CONTRIBUTION TO THE PATHOGENESIS OF HEMOPHILIA

The causes of the delayed coagulation of hemophilic blood seem to become cleareras time advances. On one side we have the investigations of the school of Minot,indicating a deficit in the globulin fraction; and on the other side are the works ofthose who maintain that there exists an excess of anticoagulant substances. Wesupport the latter theory, although in our opinion the two theories do not contradict each other, since it might be possible that this anticoagulant substancewould act on the globulin fraction diminishing its coagulant power. This substancecould be identified with the anticephalin fraction of Tocantins.

With the idea that this substance might be less stable than the coagulant fractions (fibrinogen, prothrombin, and thromboplastin) we have tried to render itinactive by keeping the blood in a refrigerator for a certain length of time. Inactivity was obtained in some of our experiments. We know that the stability ofthis substance varies from one patient to the other, but we have not been able tofix the cause of these variations.

In conclusion, we consider that other means of neutralizing the action of thisanticoagulant substance should be investigated. This inactivity once obtained, weshould have advanced far in solving the intricate problem of hemophilia.

     

APPLICATION OF MASS SPECTROMETERY TO THE SEQUENCING OF PROTEINS

Mass spectrometry is now established as a viable method for determing the sequence of amino acids in proteins. The approach involves chemical and enzymic digestion of the protein (as in classical methods) to small peptides (ten residues) which are then chemically modified to increase their volatility. The chemical modifications are (i) acetylation of basic nitrogen sites and (ii) Permethylation. The derivatives so obtained may be sequenced in the mass spectrometer as mixtures of several peptides. This is achieved by a temperature gradient in the mass spectrometer source, which effects partial, or total separation of the derivatized peptides according to their volatilities. In this way, much tedious separation work (sometimes the rate-determining factor in classical approaches) may be avoided. The sequencing process is carried out from the N-terminal residue, utilizing the preferential cleavage of the ionized petide at the amide bonds; each amino acid is associated with a characteristic mass increment and thus the sequence can be determined. Mass spectrometry is especially useful in the detection and characterization of novel amino acids which might otherwise be difficult to identify. Although almost all work to date has used electron impact mass spectrometery, in the future it is possible that newer methods of ionization, such as field desorption and chemical ionization, might be profitably used.     

APPLICATION OF MASS SPECTROMETERY TO THE SEQUENCING OF PROTEINS

Mass spectrometry is now established as a viable method for determing the sequence of amino acids in proteins. The approach involves chemical and enzymic digestion of the protein (as in classical methods) to small peptides (ten residues) which are then chemically modified to increase their volatility. The chemical modifications are (i) acetylation of basic nitrogen sites and (ii) Permethylation. The derivatives so obtained may be sequenced in the mass spectrometer as mixtures of several peptides. This is achieved by a temperature gradient in the mass spectrometer source, which effects partial, or total separation of the derivatized peptides according to their volatilities. In this way, much tedious separation work (sometimes the rate-determining factor in classical approaches) may be avoided. The sequencing process is carried out from the N-terminal residue, utilizing the preferential cleavage of the ionized petide at the amide bonds; each amino acid is associated with a characteristic mass increment and thus the sequence can be determined. Mass spectrometry is especially useful in the detection and characterization of novel amino acids which might otherwise be difficult to identify. Although almost all work to date has used electron impact mass spectrometery, in the future it is possible that newer methods of ionization, such as field desorption and chemical ionization, might be profitably used.