng, L., Calton, G.J., Burnett, J.W., (1987) Effect of borohydride reduction on antibodies. Appl. Biochem. Biotechnol. 14, 91-­‐99. Sanderson, C.J., Wilson, D.V., (1971) A simple method for coupling proteins to insoluble polysaccharides. Immunology 20, 1061-­‐1065. Scung-­‐Ho Kim, Saburo Hara, Sumihiro Hase, Tokuji Ikenaka, Hiroko Toda, Keisuke Kitamura, Norihiko Kaizuma (1985). “Comparative study on amino avid sequences of Kunitz-­‐type soybean trypsin inhibitors, Tia, Tib and Tic.” Department of Chemistry, Osaka University College of Science Toyonaka, Osaka. Van Sommeren, A.P.G., Machielsen, P.A.G.M., Gribnau, T.C.J. (1993) “Comparison of three activated agaroses for use in affinity chromatography: effect on coupling performance and ligand leakage”, J. Chromatogr, 639, 23-­‐31. Voet Donald J., Voet Judith G., Pratt Charlotte W. (2008), Principles of biochemistry, third edition. Williams, KaCee D., Williams, LaVonn A. (2011), “Diaper Rash: Common sense remedies and treatment.” International J. Pharm. Comp., Vol 15, no 6, 464-­‐469. http://www.sigmaaldrich.com/life-­‐science/metabolomics/enzyme-­‐explorer/analytical-­‐
enzymes/trypsin/trypsin-­‐inhibitors.html, 20120522 30 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 http://www.umm.edu/altmed/articles/lipase-­‐000311.htm, 20120412 https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314729545976/litdoc
18111353AA_20110830221039.pdf, 20120611 31 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 ACKNOWLEDGEMENTS I am grateful to Gunnar Johansson, Uppsala University, and to Shabira Abbas, Senior scientist at Svenska Cellulosa Aktiebolaget, that they have been the supervisors for this project. Both Gunnar and Shabira have provided me with excellent help, discussions, support and commitment throughout the project. Without their previous work and knowledge this project would not have reached the interesting results it did. I would also like to give a special thanks to Marie-­‐Lousie Lagerstedt Eidrup and Shabira Abbas for giving me the opportunity to do my master thesis at Svenska Cellulosa Aktiebolaget, SCA. I would also like to thank Curt Pettersson for being the examiner of this project and Douglas Westerlund for being my subject examiner. During my master thesis I have received help from Marcus Kjellander in the lab. I appreciate all the help with finding materials, his help with using the equipment and ordering necessary inhibitors for my work. Also a special thanks to Kathrin Götz for nice times in the lab and fun discussions. Last but not least I would like to thank everyone at the department of biochemistry and organic chemistry for welcoming and giving me a great time. Also a very special thanks to Shabira Abbas at SCA, my project couldn’t have been more fun! 32 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University 120612 APPENDIX CALCULATIONS For each inhibitor following has been used Substrate 50 µL of 20 mM BAPA, → 𝑛 = 𝑐×𝑉 = 20×10!! ×50×10!! = 1 𝜇𝑚𝑜𝑙 Buffert Ammoniumhydrogencarbonate, NH4HCO3, 100 µL of 250 mM → 25 µmol NH4HCO3 Addition of 850 µL H2O TRYPSIN One trypsin unit will hydrolyze 1,0 µmol of BAPA 0,3 𝐴𝑏𝑠/𝑚𝑖𝑛
= 31,5 𝜇𝑀𝑚𝑖𝑛!! 𝐿!! × 0,001 𝐿 = 0,00315 𝑚𝑖𝑛/𝜇𝑚𝑜𝑙 9600𝑀 !! 𝑐𝑚 !!
𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 0,93 𝜇𝑚𝑜𝑙/𝑚𝑖𝑛 𝑚 !"#$%&' = 0,00315 ×0,93 𝑚𝑔 𝑝𝑒𝑟 𝑡𝑖𝑚𝑒 = 0,0029295 𝑚𝑔 𝑝𝑒𝑟 𝑡𝑖𝑚𝑒 APROTININ One unit aprotinin reduce the activity of 2 trypsin units with 50 %. Amount trypsin 𝑚 = 2,52 𝑚𝑔 = 2,52×10!! 𝑔 𝑉 = 4 𝑚𝑙 = 4 ∗ 10!! 𝑔 𝑔
𝑀 = 23,4 𝑘𝐷𝑎 = 23,4 ∗ 10!
