THE VITAMIN B12-BINDING POWER OF PROTEINS

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1 THE VITAMIN B12BINDING POWER OF PROTEINS BY 0. D. BIRD AND BARBARA HOEVET (From the Research Laboratories of Parke, Davis and Company, Detroit, Michigan) (Received for publication, December 26, 1950) Ternberg and Eakin (1) showed that normal gastric juice or an aqueous extract of hog mucosa when added unheated to vitamin Blz rendered the vitamin nondializable and unavailable to assay microorganisms, including Lactobacillus leichmannii. They concluded that this principle which combined with vitamin Blz was the intrinsic factor of Castle or an important component thereof. Meyer et al. (2) noted a similarity between intrinsic factor from hog intestines and lysozyme. They also observed that lysozyme derived from egg white combined with vitamin Blz, thus rendering it unavailable to Lactobacdlus lactis Dorner and Escherichia co&i. Prusoff et al. (3) fractionated the intrinsic factor from desiccated hog stomach mucosa and checked the ability of these fractions to prevent the utilization of vitamin Blz by L. leichmannii Shaw (4) described the preparation of a protein fraction from swine intestines which was an inhibitor for the growth effect of vitamin Blz on L. leichmannii. Most of his preparations possessed some growth activity for L. leichmannii; this was apparently due to the presence of growth factors not inhibited by the inhibitor fraction, thus complicating the accurate determination of the amount of true vitamin Blz inhibitor. No vitamin Blz inhibitor was found in extracts of animal tissues other than those from the gastrointestinal tract. The work reported here was undertaken to study the specificity of vitamin Blzbinding by proteins, to determine whether it is a stoichiometric relationship, and to establish some of the conditions necessary for its accurate estimation. EXPERIMENTAL Methods The assay medium used was that of Skeggs et al. (5). It was modified by adding 50 mg. of thioglycolic acid per 100 ml. and omitting enzymatic casein. The acidhydrolyzed casein used was Difco Casamino acids. When the proteins were added without heating, they were dissolved in and diluted aseptically with sterile distilled water, and added aseptically to the tubes after the latter had been autoclaved and cooled. By this method good results were obtained, with only an occasional contaminated tube, whereas, if the proteins were sterilized by filtration or with alcohol, 181

2 182 VITAMIN BlzBlNDING OF PROTEINS some denaturation apparently took place and the results were unsatisfactory. The assay organism was L. leichmannii 4797 and cultures were incubated 40 hours at 37. Turbidity readings were made with the Evelyn calorimeter. Vitamin BlzBinding Power of Various ProteinsFollowing in general the method suggested by Ternberg and Eakin (l), we determined the vitamin Blzbinding power of intrinsic factor concentrates and of crystalline lysozyme. This consisted of adding graded levels of these proteins aseptically to a series of autoclaved tubes, each containing assay medium and 0.5 mpgm. of vitamin Blz. The minimum amount of the protein per Relative Binding Power of Various Proteins for Vitamin Blz Protein solution TABLE I Protein solution per tube, galvanometer readings 0 ml. 0.5 ml. 1 ml. 2 ml. 3 ml. 4 ml. 5 ml. Intrinsic factor concentrate (100 y per ml.) Crystalline lysozyme (40 y per ml.) Concanavalin B (2 mg. per ml.) t A (10 mg. per ml.)t Globulin 1 (10 mg. per ml.) (10 )$ Crystalline egg albumin (10 mg. per ml.) Urease (4 mg. per ml.) * All tubes contained 0.5 mpgm. of vitamin Bis. t Globulin fractions from soy bean protein prepared by Dr. Laboratories. I. GI.ant Smith (,f these $ Globulin fractions from blood prepared by Dr. H. B. Devlin of these Laboratories. tube required to prevent practically all growth due to vitamin Bit was designated as the amount bound by 0.5 mpgm. of vitamin B,,. In order to learn whether this property of binding vitamin B12, as measured by this test, was confined to IFC and lysozyme, tests of other soluble proteins were made. The results are given in Table I. They show that all the proteins tested have this property of binding vitamin Blz so as to make it unavailable for the nutrition of bacteria, but that IFC and lysozyme are considerably more potent than the other proteins tried. When large amounts of IFC prepared as indicated are added aseptically 1 For the sake of brevity these will be referred to as IFC. They were kindly supplied by R. C. Peterson of these Laboratories. These concentrates were prepared by saturating with (NH,)2SO( a saline extract of dried unheated hog mucosa, dialyzing the precipitate free from salts, and drying it from the frozen state.

