Carlos A. Bonilla

Defibrinating enzyme from timber rattlesnake (Crotalus H. Horridus) venom: A potential agent for therapeutic defibrination I. Purification and properties

Abstract A defibrinating (thrombin-like) enzyme, isolated from timber rattle-snake ( C. h. horridus ) venom by gel filtration, ion exchange and adsorption chromatography was found to be homogeneous by (i) analytical ultracentrifugation, (ii) electrophoresis in highly cross-linked (12.8%), acidic, 6M urea polyacrylamide gels. The molecular weight was 19,610 based on amino acid composition, 19,500 by sedimentation velocity and 19,500 by sedimentation equilibrium. The enzyme is specific for the fibrinogen-fibrin conversion and does not affect other blood clotting factors; it exhibits an unusually high stability to acid pH and high temperature and it is partially inhibited by heparin only in very high concentrations. The ease of isolation of this enzyme and its apparent lack of side effects “in vivo” warrant further investigation into its mechanism of action and its potential use as an agent for therapeutic defibrination.

Comparative biochemistry and pharmacology of salivary gland secretions : II. Chromatographic separation of the basic proteins from some North American rattlesnake venoms

Abstract The highly basic proteins from three North American rattlesnake venoms have been isolated by a simple combination of Bio-Gel P-2 recycling adsorption chromatography and ion exchange on carboxymethyl-cellulose. These basic proteins constitute less than 10% of the total protein in the crude venoms and are characterized by having small molecular weights, isoelectric points above ≈ pH 10.8 and pharmacological activities ( in vivo and in vitro ) resembling those of crotamine, the neurotoxin from Crotalus durrisus terrificus, the South American tropical rattlesnake.

Comparative biochemistry and pharmacology of salivary gland secretions. III. Chromatographic isolation of a myocardial depressor protein (MDP) from the venom of Crotalus atrox.

A protein has been isolated from the venom of the western diamondback rattlesnake (Crotalus atrox) which induces acute myocardial depression when administered to experimental animals. Purification was achieved by gel filtration on Sephadex G-100, DEAE- and CM-cellulose ion-exchange chromatography, ultra-filtration, and adsorption chromatography on hydroxyapatite. Amino acid analysis of the highly purified protein indicated N-terminal isoleucine and C-terminal tyrosine residues, and the absence of free sulfhydryl groups. Rabbits were immunized against the myocardial depressor protein (MDP) and a highly specific antiserum prepared which made it possible to study other snake venoms for the presence or absence of MDP. All of the North American Crotalid species of snakes contain MDP in varying degrees of concentration, but none of the Asiatic snake venoms tested reacted with the antiserum to the myocardial depressor protein. Intravenous administration of MDP to experimental animals (dogs, cats) produces an immediate and profound decrease in the cardiac output, the left ventricular systolic and mean pressures, the velocity of shortening of the contractile element, the systemic arterial pressure and an elevation in the left ventricular end-diastolic and pulmonary wedge pressures. These hemodynamic changes indicate that MDP administration induces an acute myocardial failure which is does dependent. The potential use of this protein for the reproducible causation of left ventricular failure, obviating the need for the more commonly used surgical ligation of the coronary arteries, warrants a full investigation into its structure, active site and its mechanism of action on the myocardial cell.

Human Mixed Saliva Protein Concentration

The concentration of protein in human mixed saliva normally is about 100 to 300 mg%, and these values are not modified significantly by stimulation (H. E. SCHUJLTZE and J. F. HEREMANS, Molecular Biology of Human Proteins, vol I, 1966, pp 762-772). Because of inherent contamination of mixed saliva by bacteria and food debris normally present in the oral cavity, it is advantageous to study isolated secretions from individual salivary glands. Because of the rapid formation of mucin clot, which tends to render most of the existing chromatographic procedures useless, mixed saliva is difficult to fractionate; consequently, most of our knowledge of its isolated protein components has been derived from selectively collected parotid or submandibular fluids. While attempting to find physiologic correlations between the oral cavity and systemic disease, however, we have thought it more appropriate to use (in spite of its drawbacks) the mixed salivary fluid. This study reports the protein concentration in mixed saliva of a normal male studied over a period of two years and is part of an investigation of the isolation, characterization, and physiologic significance of proteins in human mixed saliva. Saliva was collected without stimulation once every two weeks (beginning October 1968) by expectoration into a graduated cooled vessel (4 C) that contained thymol crystals to inhibit bacterial growth. To avoid excessive variation caused by diurnal fluctuations, samples were obtained in the evening (8:30 to 10:00 PM), two hours after the evening meal, after normal dental hygiene. Before sample collection the oral cavity was rinsed thoroughly with distilled water. Immediately after collection samples were cleared by centrifugation at 27,000 X g for 30 minutes in a refrigerated centrifuge* and the supernatant was analyzed for protein with a spectrophotometer. t In some instances, for comparative purposes, the cleared saliva was dialyzed against deionized, glass-distilled water at 4 C for 18 hours before protein determination. No attempt was made to measure the flow rate, but in general the volume of saliva collected was kept fairly constant: mean, 125.5 ml; range, 60 to 210 ml. Forty-five samples (of which 12 were di-