𝑚𝑜𝑙
0,00252
𝑚
𝑀=
234000 = 0,0000026923075 = 2,69 µμM 𝑐=
𝑉
4×10!!
Added 465 µL which gave a trypsin mass of: 𝑚 = 𝑐×𝑉×𝑀 = 2,69 𝜇𝑀×465 µμL×23,4×10! = 29269,89×10!! = 29,3 𝜇𝑔 𝑛 = 𝑐×𝑉 = 2,69 𝜇𝑀×465𝜇𝐿 = 1,25 𝑛𝑚𝑜𝑙 Amount Aprotinin 𝑚 = 1,3 𝑚𝑔 = 1,3×10!! 𝑔 𝑉 = 3 𝑚𝑙 = 3×10!! 𝑔 𝑀 = 6511,44 𝑔 𝑚𝑜𝑙 0,00130
𝑚
6511,44
𝑀
𝑐=
=
= 6,655×10!! = 66,6 µμM 𝑉
3×10!!
33 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University Added 60 µL trypsin solution that gave a mass of: 120612 𝑚 = 𝑐×𝑉×𝑀 = 2,69 𝜇𝑀×60 µμL×23,4×10! = 3,777×10!! = 3,8 𝜇𝑔 Addition of aprotinin: Aprotinin:Trypsin, 2:1 𝑉=
𝑚
3,8𝜇𝑔×2
=
= 17,5 𝜇𝐿 𝑐×𝑀 66,6𝜇𝑀×6511,44
Aprotinin:Trypsin, 3:1 𝑉=
𝑚
3,8𝜇𝑔×3
=
= 26,3 𝜇𝐿 𝑐×𝑀 66,6𝜇𝑀×6511,44
SOYBEAN TRYPSIN INHIBITOR (STI) One mg of STI will inhibit 1.0-­‐3.0 mg of trypsin. STI forms a 1:1 stoichiometric complex with trypsin. Amount trypsin 𝑚 = 1,58 𝑚𝑔 = 1,58×10!! 𝑔 𝑉 = 3 𝑚𝑙 = 3 ∗ 10!! 𝑔 𝑔
𝑀 = 23,4 𝑘𝐷𝑎 = 23,4 ∗ 10!
𝑚𝑜𝑙
0,00158
𝑚
𝑀=
234000 = 0,0000022507 = 2,25 µμM 𝑐=
𝑉
3×10!!
Volume added 𝑉 = 60 𝜇𝐿 𝑛 = 𝑐×𝑉 = 2,25×10!! ×60×10!! = 1,35×10!!" 𝑚𝑜𝑙 = 13,5 𝑛𝑚𝑜𝑙 Amount STI 𝑚 = 4,23 𝑚𝑔 = 4,23×10!! 𝑔 𝑉 = 6 𝑚𝑙 = 6 ∗ 10!! 𝑔 𝑔
𝑀 = 20,09 𝑘𝐷𝑎 = 20,09 ∗ 10!
𝑚𝑜𝑙
0,00423
𝑚
𝑀
20090 = 0,00003509 = 35,1 µμM 𝑐=
=
𝑉
6×10!!
Volume added (Inhibitor:Trypsin) -­‐
1:1 𝑉=
-­‐
𝑛 1,35×10!!"
=
= 3,846×10!! = 3,9 𝜇𝐿 𝑐
35,1×10!!
2:1 𝑉=
-­‐
2𝑛 2×1,35×10!!"
=
= 7,692×10!! = 7,7 𝜇𝐿 𝑐
35,1×10!!
3:1 34 Erika Billinger -­‐
-­‐
-­‐
Svenska Cellulosa Aktiebolaget & Uppsala University 𝑉=
3𝑛 3×1,35×10!!"
=
= 11,54×10!! = 11,5 𝜇𝐿 𝑐
35,1×10!!
𝑉=
4𝑛 4×1,35×10!!"
=
= 15,38×10!! = 15,4 𝜇𝐿 𝑐
35,1×10!!
𝑉=
5𝑛 5×1,35×10!!"
=
= 19,23×10!! = 19,2 𝜇𝐿 𝑐
35,1×10!!
