Characters Of Protein Migrating Towards Cathode In SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)was the most used method in mensurating the composing of protein subunits.In the course of research of the glycolate oxidase (GO), GO was preparedfrom leaves of Brassica parachininensis Bailey and revealed just oneprotein band at 40 kD in SDS-PAGE, and this 40 kD band migrated towardsthe anode. Whereas, the same GO revealed two protein bands migrated towardsthe anode and cathode, respectively, in SDS-acetate cellulose membraneelectrophoresis. The 40 kD protein band is the GO subunit and this GO 40kD subunit migrated towards the anode, but the protein migrated towardscathode is an unknown protein. So the relation between GO and this unknownprotein need to research. In this paper, the protein migrated towardscathode was called cathode protein. The content ofphenylalanine of thecathode protein had been assayed. It was as high as 60%. Whether the datawas credible still need us to validate. Whether the cathode protein existedin the animal was still unknown. The characters of the cathode protein andthe mechanism of the cathode protein migrating towards cathode were unknown.By the experiment, some further study was done.The crude protein extracted from green leaves of Brassicaparachininensis Bailey was subjected to prepared SDS-PAGE. The cathodebuffer was collected, in which the cathode protein was obtained. Then thecomposing of anmino acids of the cathode protein was assayed. The resultshowed that the content of phenylalanine of the cathode protein was as highas 75%. It validated the previous results. The crude protein of rabbit liverwas subjected to SDS-acetate cellulose membrane electrophoresis. Proteinbands appeared at both anode and cathode. So the cathode protein alsoexisted in animal protein.Crude extract from green leaves of Brassica parachininensis Baileywas precipitated with 10% acetic acid, 15% and 35% (NH₄)₂SO₄. The precipitation was collected and dissolved in 5mmol/L Tris-HCl (pH 8.3).Then the solution was applied to Sephadex G-50 column. The first fractionwas applied to DEAE-Cellulose-52 column. By the method, GO of high specificactivity was extracted. Proteins of each step in the extracting course ofGO were subjected to SDS-acetate cellulose membrane electrophoresis. Boththe anode and cathode had protein bands. So there must be cathode proteincombining with GO.The extracted GO existed by liquid state in 80mmol/LTris-HCl(pH8.3).The SDS-PAGE of the extracted GO had to do with theconservation time of proteins: SDS-PAGE band was 54 kD at the beginning, butsoon changed for 40 kD. But the extracted GO existed by the form ofprecipitation after precipited by 50% (NH₄)₂SO₄. Its band was 40 kD inSDS-PAGE, and its activity augmented distinctly. We conjectured that GOcombined with cathode protein close. Although SDS could denature protein,GO still combined with cathode protein close, which were at 54 kD inSDS-PAGE. The subunits size of GO was 40 kD, so the subunits size of cathodeprotein may be 14 kD (54 kD-40 kD=14 kD). After conserved for 24h at 4℃, GO and cathode protein separated one another, and they migrated towardsanode and cathode respectively. The extracted GO was subjected to SDS-PAGEand the cathode protein was collected. The component of amino acids of thecathode protein was determined. The content of phenylalanine was 75%. Butthe content of cysteine was few. This result was different from that of40 kD of GO: 40 kD of GO was rich in cysteine, but phenylalanine was few.The result showed that the cathode protein was different from GO. But thecathode protein was not a basic protein. Its basic protein to the totalamino acid was just 0.65%.The combination rates of SDS with proteins of the BSA, lysozyme andextracted GO sample were assayed. BSA and lysozyme could combine with SDSby 1:1.4. But the combination of SDS and GO is not steady. This may be one of the reasons of the protein transferring to cathode. Because: 1), thecontent of basic protein of cathode protein was under that of protamine.The positive charge of cathede protein was fewer than that of protamine.People had found that the content of basic protein of protamine was 40%.Protamine took much positive charge. 2), protamine could combine with SDSat 1:1.4 and form SDS-protein combo. 3), people had approved thatSDS-protamine combo could subside in the SDS-PAGE. So the positive chargeof protamine was correspond with the negative charge of SDS. Thus theSDS-protamine took no charge and its solubility became feeble. 4), we hadapproved that GO combined with SDS at 1:0.23, not 1:1.4.Gel filtration was used to define the range of the size of the cathodeprotein. The extracted GO was denatured by SDS and subjected to SephadexG-100. BSA and lysozyme were used to defined the wash volume of SephadexG-100. rhe extracted GO subjected to Sephadex G-100 had two protein peaks:GC-â… and GC-â…¡. The two protein peaks were subjected to SDS-PAGE. GC-â… wasat 40 kD, but GC-â…¡hadn't migrated towards anode. We considered that theGC-â…¡was the cathode protein of GO and its size was 14 kD. The GC-â… wasthe combo of GO and cathode protein(40 kD+2×14 kD).Then we use SDS-CEto confirm the size of the protein. There were two protein peaks: 35 kDand 18 kD. We guessed that 35 kD protein was GO and 18 kD protein was thecathode protein.