Stanley E. Charm

Shear effects on enzymes

Abstract Shear inactivation of enzymes has been found to be associated with flow, pumping and mixing. From data taken in a viscometer, shear inactivation in flow could be calculated. Among the materials experiencing this effect are catalase, rennet, carboxypeptidase A, interferon, heparin, fibrinogen, and phage lysozyme. Turbulent flow has greater losses than would be expected from streamline flow under the same dynamic and geometric conditions. Shear conditions also cause changes in the kinetics of reactions by reducing the reaction rate. It is thought that the shear field causes molecular distortions that result in breaking molecular bonds (inactivation) or stretching without breaking within an elastic limit that permits recovery when the shear field is removed (causing reduction of reaction rate.) There are conflicting data from different laboratories in that shear effects are not observed to the same extent. It may be that the presence of oxygen and metal ions and their reactions with disulphide bonds have an influence.

Shear degradation of fibrinogen in the circulation.

Plasma fibrinogen in vitro suffers a loss in clottability due to shearing. From calculation of the mass average shear to which plasma is subjected in the circulation, it is estimated that the half-life of fibrinogen is 4 days. This is in excellent agreement with reports that the half-life of fibrinogen in the circulatory system is 4 to 5 days. Hence shearing may be the mechanism for fibrinogen degradation in the circulatory system.

The separation and purification of enzymes through foaming

Abstract Mixtures of catalase and amylase are concentrated and purified through foaming. The relationship of the surface tension-concentration diagram to foam formation is examined. Various agents may be employed to control the foam separation process. A number of other enzymes subjected to foaming are found not to be inactivated. It is concluded that this process should be investigated further as a means of large-scale purification and concentration of enzymes.

A simple system for extending refrigerated, nonfrozen preservation of biological material using pressure.

Abstract High pressure (above 238 atmg) substantially extends the refrigerated storage of highly perishable biological material in a nonfrozen state. 5 . Calculated Equivalent Values of Pressure and Temperature for Reducing Reaction Rates of Horseradish Peroxidase Pressure (atmg) Temperature (°K) T T 0 a 0 296 0.999 272 289 0.975 340 287 0.969 408 285 0.963 a T 0 = 296 ° K It has been known for some time that high pressure stops microbial growth. The effect of high pressure is to reduce further the enzyme activity at refrigerated temperatures. Two enzymes studied, peroxidase and crude trypsin from red crab intestine, demonstrated this effect. A number of food materials such as fish, beef, and chicken were tested for microbial growth and organoleptic qualities after high-pressure storage in a simple 14-liter pressure chamber. Pressure was generated by a hand pump. The results indicated that after 30 days those items held in a non-frozen state at −3 °C and 238 atmg were not significantly different microbiologically and organoleptically from frozen controls at atmospheric pressure and −20 °C. This system should be useful for the preservation of biological materials where freezing or thawing effects are undesirable or unknown. The energy saved compared to freezing should also be considered. Only 62% of the energy is required for storage at −3 °C as compared with frozen storage at −20 °C, and about 28 cal/g must be removed in cooling to −3 °C as compared with 120 cal/g in cooling to −20 °C.

[37] Scale-Up of protein isolation☆

Publisher Summary This chapter presents operations that are applicable to the processing of a wide range of biological materials. The pilot plant isolation procedure includes preparation of tissue, tissue comminution and cell breakage, extraction, separation of solids, and fractionation of protein by solubility. It is possible to purify a mixture of proteins by exposure to heat. If extraneous protein denatures more rapidly than the desired protein, it precipitates, leaving the desired protein in solution. The precipitated protein is then separated by filtration or centrifugation. Solid–liquid chromatography is one of the most powerful tools available for the fractionation of proteins. Gels used for molecular-sieve chromatography are swollen at 60–80° to shorten the time required for hydration to a few hours. Resins and gels of the smaller particle sizes are normally avoided in large-scale use because flow rates of the resultant packed columns are too slow. For the same reason, resins are extensively freed of “fines” before use. Foaming is a purification technique for the separation of soluble proteins based on differences in their effect on surface activity. If an inert gas is bubbled through a solution, the most surface-active components collect at the gas–liquid interface.