𝑉=
6𝑛 6×1,35×10!!"
=
= 23,07×10!! = 23,1 𝜇𝐿 𝑐
35,1×10!!
120612 4:1 5:1 6:1 α-­‐1-­‐ANTITRYPSIN 5-­‐10 mg of α-­‐1-­‐antitrypsin will inhibit 1.0 mg of trypsin Amount trypsin 𝑚
𝑐!"#$%&' =
𝑀=
𝑉
0,00158
23400 = 22,5 µμM 3×10!!
𝑚 = 𝑐×𝑉×𝑀! = 22,5 µμM × 60 µμL × 23 400 = 31,6 µμg 𝑚
31,6 𝜇𝑔
n = = = 1,35 nmol 𝑀
23400 𝐷𝑎
Amount α-­‐1-­‐antitrypsin 𝑚 = 6,3 𝑚𝑔 = 6,3×10!! 𝑔 𝑉 = 4 𝑚𝑙 = 4 ∗ 10!! 𝑔 𝑀 = 44,3 𝑘𝐷𝑎 = 44,3245 ∗ 10!
𝑚
𝑐=
𝑀=
𝑉
0,0063
𝑔
𝑚𝑜𝑙
44325,4
= 0,00003553 𝑀 = 35,5 µμM 4×10!!
Experiment 1.
2.
3.
4.
5.
6.
7.
60 µl Trypsin 60 µl Trypsin + 30 µl α-­‐1-­‐antitrypsin + 770 µL dest. H2O 60 µl Trypsin + 60 µl α-­‐1-­‐antitrypsin + 710 µL dest. H2O 60 µl Trypsin + 120 µl α-­‐1-­‐antitrypsin + 710 µL dest. H2O 60 µl Trypsin + 240 µl α-­‐1-­‐antitrypsin + 650 µL dest. H2O 60 µl Trypsin + 300 µl α-­‐1-­‐antitrypsin + 590 µL dest. H2O 60 µl Trypsin + 340 µl α-­‐1-­‐antitrypsin + 450 µL dest. H2O Amount α-­‐1-­‐antitrypsin at each experiment •
•
•
𝑚 = 𝑐×𝑉×𝑀! = 35,5×10!! ×30×10!! ×44324,5 = 47,2 𝜇𝑔
𝑛 = 𝑚 𝑀 = 47,2 𝜇𝑔 44 325 𝐷𝑎 = 1,06 𝑛𝑚𝑜𝑙 𝑚 = 𝑐×𝑉×𝑀! = 35,5×10!! ×60×10!! ×44324,5 = 94,4 𝜇𝑔
𝑛 = 𝑚 𝑀 = 94,4 𝜇𝑔 44 325 𝐷𝑎 = 2,13 𝑛𝑚𝑜𝑙 𝑚 = 𝑐×𝑉×𝑀! = 35,5×10!! ×120×10!! ×44324,5 = 188,8 𝜇𝑔
35 Erika Billinger •
•
•
Svenska Cellulosa Aktiebolaget & Uppsala University 𝑛 = 𝑚 𝑀 = 188,8 𝜇𝑔 44 325 𝐷𝑎 = 4,26 𝑛𝑚𝑜𝑙 𝑚 = 𝑐×𝑉×𝑀! = 35,5×10!! ×240×10!! ×44324,5 = 377,6 𝜇𝑔
𝑛 = 𝑚 𝑀 = 377,6 𝜇𝑔 44 325 𝐷𝑎 = 8,52 𝑛𝑚𝑜𝑙 𝑚 = 𝑐×𝑉×𝑀! = 35,5×10!! ×300×10!! ×44324,5 = 472,1 𝜇𝑔
