Changes in apparent pH occurring during fast freezing of aqueous buffer solutions and cooling to -196 degrees C were studied by various semiquantitative methods, including simple visual measurements of colour changes with pH indicators, as well as measurements of pH-dependent changes in the e.p.r. (electron paramagnetic resonance) spectra of solutions of three different metalloenzymes. It is concluded that apparent pH changes of up to about 3pH units may occur under particular conditions. Such changes were independent of the time taken to freeze the samples, when this was varied from about 3ms t0 20s, but were affected by the presence of some proteins in solution. Recommendations on the buffers that should be used to avoid such apparent pH changes in e.p.r. spectroscopy and other low-temperature biochemical work are made. Phosphate and pyrophosphate buffers, which gave large decreases (2-3 pH units), and Tris, which under some conditions gave increases of about the same magnitude, are to be avoided. Certain zwitterionic buffers such as Bicine [NN-bis-(2-hydroxyethyl)glycine] are satisfactory. Apparent pH effects were found to depend on buffer and protein concentration. It is therefore recommended that as a prelude to future detailed low-temperature biochemical work, appropriate tests with an indicator system should be performed.
The nuclear enzyme poly(ADP-ribose) polymerase has been purified about 9200-fold from pig thymus nuclei with a 46% yield. An aqueous organic solvent system was used for the isolation of the polymerase from nuclei and for its purification by chromatography at sub-zero temperatures. Electrophoretic analysis under both denaturing and non-denaturing conditions revealed a single protein band suggesting that the preparation was homogeneous and that the enzyme is composed of one polypeptide chain. The molecular weight estimated from sodium dodecyl sulphate-/polyacrylamide gel electrophoresis was 63 500 and from gel filtration through columns of Sephadex G-100, 58 000. The enzyme preparation was free from poly(ADP-ribose)-degrading enzymes and from DNA. The purified polymerase showed an absolute requirement for both DNA and histones. The maximal specific activity of the homogeneous preparation measured by the standardized assay, was 20.7 pmol NAD* incorporated x min-' x mg-' of protein at 37 "C. Amino-terminal group analysis with dansyl chloride did not reveal a terminal amino acid suggesting that the amino-terminal group may be blocked. In the presence of histones, the K,,, for NAD' was 23 pM.
Difficulty may arise in distinguishing between ping-pong and sequential enzyme mechanisms, when using double-reciprocal plots of kinetic data obtained at several fixed values of the concentration of the second substrate; this is because the parallel lines diagnostic of the first case may actually be slowly converging as required by the second. An alternative approach is to choose substrate concentrations so that the second substrate concentration occurs in a number of fixed ratios to the first. A ping-pong mechanism predicts a set of straight lines and a sequential mechanism a set of curved lines (parabolas) in double-reciprocal plots. The method is illustrated by its application to bovinemilk lactose synthetase which requires UDP-galactose and N-acetyl-glucosamine as substrates in a sequential mechanism.In enzyme systems where there are two or more substrates, steady-state kinetic data plotted as the reciprocal of the initial velocity against the reciprocal of the concentration of one substrate (a doublereciprocal plot), repeated at several fixed concentrations of a second substrate, will in many cases show one of two basic patterns, either a set of parallel lines or a set of intersecting straight lines. The parallel set is indicative of a ping-pong mechanism and the intersecting set of a sequential mechanism [l].However, occasions arise where it is experimentally difficult to distinguish in double-reciprocal plots between a set of parallel lines and a set of lines which, though intersecting, is converging very slowly in the region of experimental accessibility. Examples of measurements on enzymes with sequential mechanisms, where the data appear consistent with a pingpong mechanism, include pig kidney D-amino acid oxidase [2] and bovine brain hexokinase [3,4].Lactose synthetase from human milk has also shown this phenomenon when the variation of rate with the concentrations of two of the substrates, UDP-galactose and N-acetyl-glucosamine was examined [ 5 ] . In this case the part of the mechanism involving these two substrates was shown to be sequential by using the stratagem of Koster and Veeger [2] which consists of repeating the experiment in the presence of a fixed concentration of an inhibitor (UDP-glucose) that competes with the earlier-adding of the two substrates (UDP-galactose).The purpose of the present report is to describe an alternative method of distinguishing between ping-pong and sequential mechanisms by carrying out experiments in which the concentrations of the two substrates being investigated are varied in a constant ratio, at several different values of the ratio. The practical problem becomes one of deciding whether the lines of a set are straight or curved (parabolic).The method is illustrated by its application to the galactosyl transferase of bovine milk. This enzyme is one of the two protein components of lactose synthetase [6] and catalyses the synthesis of N-acetyllactosamine (LacNAc) :The second component is the milk protein, a-lactalbumin [7], which modifies the monosaccharide-bin...
Xanthine oxidase is stable and active in aqueous dimethyl sulphoxide solutions of up to at least 57% (w/w). Simple techniques are described for mixing the enzyme in this solvent at--82 degrees C, with its substrate, xanthine. When working at high pH values under such conditions, no reaction occurred, as judged by the absence of e.p.r. signals. On warming to--60 degrees C, for 10 min, however, the Very Rapid molybdenum(V) e.p.r. signal was obtained. This signal did not change on decreasing the pH, while maintaining the sample in liquid nitrate reductase, caused its molybdenum(V) e.p.r. signal to change from the high-pH to the low-pH form. These findings are not compatible with the conclusions of Edmondson, Ballou, Van Heuvelen, Palmer & Massey [J. Biol. Chem. (1973) 248, 6135-6144], that the Very Rapid signal is in prototropic equilibrium with the Rapid signal, and should be important in understanding the mechanism of action of the enzyme. They emphasize the unique nature of the intermediate represented by the Very Rapid e.p.r. signal. The possible value of the pK for loss of an exchangeable proton from the Rapid signal is discussed.
The role of Mn2+ in the reaction catalysed by the galactosyl transferase of bovine milk and colostrum was studied by steady-state kinetic methods at pH 7.4 and 37 "C. The association constants for the binding of Mn2+ by UDP-galactose and by the buffer species used (3-[N-morpholino]propanesulphonate) were measured by electron spin resonance spectroscopy and the concentrations of all the species present in the substrate mixture of Mn2+, UDP-galactose and N-acetylglucosamine were calculated. Previous evidence indicated that high Mn2 + concentrations activate the reaction beyond what is expected from the Michaelis-Menten equation. At such levels (up to 40 mM), the concentration of free Mn2+ was found to be proportional to total Mn within 3%. Experimental data produced as a function of total Mn concentration and total UDP-galactose (i.e. free UDP-galactose plus MnUDP-galactose) concentration could be interpreted in terms of rate equations written in relatively simple form with free Mn2+ and total UDP-galactose concentrations as variables, corresponding to a large variety of kinetic models involving Mn2 +, UDP-galactose and MnUDP-galactose as substrates. A group of related mechanisms could not be eliminated at this stage and experiments were carried out in which the concentrations of two of the substrates were varied at either a constant ratio or a constant product depending on how they were involved in the Mn2+ : UDP-galactose association reaction. A choice between the two final mechanisms was made by applying statistical non-linear least-squares methods. The most likely mechanism was one in which, after the addition of Mn2+ to the enzyme, the substrates UDP-galactose and MnUDPgalactose added as alternatives to produce two branches, in each of which was added N-acetylglucosamine followed by the release of N-acetyllactosamine, then the Mn2 + complex of UDP, and finally (in the branch in which MnUDP-galactose had been the substrate) free Mn2+.
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