Serum enzyme activities following administration of purified basic proteins from rattlesnake venoms

Abstract The effects of two small-molecular-weight basic protein toxins (SBPT) isolated from C. adamanteus (Eastern diamondback rattler) and C. h. atricaudatus (Canebrake rattler) on serum enzyme levels in mice were studied. For comparative purposes, a third, larger protein, the anticoagulation fraction from C. h. atricaudatus venom (fraction CMC-I), was also tested. Single, sublethal injections of both SBPT elicited dose-dependent rises in glutamic oxalacetic transaminase (SGOT), hydroxybutyric dehydrogenase (α-HBD) and aldolase (ALD). No changes in the level of alkaline phosphatase (AP), packed red cell volume (HTC, hematocrit) or total serum protein (TP) were, however, observed. The onset and duration of the elevated serum enzyme levels induced by the two toxins was consistent with changes seen following administration of the β-adrenergic stimulating agent, isoproterenol, to rats. The results support the hypothesis that the isolated SBPT from rattlesnake venoms exert their lethal action by producing minimal to massive myocardial infarction in animals depending on the dose employed.

Hypotensin — a hypotensive peptide isolated from Crotalus atrox venom: Purification, amino acid composition and terminal amino acid residues

Arterial hypotension is one of the most common clinical findings following severe bites by the North American rattlesnakes [I] . As has been indicated by Russell et al. [2] the immediate and profound decrease in systemic arterial pressure can not be attributed to direct cardiac effects since neither the heart rate nor the force of contraction are directly altered by intravenous administration of Crotalus venoms to a wide variety of experimental animals. Indirect evidence for the potential role of bradykinin in snake venom-induced hypotension has been reported by Russell [3] who showed decreased bradykininogen levels in three patients following rattlesnake bites. Only one of the patients, however, was reported to be in shock when reporting to the hospital while the other two were normotensive and/or slightly hypotensive. It is obvious, then, that because of the great complexity of the venom secretion from the rattlesnakes little is known about the specific components which may induce the profound fall in blood pressure following envenomation. In this paper the isolation of a potent hypotensive peptide* (heretofore called hypotensin) from the venom of the Western diamondback rattlesnake (CFotalus &0x), its amino composition, terminal residues and some of its biological properties are reported.

Fatty acid and prostaglandin composition of left ventricular myocardium from dexamethasone-treated dogs with severe low output syndrome (LOS)

