Elemer Mihalyi

Studies on the tryptic digestion of myosin

Abstract The digestion of myosin by trypsin has been followed by measuring: (1) the uptake of base in the pH-stat, (2) changes in viscosity, (3) non-protein N formation, (4) changes in sedimentation properties and (5) changes in optical rotation. The pH-stat studies point to the existence of 2 parallel first-order reaction classes with markedly different velocity constants. Approximately 64 peptide bonds are cleaved in the fast reaction and 240 peptide bonds in slow reaction. The kinetic data obtained by means of the different physicochemical methods are consistent with a model of myosin in which a small number polypeptide chains are arranged parallel to one another throughout the lenght of the myosin rod. Some segments of this rod structure are highly ordered and some are more randomly disposed. The enzyme attacks the random regions at a much higher rate than the folded areas. Thus the early appearance of large intermediates in proteolysis would appear to be a reflection of the difference in the rates of attack of these different segments of the molecule, and does not imply the pre-existence of any sort of subunit structure.

Digestion of fibrinogen by trypsin: I. Kinetic studies of the reaction

Abstract The digestion of fibrinogen by trypsin, as followed in the pH-stat, can be described in terms of three distinct reaction classes: (a) A fast reaction involving the splitting of 12 peptide bonds during which clottability is lost, (b) a slower reaction involving 80 peptide bonds, associated with the appearance of a slower sedimenting component in the ultracentrifuge and indicating the production of large fragments, and (c) a slow reaction which covers the remaining bonds susceptible to trypsin and which results in the fragmentation of the molecule into small peptides. The digestion was also followed in the viscosimeter, ultracentrifuge and polarimeter, and the liberation of non-protein nitrogen measured. The disappearance of clotting ability associated with the first phases of the reaction was studied, and certain conclusions were drawn concerning the mechanism of this inactivation process.

Physicochemical studies of bovine fibrinogen III. Optical rotation of the native and denatured molecule

Abstract The optical rotation and the rotatory dispersion of fibrinogen have been determined in the native and in various denatured states. 1. 1. The stability of the molecule, revealed by changes of [α]D, has been examined as a function of pH, urea concentration, guanidine · HCl concentration, and cleavage of the disulfide bonds. The pH range for stability of fibrinogen is narrow (pH 5.5–10) and there is an apparent correlation between [α]d and net charge. 2. 2. The dispersion studies showed coherent variations of [α]D, λc and b0 and consistent values of the helical content could be calculated from these parameters. Native fibrinogen appears to have a helical content of 33%, which is reduced to approx. 20% by acid, alkali, and 5 M urea denaturation, and disappears completely at 5 M guanidine · HCl, or 10 M urea concentration. 3. 3. The molecule in the denatured state shows reversible changes in rotatory properties within the concentration range of the denaturing agent where aggregation does not occur.

Digestion of fibrinogen by trypsin. II. Characterization of the large fragment obtained.

Abstract The large fragment of the fibrinogen molecule produced by short tryptic digestion has been isolated in a homogeneous state. The physico-chemical parameters of the fragment were determined: s = 5.22, D = 5.12 · 10 −7 , β − = 0.735 . From these values and from the results of approach to sedimentation-equilibrium studies an average molecular weight of 95 000 was calculated. The complete amino acid and hexose content of the fragment was also determined. The relationship of the fragments to the parent fibrinogen molecule are discussed.

Physicochemical studies of bovine fibrinogen I. Molecular weight and hydrodynamic properties of fibrinogen and fibrinogen cleaved by sulfite in 5 M guanidine · HCl solution

The molecular weight, sedimentation behavior and intrinsic viscosity of native fibrinogen, fibrinogen denatured in 5 M guanidine · HCl, and sulfite-treated fibrinogen in 5 M guanidine · HCl were studied. The molecular weight remains unchanged in 5 M guanidine · HCl, but decreases to approx. one-sixth of its original value after cleavage of the disulfide bonds. Both fibrinogen and fibrinogen cleaved by sulfite show one single component in the sedimentation patterns. The s20,w0 values were 6.86 and 2.44 S for fibrinogen and cleaved fibrinogen in 5 M guanidine · HCl, and the corresponding intrinsic viscosities were 0.51 and 0.35. From the data, it follows that in guanidine · HCl the effective volume of fibrinogen is approximately doubled, without any appreciable change of the molecular asymmetry, while the smaller fragments resulting from the cleavage by sulfite may have a more compact conformation than that of a random coil.

THE AMINO ACID COMPOSITION OF BOVINE FIBRINOGEN.

Abstract The amino acid composition of highly purified bovine fibrinogen has been determined with the automatic amino acid analyzer of Spackman, Stein, and Moore. Tyrosine, tryptophan, amide, and hexose were also determined by independent methods. The total nitrogen content of the protein has been estimated. The refractive index and partial specific volume have been calculated from the composition of the molecule and compared with experimental values.

Physicochemical studies of bovine fibrinogen II. Depolarization of fluorescence studies

Abstract 1. 1. Depolarization of fluorescence measurements of fibrinogen labeled with dimethylaminonaphthalene sulfonate yielded a rotational relaxation time of 195 nsec, lower than the value calculated for an unhydrated sphere of a volume equal to that of the fibrinogen molecule. The tryptic fragment of fibrinogen, by the same method, gave a relaxation time of 178 nsec, corresponding to the molecular shape and size of the fragment. The conclusion is drawn that the molecular segments which yield the fragments have rotational freedom within the native molecule. 2. 2. The rotational relaxation time of fibrinogen was determined as a function of pH and urea, or guanidine · HCl concentration. The stability range of the molecule, judged from these results, is very similar to that found by optical rotation measurements. Above pH 10, and at concentrations of urea higher than 3 M, or of guanidine · HCl higher than 1 M, at neutral pH, ϱh decreases markedly, indicating the loosening up of the molecule. At lower urea, or guanidine · HCl concentrations, ϱh increases slightly, indicating aggregation of the denatured fibrinogen molecules. This phenomenon was demonstrated also in sedimentation experiments.

Nucleic acid and nucleotide content of myosin preparations

Abstract Myosin preparations contain 0.5–0.8% of ribonucleic acid, which cannot be separated from the protein by the usual purification procedures, but can be extracted with hot 10 % NaCl solution and also removed partly after digestion with ribonuclease. The physicochemical and enzymic properties of myosin are unaffected by the depolymerization of the nucleic acid by ribonuclease. Hot TCA extracts from myosin contained the hydrolysis products of the nucleic acid, and an extra amount of adenine. The amount of the extra adenine is small in myosin A, but amounts to 0.1–0.4 group/105 g. protein in myosin B preparations. After extraction of the nucleic acid, the residual phosphorus amounts to 0.5–0.8 mole/105 g. protein.

KINETICS AND MOLECULAR MECHANISM OF THE PROTEOLYTIC FRAGMENTATION OF FIBRINOGEN

The events of the fragmentation of fibrinogen by proteolytic enzymes have to be related to the primary process, that is, the cleavage of peptide bonds. This gives a frame of reference independent of the kinetic variables of the digestion. Various aspects of this facet of the reaction will be discussed in the first section of this paper. The discussion of the fragmentation of the molecule will follow, with the second section dealing with the large fragments that have a compact, protein-like secondary structure, and the last section with the peptide fragments of various sizes.

Estimation of stray-light and fluorescence effects in differential spectroscopy

Abstract A simple method is described to ascertain the effects of stray-light and fluorescence in differential spectroscopy.