Enzyme Fractionation by Salting Out

Dll. Dixon, M., and Webb, E. C., Enzyme fractionation by salting-out A theoretical note. Advan. Protein Chem. 16, 197-219 (1961). 

For many years salting-out by high concentrations of ammoniiun sulfate has been one of the classical methods of protein separation. There is very little literature on the theoretical basis of the method, particularly as applied to the isolation of enzymes, where it has mainly been used quite empirically. The underlying assumption in most cases seems to have been that the different proteins are precipitated at different fixed ammonium sulfate concentrations, provided the pH and temperature are fixed. For example one may commonly read in instructions for the piuification of an enzyme that the enzyme is precipitated at 65% saturation with ammonium sulfate or that the fraction precipitating between 0.62 and 0.68 saturation should be taken. It is, however, a fairly common experience that when one repeats a published method the enzyme fails to precipitate within the limits given. Furthermore, where the purification of a protein involves more than one salt-fractionation stage, the limits are usually found to be different for the different stages. 

Preparation of Enzyme. Separation and purification of the enzyme from the liver of the common squid were as follows Squid livers were mashed and dispersed in five volumes of distilled water, then acidified to pH 4.0. Much oil was eliminated. This was followed by ultrafiltration, salting out with ammonium sulfate (50% saturation), dialyzing and freeze-drying in vacuo, to yield a crude enzyme. A purified enzyme was obtained from this crude enzyme by using column chromatography. Active fractions were separated by cation exchange resin. Mono S (Pharmacia) and further purified by gel-flltration column of Superdex 75 (Pharmacia). The active fractions were collected as the purified enzyme. 

This conception works out as shown in Scheme 10 CTP 23 formed by the above described sequence is directly consumed by -acetyl neuraminic acid 26 under the catalytic influence of cytidine-5 -monophosphosialate synthase (E.C. 2.7.7.43). This enzyme is isolated from calf brain by ammonium sulfate precipitation (2 5) and subsequent affinity chromatography. The stationary phase consists of CNBr-activated Sepharose 4B reacted with p-[3-(2-amino ethylthio)propyl]-iV-acetyl neuraminic acid 27, which is synthesized by radiating a mixture of the allyl glycoside and cysteamine to achieve radical C-S bond formation (24), The behavior of methyl p-N-acetyl-neuraminic acid as an inhibitor is in accordance with Zbiral s findings (25), where the methyl a-glycoside has been shown to compete with the native substrate for the enzyme, and thus 27 is recommended to be an ideally suited ligand (Scheme 9). A typical analytical run is shown in Scheme 9. Due to elution of the protein fraction by a salt gradient, the transfer to a preparative scale is rather difEcult denaturation occurs and thus a drop in activity down to 6% is observed. 

Of the two fractions into which the phenylalanine hydroxylase is separable, I is salted out between 0-40% (NH4)2S04 saturation and II, at 40-80 % saturation. Rat liver is rich in I and it has been purified from this source about seventy- to a hundredfold by Kaufman and Levenberg 188, 189). Fraction II is rich in rabbit liver which, conversely, is low in I. This enzyme has also been purified to about the same degree as I by variations of the procedures used in the purification of enzyme I. 

Nucleoids are nuclei-like structures produced from cells lysed with nonionic detergents in the presence of high salt concentrations. By this treatment, histones and most of the nonhistone proteins are removed [3], They contain naked, histone-free DNA, RNA, and a few proteins [1]. DNA is supercoiled and attached to a cage of residual proteins and RNA [5]. This relationship continues during replication and transcription and seems to be sequence specific [5]. Poly(ADP-ribose) is involved in the regulation of the activities of various enzymes, as well as structural proteins, and seems to be involved in the control of DNA and RNA synthesis [9,11]. It was interesting for us to examine whether ADP-ribosylated proteins remained after generation of nucleoids. We foimd out that one of the residual protein fractions (possibly bound to DNA) was ADP-ribosylated. 

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