[Two-dimensional gel electrophoresis of matrix metalloproteinases in subretinal fluids]

In this work, the experimental conditions for two-dimensional gel electrophoresis of subretinal fluids (SRF) matrix metalloproteinases were established. The conditions tested included the composition of lysis solution and lysis method, the composition of rehydration solution and isoelectric focusing program (IEF), the composition of equilibration buffer and equilibration process and the composition of incubation solution and incubation methods. The main equipments used were IPGphor isoelectric focusing system from Amersham pharmacia and PROTEAN II xi cell from Bio-Rad, the gel strips used were the 18 cm long, pH 3 - 8 Linear immobiline DryStrips. Among the 9 samples analyzed, 2 were PVR-A, 2 were PVR-C1, 2 were PVR-C2, 2 were PVR-C3 and the remaining one could not be classified definitely. The new 2-DE MMPs method is better than Gelatin SDS-PAGE zymograhpy method, as it is higher in resolution, sensitivity and reproducibility. The experimental results suggested that the four types of MMPs expressed differently at different stages of PVR. Two of the MMPs isomers have same molecular weight (MW) but different in isoelectic points (pI). The four MMPs are determined to be MMP-1, MMP-2, MMP-9 and MMP-9, with MMP-9 has two active forms. In addition, MMP-9 and MMP-1 may be present in PVR-A samples but not in PVR-C samples, whereas MMP-2 is present in PVR-C but not in PVR-A samples. These results revealed the complex profiles of MMPs' expression in PVR. The new method can be applied to test MMPs expression in tissues, cells and other types of samples with a little modification in the protocol, and can be followed by mass spectroscopic analysis of MMPs.