Permeability of renal capillaries. I. Preparation of neutral and charged protein probes

Acta Physiologica Scandinavica
G OjtegM Wolgast

Abstract

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. T...Continue Reading

References

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Citations

Nov 1, 1995·Archives of Oral Biology·L A LindénM Wolgast
Jul 2, 1998·Acta Physiologica Scandinavica·J SörenssonB Haraldsson
Sep 1, 1988·Acta Physiologica Scandinavica·A BayatiM Wolgast
Oct 1, 1988·Acta Physiologica Scandinavica·A StenbergA E Persson
Mar 1, 1993·Acta Physiologica Scandinavica·G Ojteg, M Wolgast
Nov 1, 1993·Acta Physiologica Scandinavica·E R Zakaria, B Rippe
Jan 1, 1995·Upsala Journal of Medical Sciences·M Wolgast
May 25, 2005·European Journal of Pharmaceutical Sciences : Official Journal of the European Federation for Pharmaceutical Sciences·Corine C VisserAlbertus G de Boer
Mar 16, 2001·American Journal of Physiology. Renal Physiology·J SörenssonB Haraldsson

Related Concepts

Vascular Permeability
Molecular Sieve Chromatography
High Pressure Liquid Chromatography Procedure
SDS-PAGE
Alpha-Peroxidase
Iodine Radioisotopes
Isoelectric Focusing
Lactate Dehydrogenase
Myoglobin
Protein Conformation

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