𝑛 = 𝑚 𝑀 = 47,2 𝜇𝑔 44 325 𝐷𝑎 = 10,65 𝑛𝑚𝑜𝑙 𝑚 = 𝑐×𝑉×𝑀! = 35,5×10!! ×340×10!! ×44324,5 = 535 𝜇𝑔
𝑛 = 𝑚 𝑀 = 535 𝜇𝑔 44 325 𝐷𝑎 = 12,07 𝑛𝑚𝑜𝑙 Ratio Trypsin: α-­‐1-­‐antitrypsin With 30 µL α-­‐1-­‐antitrypsin = 0,79 With 60 µL α-­‐1-­‐antitrypsin = 1,58 With 120 µL α-­‐1-­‐antitrypsin = 3,16 With 240 µL α-­‐1-­‐antitrypsin = 6,31 With 300 µL α-­‐1-­‐antitrypsin = 7,89 With 340 µL α-­‐1-­‐antitrypsin = 8,94 LEUPEPTIN Amount trypsin 𝑚 = 6,38 𝑚𝑔 𝑉!"!!"#! !"# !" = 5 𝑚𝑙 𝑚 𝑀
6,38 𝑚𝑔 23,4 𝑘𝐷𝑎
𝑐=
= = 0,0000545299 = 54,53 𝜇𝑀 𝑉
5 𝑚𝑙
A volume of 18 µL trypsin solution give 𝑛 = 𝑐×𝑉 = 54,53 𝜇𝑀×18 𝜇𝐿 = 981,54 𝑝𝑖𝑘𝑜𝑚𝑜𝑙 = 0,982 𝑛𝑚𝑜𝑙 Amount leupeptin 𝑚 = 𝑐𝑉𝑀 = 100×10!! ×4×10!! ×463,01 = 0,1852 𝑚𝑔 = 185,2 𝜇𝑔 Ratio 1:1, a volume of leupeptin that needs to be added is 𝑉=
𝑛
0,982×10!!
= = 0,00982 𝑚𝑙 = 9,82 µμL 𝑐
100×10!!
Ratio 1:2 a volume of leupeptin that needs to be added is 𝑉=
𝑛
2×0,982×10!!
= = 0,01964 𝑚𝑙 = 19,64 µμL 𝑐
100×10!!
Ratio 1:3 a volume of leupeptin that needs to be added is 𝑉=
𝑛
3×0,982×10!!
= = 0,02946 𝑚𝑙 = 29,46 µμL 𝑐
100×10!!
36 120612 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University IMMOBILIZATION IMMOBILIZED α-­‐1-­‐ANTITRYPSIN Coupling solution Mw(NaHCO3) = 84,01 g/mol Mw(NaCl) = 58,44 g/mol V = 400 ml 𝑚(𝑁𝑎𝐻𝐶𝑂! ) = 𝑐×𝑉×𝑀! = 0,2×0,4×84,01 = 6,7208 𝑔 𝑚(𝑁𝑎𝐶𝑙) = 𝑐×𝑉×𝑀! = 0,5×0,4×58,44 = 11,688 𝑔 Weighed quantity 𝑚 𝑁𝑎𝐶𝑙 = 11,699 𝑔 𝑚 𝑁𝑎𝐻𝐶𝑂! = 6,720 𝑔 Amount α-­‐1-­‐antitrypsin 𝑚 = 𝑐×𝑉×𝑀! = 20 𝜇𝑚𝑜𝑙×5 𝑚𝑙×44,3 𝑘𝐷𝑎 𝑚 𝐴1𝐴𝑇 = 4,03 𝑚𝑔 𝑉 𝑏𝑢𝑓𝑓𝑒𝑟𝑡 = 4,55 𝑚𝑙 Tris-­‐HCl buffert 𝑐 = 0.1𝑀 𝑚 = 𝑐×𝑉×𝑀! = 0,1×0,4×121,1 = 4,844 𝑔 𝑊𝑒𝑖𝑔ℎ𝑒𝑑 𝑚 = 4,897 𝑔 Dissolved in dest. Water, pH around 10, 6M HCl is added until pH reaches 8,5. 