3 0. D. BIRD AND B. HOEVET 183 to the vitamin B,, assay medium (containing no vitamin Bvz), there is a growth response. This response is much greater if the same amounts of IFC are autoclaved with the medium prior to assaying. These results are shown in Fig. 1, along with curves for the response of crystalline vitamin Blz added aseptically and autoclaved with the medium. These response curves indicate that, even before these concentrates have had any vitamin Blz added to them, they contain some vitamin Blzlike material. The increase in activity caused by autoclaving may be looked upon as SIYG IFC a5/ P?;YB/2 FIQ. 1. Response curves for vitamin B 12 added aseptically (Curve A) ; vitamin Bit autoclaved with medium (Curve B); intrinsic factor concentrate added aseptically (Curve C); intrinsic factor concentrate autoclaved with medium (Curve D). vitamin Blz already bound to IFC, but this does not account for the response given by IFC when added aseptically. AlkaliLabile and AlkaliStable Activity in Intrinsic Factor Concentrates In Fig. 2 is shown a response curve (A) for IFC when added aseptically to assay tubes each containing 0.5 mpgm. of vitamin Blz. It is seen that growth decreases steadily to a certain point and then increases with increasing doses of IFC. Curve B shows the response to the same amounts of IFC when no vitamin Blz is present in the assay tubes. The latter part of Curve B coincides with the latter part of Curve A. Subtracting Curve B from Curve A gives a third or difference Curve (C), which we feel represents more closely the inhibition of growth due to IFC. As further substantiation of this, Curve D, representing the addition of the same

4 184 VITAMIN B12BINDING OF PROTEINS amounts of IFC after autoclaving at ph 11 to destroy vitamin B12, is seen to coincide quite well with the ascending sections of Curves A and B. Thus it seems probable that the vitamin Blzlike activity in IFC which gives a response when added to the assay medium, either aseptically or after being autoclaved at alkaline ph, is due to desoxyriboside. Analyses of three lots of IFC made as described above are given in Table II. The total vitamin B,, activity is shown in Column 1. This is NG IFG/TUBE FIG. 2. Showing that intrinsic factor concentrate binds vitamin Br2 but also gives a growth response itself. The curves represent responses to the following: intrinsic factor concentrate mpgm. of vitamin Blz per tube (Curve A); same as Curve A with no vitamin B12 added (Curve B); difference curve, Curve A minus Curve B (Curve C); intrinsic factor concentrate after autoclaving at ph 11 (Curve D). the figure obtained when the samples are autoclaved with the medium. In Column 2 is shown the activity obtained when the samples are autoclaved at ph 11 before assaying. These figures presumably represent desoxyriboside but are expressed in terms of vitamin Bl2 equivalent. In Column 3 are shown the values representing the vitamin Blzbinding power of these IFC preparations. These values were determined by projecting the first three turbidity readings of the response curves (as shown in Fig. 2) to the baseline. These data indicate that IFC as prepared already contains about 1 y of bound vitamin Bl2 per gm. and is capable of binding 3 to 4 y more per gm.

5 0. D. BIRD AND B. HOEVET 185 Diference in Binding Power of Intrinsic Factor Concentrate and LysoxymeIn Table III are shown data indicating the binding power of IFC and lysozyme for vitamin Blzlike substances as measured by the method of Ternberg and Eakin (1). IFC binds vitamins Blz and BIZo equally well TABLE II Comparative Vitamin Blz Content and Vitamin BlzBinding Power of Intrinsic Factor Concentrates Intrinsic concentrate factor No. (1) (2) (3) WV?= WGm. m/am * As measured in the regular vitamin Brs assay in which the tubes are autoclaved 20 minutes. t Samples autoclaved 30 minutes in solution at ph 11, then diluted for assay, and autoclaved again as in Column 1. $ In addition to vitamin Brz naturally bound as indicated in Column 1. TABLE III Binding of Vitamin Bl2, Vitamin B1za, and Thymidine by Intrinsic Factor Concentrate and Lysozyme IFC Galvanometer readings with following added per tube 0.5 gl;w. p.5 g$y Y I tbymidiie Y Galvanometer readings with following added per tube D.5 Bzaw. 10 Y thymidine but does not bind thymidine at all. However, lysozyme binds all three substances. Another difference between the complexes formed with lysozyme and the intrinsic factor is indicated in Table IV. When either vitamin Blz or thymidine is combined with lysozyme in proportions which prevent the utilization of these growth factors by L. kichmannii, they are easily dialyzed from these complexes. But when vitamin BU combines with IFC

