Domuz ciğeri ve bezelye tohumundan diamin enziminin izolasyonu saflaştırılması ve özellikleri

Abstract

Son yirmibeş yıl içinde pekçok sanayii dalında uygulama alam bulan enzimler, günümüzde yeni kullanım alanlarının ortaya çıkması ile giderek önem kazanmaktadır. Enzimler 103-106 aralığında molekül ağırlığına sahip makromoleküllerdir. Bu çalışma, DAO enziminin izolasyonunu, önemli ayırma metodlarının prensipleri doğrultusunda enzim konsantrasyonunun düzenlenmesini ve saflaştırılmasında hangi metodların nasıl kullanılacağını esas al maktadır. Çalışmada bezelye tohumu DAO enzimini yüksek verimde saflaştırma işlemini gerçekleştirmek ve çok miktarda elde etmek amaçlanmıştır. Değişik koşullar altında büyüyen tohumların aktivite veriminin karşılaştırılması yapılarak enzim için büyüme koşulları tayin edilmiştir. Yüksek verimdeki ürün 7-10 günlük periyotta büyüyen ağarmış bezelyelerin kullanılması ile mümkün olmuştur. Enzimin molekül ağırlığı, bağlanma ayıracı ile ön işleme uğratılarak ve ön işlemsiz olarak SDS-akrilamid jel elektroforez ile ve düşük iyonik kuvvette (non-denature koşullar) ve yüksek iyonik kuvvette (de nature koşullar) jel filtrasyonu ile tayin edilmiş ve sonuçlara göre 9x10.4 dalton ağırlığında özdeş iki alt birim zincirden ibaret olduğu saptanan enzimin, molekül ağırlığının yaklaşık olarak 18x104 dalton olduğu tespit edilmiştir. Aktif proteini çöktürmek için amonyum sülfat yüzde grafiği, pH, sıcaklık-kararlılık ve iyonik kuvvet-kararlılık grafikleri oluşturularak, saflaştırılmış enzimin izoelektrik noktası, kararlılığı ve aktivite değişikliği tayin edilmiş ve sonuçlar irdelenmiştir.