100 µL A1AT+NHS give 𝑛 = 𝑐×𝑉 = 20 𝜇𝑀×100 𝜇𝐿 = 0,002 𝜇𝑚𝑜𝑙 = 2 𝑛𝑚𝑜𝑙 10 % av A1AT 10
→ 0,2 𝑛𝑚𝑜𝑙 𝑡𝑟𝑦𝑝𝑠𝑖𝑛 100
𝑚 = 𝑛×𝑀 = 0,2 𝑛𝑚𝑜𝑙 × 23,4 𝑘𝐷𝑎 = 4,68 𝜇𝑔 50 % av A1AT 50
→ 1 𝑛𝑚𝑜𝑙 𝑡𝑟𝑦𝑝𝑠𝑖𝑛 100
𝑚 = 𝑛×𝑀 = 1 𝑛𝑚𝑜𝑙 × 23,4 𝑘𝐷𝑎 = 23,4 𝜇𝑔 Amount trypsin 𝑚 = 6,38 𝑚𝑔 𝑉!"!!"#! !"# !" = 5 𝑚𝑙 𝑚 𝑀
6,38 𝑚𝑔 23,4 𝑘𝐷𝑎
𝑐=
= = 54,5 𝜇𝑀 𝑉
5 𝑚𝑙
37 120612 Erika Billinger Svenska Cellulosa Aktiebolaget & Uppsala University IMMOBILIZED LEUPEPTIN The same coupling buffert and tris-­‐HCl was used as in the immobilization for α-­‐1-­‐antitrypsin. Trypsin solution 𝑐 = 100 𝜇𝑀 𝑉 = 4 𝑚𝑙 𝑛 = 𝑐×𝑉, 𝑚 = 𝑀×𝑛 𝑚 = 𝑐×𝑀×𝑉 = 100 𝜇𝑀×23,4 𝑘𝐷𝑎×4 𝑚𝑙 = 9 360 𝜇𝑔 = 9,36 𝑚𝑔 𝑢𝑝𝑝𝑣ä𝑔𝑑 𝑚 𝑡𝑟𝑦𝑝𝑠𝑖𝑛 = 7,46 𝑚𝑔 𝑚
7,46 𝑚𝑔
𝐺𝑒𝑟 𝑒𝑛 𝑣𝑜𝑙𝑦𝑚 𝑝å 𝑉 = =
= 3,19 𝑚𝑙 𝑐×𝑀 100 𝜇𝑀 × 23 400 𝐷𝑎
𝑉!"#$%&' = 50 𝜇𝑙 → 𝑛 = 𝑐×𝑉 = 100 𝜇𝑀 × 50 𝜇𝑙 = 5 000 𝑝𝑚𝑜𝑙 = 5 𝑛𝑚𝑜𝑙 1:1 (leupeptin:trypsin) 𝑛!"#$"$!"# = 5 𝑛𝑚𝑜𝑙 → 𝑉!"#$%&' = 𝑛 5 𝑛𝑚𝑜𝑙
=
= 31,25 𝜇𝑙 𝑐
160 𝜇𝑙
2:1 (leupeptin:trypsin) 𝑛!"#$"$%&' = 5 𝑛𝑚𝑜𝑙 → 𝑉!"#$%&' = 𝑛 2×5 𝑛𝑚𝑜𝑙
=
= 62,5 𝜇𝑙 𝑐
160 𝜇𝑙
Immobilized with NHS-­‐activated Sepharose 𝑛 𝑁𝐻𝑆 = 16 − 23 𝜇𝑚𝑜𝑙 𝑚𝑙 𝑑𝑟𝑎𝑖𝑛𝑒𝑑 𝑚𝑒𝑑𝑖𝑢𝑚 1: 1 𝑎𝑚𝑖𝑛𝑒: 𝑁𝐻𝑆 𝑔𝑖𝑣𝑒𝑠 𝑛 = 20 𝜇𝑚𝑜𝑙 𝑔
𝑚 = 𝑛×𝑀! = 20 𝜇𝑚𝑜𝑙× 60,10
= 120,20 𝜇𝑔 𝑚𝑜𝑙
0,5 ml NHS-­‐activated sepharose Ethylenediamine 897 𝑔 𝐿 𝐸𝑡ℎ𝑦𝑙𝑒𝑛𝑑𝑖𝑎𝑚𝑖𝑛, 𝐶! 𝐻! 𝑁! 𝑀! = 60,1 𝑔 𝑚𝑜𝑙 897 𝑔 𝐿
𝑐=
= 14,93 ~ 15 𝑀 60,10 𝑔 𝑚𝑜𝑙
𝐷𝑖𝑙𝑢𝑡𝑒𝑑 𝑡𝑜 1 𝑀 → 6,67 𝑚𝑙 𝑒𝑡ℎ𝑦𝑙𝑒𝑛𝑑𝑖𝑎𝑚𝑖𝑛 & 93,33 𝑚𝑙 𝑑𝑖𝑠𝑡𝑖𝑙𝑙𝑒𝑑 𝑤𝑎𝑡𝑒𝑟 38 120612