6 186 VITAMIN BwBINDING OF PROTEINS in these proportions, the vitamin B,, is no longer dialyzable. Still another difference in the action of these two proteins toward vitamin B,, is shown in, Fig. 3. In this experiment constant amounts of IFC or lysozyme were added aseptically to tubes containing increasing amounts of vitamin Blz. Eventually enough vitamin Blz was added to the tubes containing IFC so that growth started, although the ratio of vitamin Blz to TABLE E$ect of Dialysis on Bound Vitamin BU and Thymidine IV Protein Lysozyme IFC (40 mg.) Lysoayme used (20 mg.) (20 mg.) I I Activity recovered after dialysis Substance added Inside Outside I sac Total Per cent Per cent sax inside outside m%=fl. Vitamin Blz ( mam.) Vitamin Bit ( wgm.) Y Thymidine (1000 y) 90 M ;Y B/e/ TUBE +w.w. %a~ ; 1 lo;3 / FIG. 3. Response curves for vitamin B~z alone (Curve A); vitamin BH, y of lysosyme per tube (Curve B); vitamin Bl y of intrinsic factor concentrate per tube (Curve C). IFC required to initiate growth was about 3.5 times that at which growth ceased in experiments in which increasing amounts of IFC were added to constant amounts of vitamin Blz. The ratio of vitamin B1, to lysozyme was carried even further and no growth occurred. Measurement of Vitamin BlzBinding Power by Dialysis Experiments Another method of measuring the vitamin Blzbinding power of IFC is illustrated by Table V. In this series of experiments increasing amounts of crystalline vitamin Blz were added to successive portions of the water solution of IFC. These mixtures were transferred to dialyzing sacs of Visk

7 0. D. BIRD AND B. HOEVET 187 ing cellulose casings and dialyzed 24 hours against frequent changes of distilled water. Then the vitamin Blz content of the residues inside the sacs and the combined dialysates from each sac were assayed microbiologitally for vitamin B,z. In the last column are shown the percentages of the total vitamin Bls which dialyzed through the membrane. Essentially all the vitamin Blz was held by the protein and did not dialyze in Experiments 1 to 8, or until a total of 2400 mpgm. of vitamin Blz had been added to each 40 mg. of IFC. Beginning with Experiment 9 there was a gradual increase in the dialyzable vitamin Blz. On the other hand, the amount of Experiment TABLE Amount of Vitamin Blz Bound by Intrinsic Factor Concentrate As Indicated by Dialysis No. itamin Be addec to 40 mg. IFC w.gm Inside Wm sac, V jutside Activity recovered after dialysis mgm sac Total recovered fw&w O vitamin Blz retained bound to the protein inside the sac (Column 3) remained relatively constant from Experiment 9 on, regardless of the excess of vitamin added. In Experiments 9 to 13, in which there was an excess of vitamin Blz over IFC, there was an average of 3334 mfgm. of vitamin Blz bound by 40 mg. of IFC, or 1 gm. of IFC bound 83 y of vitamin B,z. The three methods described above for determining the vitamin Blzbinding power of proteins give widely differing results. The values obtained for one preparation of IFC by these three methods are summarized in Table VI. DISCUSSION Measured by Method 2 (Table VI), IFC appears to bind nearly 3 times as much vitamin Blz as when measured by Method 1. Thus these methods of measuring vitamin Blzbinding by determining the availability of

