The Application of Freeze-Cleaving Technics to Studies on Red Blood Cell Fine Structure

A simplified freeze-cleave and replication method of tissue preparation forexamination in the electron microscope is applied to studies on red blood cellfine structure. With this technic, the cytoplasm of red blood cells appearsto have a uniform pattern of packed granularity, with individual particles approximating the dimensions anticipated for replicas of individual hemoglobinmolecules. The cell surface is smooth and partially covered with small particles which may represent antigens, enzymes, or some structural proteins.The possibility that particles seen in cells and on cell membranes may represent an artifact is discussed. Pretreatment of cells prior to freezing influencesthe plane of cleavage through packed cells so that the plane of cleavage canbe preferentially directed either through the cytoplasm or along red cellmembranes. The freeze-cleave technic may be of particular value in applications where extensive areas of membrane must be surveyed, such as searchingfor leukemogenic viruses budding through cell membranes.

Submitted on August 16, 1966 Accepted on November 24, 1966     

Circular dichroism and fluorescence spectroscopic analyses of a proline-rich glycoprotein from human parotid saliva

A proline-rich glycoprotein (PRG) was isolated from human parotid saliva and examined by circular dichroism and fluorescence spectroscopy. Addition of guanidine hydrochloride to PRG labeled with an extrinsic dansyl probe had no effect on the fluorescence spectra's 511 nm lambda-max location. Thermodynamic calculations supported the contention that PRG has no significant tertiary structure. Circular dichroism results for PRG were simulated by computer and a secondary structure composed of 70% random coil and 30%β-form conformation was predicted. Circular dichroism of PRG failed to detect either poly-L-proline type I or II structures. Deglycosylation of PRG had no measurable effect on the circular dichroism spectrum, indicating that the carbohydrate side chains had little influence on PRG secondary structure. Based upon mathematical calculations, β-turns were predicted around three glycosylated Asn residues of PRG. These collective data suggest that PRG is composed of a disordered polypeptide chain with at least three of the N-linked Asn residues participating in some type of β-turn.     

Peptide synthesis on the new polyoxyethylene-polystyrene graft copolymer,synthesis of insulin B 21–30

An insoluble graft copolymer composed of the covalently bound polyoxyethylene to cross-linked polystyrene-divinylbenzene was employed as the solid support for peptide synthesis. The physical properties of this new polymeric support were similar to those of the cross-linked polystyrene. Thus, identical manipulations such as shaking and filtration could be performed on this resin. The graft copolymer was used for the synthesis of the C-terminal decapeptide of bovine insulin B-chain in the usual solid-phase manner. The protected peptide was cleaved from the polymeric support by photolysis and was purified by chromatography on silica gel and Sephadex LH-20. Samples of the decapeptide were treated with liquid HF and the unprotected peptide was purified by ion-exchange chromatography. The free peptide was shown to be homogeneous by HPLC, electrophoresis and t.l.c. The identity of this peptide was also confirmed by FAB-MS and amino acid analysis. The synthesized peptides were shown to be free of racemization. The free peptide possesses a disordered conformation as revealed by the CD measurement.