Abstract— The photodynamic action of protoporphyrin on red cell ghosts is reflected by extensive cross‐linking of membrane proteins to very high molecular weight protein aggregates. This process was studied with sepharose gel chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis. Most sensitive to this photodynamic effect are spectrin and band 2. 1, 2. 2, 2.3 and 4.1. polypeptides, which are cross‐linked after very brief illumination periods, with a concomitant loss of spectrin‐associated ATPase activity. Band 6 protein, representing the monomeric form of glyceraldehyde‐3‐phosphate dehydrogenase, is also very sensitive to protoporphyrin‐induced cross‐linking. The enzymatic activity decreased even faster than the amount of band 6 polypeptides, suggesting that modification(s) of the enzyme other than cross‐linking, possibly by rapid photooxidation of a thiol group, may be responsible for inactivation. Extracted and purified spectrin was cross‐linked with about the same velocity as membrane‐bound spectrin, reinforcing our previously drawn conclusion that membrane lipids are not involved in the cross‐linking reaction. Eluted band 6 polypeptides on the other hand exhibited a relatively fast photo‐oxidative modification but a much slower cross‐linking to dimers and tetramers. This suggests that the membrane structure, e.g. the spectrin matrix may play an essential role in the incorporation of membrane‐bound band 6 polypeptides in the high molecular weight cross‐linked complex.
Two extraction methods for the isolation of DNA from formalin-fixed, paraffin-embedded tissue samples from colonic carcinomas were compared. The processed DNAs were compared with DNAs from fresh specimens of the same tumors. The two extraction methods gave similar results. Formalin-fixation and paraffin-embedding irreversibly denatured DNA and consequently decreased the extraction yield and interfered with the quantitative measurement of DNA. Southern blot and dot blot analysis of processed and native DNA was performed using a c-myc and an actin probe. The results show that for Southern analysis processed DNA can be used but, due to the generation of random breaks, the restriction fragments have to be small. Furthermore, the fixation-induced crosslinking of DNA appears to hamper hybridization. For these reasons processed DNA can be analyzed better by dot blot rather than Southern blot hybridization.
Virtually all protoporphyrin in erythrocytes of patients with erythropoietic protoporphyria is bound to hemoglobin. The maximum of the fluorescence excitation spectrum of this protoporphyrin-hemoglobin complex shifted, with increasing concentration, from 405 nm to 389 nm. A similar shift was observed when titrating a solution of free protoporphyrin with hemoglobin. The Soret maximum of free protoporphyrin itself, on the other hand, was not concentration-dependent. These observations indicate that spectrofluorometric measurements do not allow conclusions concerning the mode of protoporphyrin binding to hemoglobin. Experiments on protoporphyrin exchange between the hemoglobins A, F and S reinforced the previously drawn conclusion that protoporphyrin is bound to hemoglobin at the heme-binding sites.