The effects of dexamethasone (DEX) administration on the left ventricular myocardial content of fatty acids and prostaglandins E1, E2 and F2alpha were studied. Following a complete right and left cardiac catheterization, either DEX (8 mg/kg) or an equivalent volume of its vehicle was given intravenously 30 minutes prior to low output syndrome (LOS induction, and supplemental doses of DEX (4 mg/kg) or vehicle administered at 15 and 75 minutes post-LOS induction. Low output syndrome was induced by intravenous administration of a myocardial depressor protein (MDP) which has been isolated from the venom of the Western diamondback rattlesnake, Crotalus atrox. Neither DEX nor its vehicle had a significant effect during the entire experiment, that is, in the normal or low cardiac output state in most of the hemodynamic parameters investigated. The three hour mortality rate for the DEX-treated animals was 22% (n=10) while that of the control group was 41% (n=26) indicating that the beneficial effects of this corticosteroid are not really apparent from hemodynamic evaluation alone. Since DEX only had a significant post-LOS induction effect in maintaining a lower left ventricular end-diastolic and pulmonary capillary wedge pressures, a higher arterio-venous oxygen saturation difference, and a more efficient contractile state of myocardial fibers (Vmax), an indirect correlation to coronary arterial blood flow at the subcellular level was sought. To this effect, prostaglandins and specific lipid classes of left ventricular myocardium (LVM) from control and LOS animals receiving either vehicle or DEX were analyzed. Low output state induction alone raised myocardial PG levels above those of sham-catheterized animals; on the other hand, dexamethasone induced a significant decrease in the three prostaglandins studied when administered to control (no LOS) animals. In the presence of LOS, however, dexamethasone overrode in part the increase in PGE1 and PGE2 brought about by LOS while in the case of PGF2alpha the LOS effect was totally prevented and its concentration was not significantly higher than in control animals receiving dexamathasone. LOS induction led to an increase in myristic and arachidonic acids and a decrease in palmitic and linolenic acids. Dexamethasone administration to control animals increased the concentration of stearic acid above all the other groups but decreased the concentration of linolenic acid when compared to DEX-treated animals with LOS or sham-catheterized animals. There were no significant differences in the total myocardial lipid among the four groups of animals studied. It is suggested that the potentially beneficial effects of corticosteroid administration to animals with low output syndrome are related to their effects on fatty acid and prostaglandin content of myocardium.

Purification and partial characterization of hypocalcemic factor from human saliva.

Abstract This study reports the isolation and partial purification of a polypeptide from human saliva which causes a significant serum calcium lowering when administered to mice. Purification was achieved by preparative electrophoresis, dialysis, two gel filtration steps on Sephadex G-150, and ion exchange chromatography on DEAE-cellulose. Homogeneity was determined by poly-acrylamide electrophoresis. Blood sampling was carried out by puncture of the orbital venous plexus and serum analyzed for calcium. The most active preparations lower serum calcium from 10–27% of initial value, producing tetany and convulsions in some cases. The molecular weight of this polypeptide was estimated to be 4, 260 by the use of a calibrated Sephadex G-75 column. This is a much smaller molecular weight than that expected from its initial exclusion from Sephadex G-150, and suggests that this hypocalcemic factor is associated with larger molecules through most of the purification procedure up to and including DEAE-cellulose chr...

Effect of submandibular gland sialoadenectomy and excretory duct ligation on plasma calcium levels in mice.

Although the changes in gland weight, structure, and nerve growth induced by submandibular gland duct ligation in rats and mice have been well established (A. TAMARIN, Secretory Mechanisms of Salivary Glands, 1967, pp 220237; L. C. JUNQUETRA, Exp Cell Res 2:327-338, 1951; P. WEIs and E. D. BUEKER, Proc Soc Exp Biol Med 121:1135-1140, 1966), little is known about systemic effects associated with ligation of the duct or removal of the gland. It has been reported (A. W. WASE and Y. S. L. FENG, Nature 178:1229-1230, 1956) that calcium 45 incorporation into bone and most soft tissues (except thymus and lung) is greater in sialoadenectomized mice than intact mice. Results of strontium 89 uptake by bone led the same authors to suggest that gland removal may induce a mineral depletion, probably as a result of interference with calcium absorption from the digestive tract. A significant increase in the serum calcium response to parathyroid extract in sialoadenectomized rats has also been reported (L. KRAINTZ, Ann NY Acad Sci 85:310-312, 1960). The effects of submandibular gland duct ligation and removal on the plasma calcium levels of mice are the subject of this report. Adult (105 to 115 days old) Swiss albino mice, weighing 30 + 2 gm each, from a highly inbred strain were separated at random into two experimental groups: (1) sialoadenectomized and (2) ligated. Each group was further separated into two subgroups, male and female. After obtaining control (zero time) calcium levels the mice were anaesthetized by intraperitoneal injections of pentobarbital sodium. A midline incision was made through the skin and fascia of the ventral surface of the neck, the excretory ducts and main blood vessels were tied off, and the submandibular-sublingual salivary complex was dissected free from its supporting connective tissue and removed in group 1. In group 2, the submandibular ducts were ligated with no. 4/0 silk sutures near their entrance into the mylohyoid muscle. All incisions were closed with interrupted no. 3/0 gut suture. Diet consisted of lab chow* and tap water ad libitum. Plasma calcium levels were determined again at 7, 15, and 30 days using the glyoxal bis 2-hydroxyanil (GBHA)