In this study the isolation of enzymes is discussed, concentrat ing initially on the principles of the important separation methods employed» Then the operation of these methods is illustrated by consider ing one specific example, purification of enzymes. The studies on the isolated enzyme yield information on its spe cificity for substrates, kinetic parameters for the reaction, and pos sible means of regulation. The aim of a purification procedure should be to isolate a given enzyme with the maximum possible yield, based on the percentage reco vered activity compared with the total activity in the original ex tract. In addition the preparation should posses the maximum cataly tic activity, ie. there should be no degraded or other inactivated enzyme present, and it should be of the maximum possible purity, con tain no other enzymes or large molecules. The catalytic activity of a preparation is determined by a sui table assay procedure in which the rate of disapparance of substrate or the rate of appearance of product is determined under defined con ditions of substrate concentration, temperature, pH, etc., The units of activity are usually expressed as either umol substrate consumed, or product formed, per minute ('units' or 'international units') or mol substrate consumed, or product formed, per second ('katal* in the SI system). There is no way of predicting the catalytic activity of a puri fied enzyme under a given set of conditions and so purification is carried on until the specific activity of the preparation (ie. units per mg or katal per kg) increases to reach a constant value which is not increased by further purification steps. The steps involved in the purification of an enzyme can be dis cussed in terms of the type of flow sheet shown in Fig.l. Within this general scheme, the procedure to be adopted for a given enzyme involves choices of: (i) source of enzyme; (ii) methods of homogeni- zation; and (iii) methods of separation. V1X 1 TOTAL l 1 LAROE-SCALE ı Source o Extract OCrude V 1 HOMOOENIZATION l ^ ' SEPERATION _ .. V /K Preparatıon \ EXTRACTION \ \ ÖR \ SMALL-SCAUE \ DISPERTION \ SEPERATION PART1AL \ \ HOMOOENIZATION \ AFFINITY \ l SUBCELLULAR \ 8EPERATION \ \j FRACTIONATION \ , \ V ^Purified <\ Püre Organelles Enzymes Ficure l Steps Involved in thc Purification of Enzymos. Enzymes are macromolecules with molecular weights ranging from about 1x104 to several million. The majority of present-day determina- tions of molecular weights of enzymes are performed by use öne ör more of the following techniques. (i) ultracentrifugation (ü) gel filtration (iıi) sodium dodecylsulphate polyacrylamide gel electrophoresis. Of these methods, (ü) and (iii) are 'semi-empirical' and compari- sons are made with standart molecules of known molecular weight. in (i), however, the molecular weight can be calculated using equations derived from first principles. An ultracentrifuge is capable of generating intense centrifugal fields; in a typical machine a rotor speed of 6.5xl04 rpm correspondş to a field of the order of 30x10.4 times gravity. Under these conditi- ons macromolecules will have a tendency to sediment, provided that the density of the macromolecule is greater than that of the solution (this generally holds for enzymes in aqueous solution). in gel filtration, the separation between molecules of different sizes is made on the basis of their ability to enter the pores within the beads of a beaded gel. The most widely used types of gel are sep- hadex (cross-linked desctrans) and Bio-Gel P(Cross-linked)polymers of acrylamide). viii Small molecules which can enter the pores of beads are retarted as they pass down a column containing the gel; large molecules which are unable to enter the pores pass through the column unimpeded. By varying the size of the pores (which is controlled by the degree of cross-linking in the preparation of the beaded gel) if s possible to change the range of molecular weights which can be fractioned. Sephadex 6-100, for example, can fractionate globular proteins in the molecular weight range from 4000 ^0 15xl04. Gel filtration can be carried out on a large scale but since large columns are rather time consuming to run and the gels required to fill them are expensive. A dialysis membrane such as cellophane acts as a sieve with holes large enough to permit the passage of globular protein up to about 2xl04 molecular weight but not of larger molecules. It is possible to change the pore sizes by various mechanical and chemical treatments. Although dialysis is not generally useful for the separation of enzy mes from each other, it is widely used during purification procedures to remove salts, organic solvents, or low molecular weight inhibitors from solutions of enzymes. Ion-exchange chromatography depends on the electrostatic attracti on between species of opposite charge. Ion exchangers usually consist of modified derivatives of some support material such as cellulose, sephadex, etc., as shown in the examples of DEAE-cellulose and CM-cel- lulose (Fig. 2). Cellulose-O-CHo-CHp-N. \ H.CHpCHo.CH2CH3 Cellulose-0-CH2-C02 DEAE-cellulose (Diethyl ami noethyl -cellulose) possesses a pKa ^ 10 will bind negatively charged species and is therefore an anion exchanger CM-cellulose (Carboxymethyl -eel 1 ul ose) possesses a pKa*^ 4 will bind positively charged species and is there fore an cation exchanger. Fig. 2 Ion Exchangers Commonly Used in Purifications of Enzymes During a purification procedure, the enzyme is usually applied to an ion exchanger in a solution. of low ionic strength and at a pH where the appropriate interaction will occur (i.e. the enzyme and the ion exchanger have opposite changes). Desorption of the bound species can be brought about either by changing the pH, and thus altering the charges, or by increasing the ionic strength of the solution so that the increased concentration of cations or anions will compete with the enzyme for the binding sites on the ion exchanger. Use of a gra dient of increasing ionic strength permits the separation of proteins in a mixture on the basis of their ability to bind to the ion exchan ger. ix Ion-exchange chromatography can be performed on a large or a small scale. On a large scale it is often convenient to work in a batch wise manner, i.e. adsorb the enzyme by adding the ion exchanger to a solution and' then to pour the material into a column for control led desorption using an ionic strength gradient. On a small scale, both adsorption and desorption are performed in a column. Ion-exchan ge chromatography finds very wide application in present day purifica tion procedures. Electrophoretic seperation is based on the differantial movement of charged molecules under the influence of an applied potential dif ference. The rate of movement of a species, i s governed by the charge it carries and also by its size and shape. In order to minimize con vection currents, the buffer (electrolyte) solution is soaked into a support (paper, cellulose powder, starch, or polyacrylamide gels). The position of a protein on the support can be determined by use of a stain such as Coomassie Blue which binds to proteins. Althouh the technique is normally performed on a small, analytical scale (a few mg or less), it is possible to use the method on a large prepara tive scale. After preparative electrophoresis the seperated enzymes can be eluted out of the support or run off the bottom of a column in sequence. During this study, a number of the studies reported here requi red the use of large quantities of the peaseedling diamine oxidase (DAO). In order to satisfy this requirement a purification procedure to obtain homogeneous enzyme in high yield and in large quantity had to be developed. Such an isolation procedure is described. The steps of the isolation procedure are ; -Crude homogenate, -Supernatant from homogenate heated at 60° for 10 min., -%33-60 saturation with (NH^SO^ -Precipitation at pH:5.3, -Column electrophoresis» -Column chromatography on DEAE-cellulose-» Use of a new and powerful assay technique required a complete comparison to assays commonly employed for this enzyme. A conversion factor was obtained allowing comparison of our purifications (via spe cific activity) as monitored by the spectrophotometry assay to cur rent reports still using respirometric measurements. Highes yields were possible by using etiolated peas grown for seven or eight days. This enzyme is produced to study comparing to tal activity yielded by growing the seedlings under various conditi ons. The molecular weight of the enzyme was determined by SDS-acryla- mide gel electrophoresis with a,nd without pre-treatment with a cross- linking agent and by gel filtration in both Ipw ionic strength (non- denaturing conditions!) and high ionic strength (denaturing conditions). The result indicate that the molecular weight of the enzyme is approximately 18x104 daltons and that native enzyme is composed of two identical chains of 9x1 0^ daltons. Copper analyses show that the native enzyme contains two strongly bound to Cu (II) ions, presumably one per subunit. Apoenzyme first treated with an excess of Cu (II) ions and then treated with. 1.0 m M EDTA to remove the loosely and unspecifically bound Cu(II) ions gives approxiamately 1 copper ions per molecule. Other characterizations of the purified system were the following: Isoelectric focusing of the enzyme gave a value of 6.5 for the isoelectric point. UV/VIS spectroscopy of the native (oxidized) form of the enzyme gave spectra characteristic of pea seedling DAO obtained highly purified by other current researchers. UV/VIS spectroscopy was used to obtain detailed spectra on the reduced form of the enzyme and the copper-free apoenzyme. A variety of stability and activity profiles were determined for the purified DAO. These included a percent ammonium sulphate profile for the precipitation of active protein, a pH profile, a temperature- stability profile and an ionic strength (of Pi buffer) -stability profile. Preliminary studies in the identification of the organic prosthe tic group were conducted. Use of deforming buffers and aldehyde rea gents to promote removal of the suspected pyridoxal phosphate (PLP- Like) moiety and to react in formation of the expected thiazolidine derivative proved to be inconclusive in the resolution of the cofac- tor. The repetation of freezing and thawing of holoenzyme solutions resulted in appearance of a peak. characteristic of PLP. Denaturation by heat treatment permitted isolation of a yellow chromophore from the supernatant. TLC and mass spectral studies of the yellow substances were inconclusive. Finally the analysis of the copper/carbamate complex extracted from the native enzyme presented no spectral evidence of either PLP or any other absorbing organic moiety also bound.