By running the individual purified thymus histone fractions it has been shown that the lysine-rich histones F1 and F2B are stained red whereas the arginine-rich ...
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The gels were de-stained in 40ml of 40% (v/v) ethanol at 55°C until the background colour was removed (approximately 30h). During this time, the de-staining solution was changed four times. By running the individual purified thymus histone fractions it has been shown that the lysine-rich histones F1 and F2B are stained red whereas the arginine-rich ones F3 and F2A1 stain blue. The intermediate fraction, F2A2, containing approximately equimolar proportions of lysine and arginine also stains blue. Records are made by colour photography on Kodak Ektachrome X film. The best differential staining was achieved with a mixture of Alizarine Black and Ponceau S in the ratio 40: 1. However, satisfactory results could be obtained throughout the range 35: 1 to 45:l. Outside these limits, the staining was no longer specific. Studies on a number of other proteins indicate that the basis for the differential staining is the ratio of lysine to arginine. Proteins containing both of these basic amino acids in appreciable quantities (e.g. the avian erythrocyte-specific histone F2C) appear to bind both stains. This method should be of considerable use when analysing the histones isolated from relatively obscure sources, as preliminary information regarding the amino acid composition of the individual fractions may be obtained before preparative fractionation procedures have been developed to permit normal amino acid analyses. This work was supported by grants to the Chester Beatty Research Institute (Institute of Cancer Research: Royal Cancer Hospital) from the Medical Research Council and the Cancer Campaign for Research. Dick, C. &Johns, E. W. (1969) Biochim. Biophys. Acta 174, 380-386 Johns, E. W. (1964) Biochem. J. 92, 55-59 Johns, E. W. (1967~)Biochem. J. 104,78-82 Johns, E. W. (19676) Biochem. J. 105, 611-614 Neville, D. M., Jr. (1971) J . Biol. Chem. 246, 6328-6334 Panyim, S., Bilek, D. & Chalkley, R. (1971) J. Bio/. Chem. 246,420&4215
The Immobilization of Lactoperoxidase and p-Pructofuranosidase on Glass and on Sand, by the Metal-Link Method DAVID THORNTON, ANNE FRANCIS and DESMOND B. JOHNSON Department of Biochemistry, University College, Galway, Ireland and PAUL D. RYAN Department of Geology, University College, Galway, Ireland The metal-link method is reported to be convenient and inexpensive in the preparation of immobilized enzymes with a view to their use in industrial processes (Emery et al., 1972). Other reports suggest that the method may be limited in the number of enzymes to which it can be applied. Coughlan &Johnson (1973) failed to prepare active immobilized xanthine oxidase by using this method, Johnson & Thornton (1973) reported limitations in the application of the method to lactoperoxidase, and M. J. Byrne & D. B. Johnson (unpublished work) found that alcohol dehydrogenase and lactate dehydrogenase were not successfully immobilized by the process. Further investigation indicates the source of one of the limitations of the method. We report here on this limitation, and on the application of the method to the immobilization of enzymes on sand. The solid supports used in this study were glass beads (average diameter 0.170.18mm; B. Braun, Melsungen, Germany), porous glass (CPG-10; 200-400 mesh; average pore diameter 209nm) and acid-washed sand (both from BDH Chemicals, Poole, Dorset, U.K.). The sand was a clear crystalline quartz of high purity, with less than 0.1 % of the sample contributed by rose quartz, srnokey quartz, rock fragments and
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quartz stained by iron oxides. Grain size analysis indicated that 48 % of the sample was in therange25&500pm, 51 %in therange 125-250pm, andless than0.2% below 125pm. Lactoperoxidase (a gift from Dr. D. B. Morel]) was assayed at 300nm in phosphate buffer ( 0 . 