Scaling up of elution gel filtration chromatography

Abstract Preliminary work indicates that both the ratio of column length to diameter and the Reynolds Number affect the quality of resolution in column elution. If these two dimensionless groups are held constant, the quality of the elution pattern will be the same in any column. It appears that the degree of resolution is a function of the velocity profile and dispersion in the column. Constant L D and Reynolds Number result in dynamic similarity and in similar velocity profiles and dispersion characteristics, irrespective of column diameter. Also, it appears that the lower the Reynolds Number and the greater the ratio of column length to diameter, the more effective is the purification.

Gel filtration with vibration

Abstract The fractionation of a protein mixture on a Sephadex G-100 gel was shown to be affected by vibrating the column under defined conditions. Vibrations perpendicular and parallel to the axis of the column, with one of them predominating over the other, could be produced with an air vibrometer attached to the column. The prevailing perpendicular type of vibrations improved the fractionation pattern of the high molecular weight components of a mixture. The parallel vibrations were shown to distort the fractionation at the same molecular weight level while decreasing the retention of the smaller molecules on the gel.

INFLUENCE OF ANAESTHETICS ON RHEOLOGY OF HUMAN BLOOD

SummaryCyclopropane, halothane, and methoxyflurane were investigated for their effects on the viscometry of freshly drawn human blood (group O, Rh+). A total of 43 blood and 63 plasma samples containing specific anaesthetic agents were studied under controlled conditions of temperature, pH, Pco2, Po2, and red blood cell and plasma protein concentrations. A series of three cone plate viscometers, each with different shear rate ranges from 1500 to 6.75 sec.-1, was used. The Casson viscosity of blood and apparent plasma viscosity were determined from the slope of the shear stress-shear rate relationship plotted on a square root scale. Yield stress was determined by extrapolation from the shear stress axis intercept. Our findings indicate that the three anaesthetic agents, within concentrations equivalent to those used during clinical anaesthesia, caused no discernible effect on either yield stress or the apparent viscosity of blood or plasma. However, there was a marginal increase of the yield stress of blood with high concentrations of cyclopropane (above 20 mg.%) and a trend toward reduced plasma viscosity in all of the methoxyflurane samples.RésuméNous avons étudié les effets du cyclopropane, de l’halothane et du méthoxyflurane sur la viscométrie du sang humain fraichement prélevé (groupe O, Rh positif). Nous avons fait nos études sur quarante-trois échantillons de sang et soixante-trois échantillons de plasma contenant spécifiquement ces agents anes-thésiques; l’étude a été faite aux mêmes conditions de température, de pH, de Pco2 et de concentration en protéines des globules rouges et du plasma. Nous avons fait des séries de trois viscomètres cone-plate, chacun possédant différentes échelles ”shear rate” de 1500 — 6.75 sec.-1. Nous avons précisé la viscosité Casson du sang et la viscosité apparente du plasma à partir de la courbe de relation ”shear stress-shear rate” placée sur une échelle de racine carrée. Le ”yield stress” a été obtenu par l’extrapolation à partir du croisement de Taxe du ”shear stress”. Nos résultats nous apprennent que, à des concentrations équivalentes à celles utilisées en anesthésie clinique, ces trois agents anesthésiques ne produisent aucun effet décelable ni sur le ”yield stress” ni sur la viscosité apparente du sang ou du plasma. Toutefois, avec une haute concentration sanguine de cyclopropane (au-dessus de 20 mg.%), nous avons observé une faible augmentation du ”yield stress” du sang; de plus, dans tous les échantillons avec le méthoxyflurane, nous avons observé une tendance à la diminution de la viscosité du plasma.

Influence of anesthetic agents upon plasma electrophoretic mobility and surface tension as related to blood viscosity.

Blood viscosity, at constant temperature, is a function of three main groups of factors: (1) the size, shape, number and hemoglobin concentration of the red blood cells; (2) concentration of and molecular size of the various plasma proteins; and (3) interactions between these two groups of cells which appear to be related at least partly to the cellular interfacial tension and to cell charges (figure).2

Application of Immunoadsorbents for Isolation of Placental Alkaline Phosphatase, Carboxypeptidase G-1, and Serum Hepatitis Antigen

Abstract The extraordinary specificity of antigen-antibody reactions forms the basis of a simplified isolation method for antigens. The use of antigens to isolate antibodies has been employed by immunologists for many years [1, 2]. The early use of imnunoadsorbents to isolate antibodies was not always successful because the physical adsorption used to fix the antigen permitted “leaking”. However, with the various methods available today for covalently bonding to solid supports, this is no longer a serious problem.