Permeability of renal capillaries. I. Preparation of neutral and charged protein probes.
This paper describes the preparation of charged and uncharged protein molecular probes for study of the permselectivity of renal capillaries. Horse heart myoglobin was used as a neutral myoglobin. Since it contained several fractions with different isoelectric points, it was purified by fast protein liquid chromatography (FPLC). To obtain a negatively charged myoglobin, the original horse heart myoglobin was treated with cyanate, resulting in net charge of -5.7 +/- 0.3 at physiological pH (mean +/- SEM). The charge was determined from the Donnan potential which develops over a semipermeable membrane separating the inside solution in which the protein was dissolved from a surrounding bath of equal ionic strength. Sperm whale myoglobin was similarly purified by FPLC and used as a positively (+1.7 +/- 0.2) charged isomer. Horseradish peroxidase (HRP) was purified by means of gel and ion-exchange chromatography and found to be neutral at physiological pH. Negatively charged (-14.0 +/- 0.5) HRP was obtained by succinylation. Two isomers of lactate dehyrogenase (LDH) were used, namely the slightly positive (+2) LDH-M4 and the strongly negative (-19) LDH-H4. These isomers, which occur naturally, did not require further purification. The Stokes-Einstein radii, as measured by gel chromatography, of inulin, myoglobin, HRP and LDH were 11, 17.5, 32 and 46 A, respectively. The chemical modifications did not alter the Stokes-Einstein radii. In biological studies on rat kidneys samples of both plasma and renal hilar lymph were found to contain radioactive low molecular weight degradation products in addition to the intact proteins. This necessitated separation of all individual samples on small Sephadex columns prior to analysis.
D000818:Animals; D002199:Capillary Permeability; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006735:Horseradish Peroxidase / Q000302:isolation & purification; D006736:Horses; D006801:Humans; D007457:Iodine Radioisotopes; D007525:Isoelectric Focusing; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification; D009211:Myoglobin / Q000302:isolation & purification; D011487:Protein Conformation; D011506:Proteins / Q000302:isolation & purification; D012079:Renal Circulation; D014907:Whales
D007457:Iodine Radioisotopes; D009211:Myoglobin; D011506:Proteins; D007770:L-Lactate Dehydrogenase; D006735:Horseradish Peroxidase