0 8 ~ )containing H 2 0 2 (0.166m~)and pyrogallol (58rng/lOOml). p-Fructofuranosidase (Grade 111, Sigma Chemical Co., St. Louis, Mo., U.S.A.) was assayed by measuring the release of reducing sugar from sucrose, by using the dinitrosalicylic acid method of Bruner (1964). Immobilized preparations were assayed in shaken vessels, at 25"C, containing, in the case of lactoperoxidase preparations, 20ml of the buffered substrate solution described, and for immobilizedp-fructofuranosidase, 30ml of 1M-sucrose solution. Sucrose solutions were usually unbuffered, except for the determination of the pH optimum of the immobilized preparation, when substrate was prepared in 0.02Msodium acetate buffer. Units of activity were defined arbitrarily. For lactoperoxidase a unit was taken as that activity producing an absorbance change of 10 units/min, under the conditions described. For /I-fructofuranosidase a unit was defined as that activity hydrolysing 1p~ of sucrose/h in 30ml of unbuffered 1M-sucrose solution. Enzyme supports (2g) [activated by stirring with Tic& (12.5 %, w/v) for 3 h], were stirred with enzyme solution (10ml containing 40mg of enzyme) for 18h at 4°C. Unbound enzyme was removed by washing with 1M-NaCI in the buffer used in the assay of the enzyme, followed by washing with the buffer alone. Emery etal. (1972) report that the pH at whichenzyme is attached to activated support, by overnight stirring, is usually the pH of optimum activity of the free enzyme. This suggests that enzyme may be bound successfully over a wide range of pH values. In the case of lactoperoxidase and p-fructofuranosidase immobilization we find that the pH used for attachment is critical (Table 1). The results indicate that samples prepared at pH4.5 are considerably more active than those prepared at pH6.5, the pH optimum of lactoperoxidase. Measurement of bound protein [by the method of Moore &Stein (1948) after hydrolysis in 5.8~-HC1]indicated that more protein was bound at pH4.5 than at pH6.5. Porous-glass-bound lactoperoxidase retained 3 mg of protein/g at pH4.5, and l m g of protein/g at pH6.5. However, specific activity retention was better at pH6.5 (3.779, than at pH4.5 (1.9%). It seems likely that the greater efficiency of the metal-link method at the lower pH value explains the poor results obtained with xanthine oxidase, lactate dehydrogenase and alcohol dehydrogenase, which were treated for immobilization at alkaline pH values, their pH optima. It may also be inferred that the method, at least at its most efficient, is restricted to enzymes stable at pH4.5.
Table 1 . Activity of immobilized enzymes prepared with TiCI, Activities were determined by incubation in shaken vessels, as described in the text. Enzyme Lactoperoxidase
8-Fructofuranosidase
Support Porous glass Porous glass Glass beads Glass beads Sand Sand Porous glass Porous glass Glass beads Sand Sand Sand
Attachment PH 6.5 4.5 6.5 4.5 6.5 4.5 6.5 4.5 4.5 4.5 6.5 7.5
Activity (units/g) 2.6 4.0 0.0 0.50 0.05 0.23 50 400 130 18 6 6 1974
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0'
I
I
t
I
I
25
30
40
50
60
I
70
Incubation temp. ("C) Fig. 1. Thermostability of free (0) and sand-bound
(0)lactoperoxidase
Free enzyme and samples of immobilized enzyme (lomg) were incubated at the temperatures shown at pH6.5 for 5min and then were assayed by the methods described in the text.
Although the results indicate these limitations of the metal-link method, they also indicate its applicability to the novel support, sand. As a preliminary characterization of this support we have studied some of the properties of lactoperoxidase and ,6fructofuranosidase bound to it by the metal-link method. We found that lactoperoxidase and 15-fructofuranosidase were non-specifically adsorbed on the unactivated support. At pH6.5 adsorption provided a preparation with 1OOpg of ,6-fructofuranosidase/g of sand. This is in keeping with previous observations on trypsin adsorption on quartz (Kobamoto et al., 1964). Immobilization of lactoperoxidase on sand by the metal-link method left the pH optimum unchanged, but caused a slight shift in apparent K,. values ~ 3.1 x 1 0 - 8 ~ )The . pH optimum of 8-fructofuranosidase bound to (from 2 . 6 1~ 0 - 8 to sand was 4.6, compared with 4.4 for the soluble enzyme. The results of a study on the heat stability of sand-bound lactoperoxidase are given in Fig. 1. These results indicate that optimization of enzyme immobilization on sand may be of value, because of its low cost, and suitability for industrial processes. We thank the National Science Council for support. Bruner, R. L. (1964) Methods Carbohyd. Chem. 4,67-71 Coughlan, M. P. & Johnson, D. B. (1973)Biochim. Biophys. Acta 302,200-204 Emery, A.N., Hough, J. S.,Novais, J. M. &Lyons,T. P. (1972)Chem.Eng. (London)258,71-76 Johnson, D. B. & Thornton, D. (1973) Abstr. Commun. FEBS Special Meet., Dublin, Abstr. 189 Kobamoto, N., Lofroth, G., Camp, P., Van Amburg, G. & Augenstein, L. (1966)Biochem. Biophys. Res. Commun. 24,622-627
Moore, S . & Stein, W. H.(1948)J. Biol. Chem. 176,367-388
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