8 188 VITAMIN Bl2BINDING OF PROTEINS vitamin B12 for growth of microorganisms in the presence of an active protein do not indicate a stoichiometric relationship between the amounts of vitamin Bl2 and the active protein. However, Method 3, depending on the dializability of any excess of vitamin Big over that which can be bound by the active protein present, gives constant ratios of protein to vitamin B,2 over a considerable range of concentrations of the latter. Furthermore, the much higher vitamin Blzbinding power of IFC indicated by the dialysis method shows that in the growth inhibition test the bacteria are able to utilize all but a small part of the bound vitamin. One would expect that the dialysis method might come closest to representing physiological conditions in which vitamin Bl2 would be found when taken orally in combination with the intrinsic factor. Method 1 (Table VI) indicates that 1 gm. of IFC will bind 3.5 y of vi TABLE VI Vitamin BitBinding Power of Intrinsic pactor Concentrate Indicated by Di$erent Methods Method No. Description of method Vitamin Be bound by 1 gm. IFC mpgm. vitamin Blz per tube, IFC added stepwise; end 3.5 point, 1st tube with no growth y IFC per tube, vitamin BU a dded stepwise; end 9 point, 1st tube with growth 3 40 mg. IFC + increasing amounts of vitamin Bu; end 83 point, 1st tube where vitamin Blz dialyzes tamin B12, whereas 1 gm. of lysozyme will bind 5.1 y of vitamin B12, or 1.45 times as much. When the two are compared by Method 2, 1 gm. of IFC binds 9 y of vitamin B12, while 1 gm. of lysozyme binds more than 30 y of vitamin B12, or over 3.3 times as much. In comparing the vitamin Blzbinding power of IFC with that of lysozyme, there is an even greater discrepancy when Method 2 is used than with Method 1. Dialysis experiments indicate that there is no true binding of vitamin B12 by lysozyme, and it would therefore seem that tests of growth inhibition apparently indicating such binding may be due to an inhibitory effect of lysozyme on the microorganism, and not to a binding of vitamin B12, to make it unavailable. With L. leichmannii 4797 as the test organism, the method suggested by Ternberg and Eakin (1) for measuring the vitamin Blzcombining power of unheated proteins has not been very successful in our hands for assaying such substances as crude extracts of hog mucosa. This method consists of adding increasing amounts of the unheated protein to tubes of the assay medium containing just enough vitamin B12 to cause maximum

9 0. D. BIRD AND B. HOEVET 189 growth and noting how much protein must be added to stop growth. These crude samples contain such a large percentage of desoxyribosides or other nonspecific growth factors in proportion to the active protein present that, before complete inhibition of growth is accomplished, the response curve begins to ascend again, owing to these nonvitamin B,, substances which produce growth but are not bound by the protein. One way to avoid this difficulty is to subtract from the response curve obtained by adding the unheated active protein the curve obtained by adding the identical amounts of protein to assay medium containing no vitamin B,,. However, if the protein contains large amounts of desoxyribosides; as may be the case with crude mucosa extracts, the second response curve will be almost as high as the first and the difference curve will be very inaccurate. In cases of this kind we have been able to get an accurate indication of vitamin Blzbinding power only by the dialysis procedure described above. SUMMARY A variety of proteins, including intrinsic factor concentrate and lysozyme, bind vitamin Blz when measured by adding them unheated in increasing amounts to tubes of vitamin Blz assay medium containing known amounts of vitamin B,, and noting the inhibition of growth of Lactobadus leichmannii. This inhibition method indicates that intrinsic factor concentrates bind vitamin Bls compounds but not thymidine, whereas lysozyme binds vitamin Blz and thymidine. A method of measuring vitamin Binbinding based on the nondializability of the bound vitamin indicates a much higher capacity of intrinsic factor concentrate to bind vitamin Blz than is shown by the inhibition method. This dialysis method indicates that lysozyme does not truly bind vitamin B12, whereas intrinsic factor concentrate does, since the added vitamin is dialyzed from the former but not the latter. The method is also more suitable than the inhibition method for determining the vitamin B12binding power of crude sources of active protein which contain relatively large amounts of nonvitamin Blz growth factors for L. leichmannii. BIBLIOGRAPHY 1. Ternberg, J. L., and Eakin, R. E., J. Am. Chem. Sot., 71, 3858 (1949). 2. Meyer, C. E., Eppstein, S. H., Bethell, F. H., and Hall, B. E., Federation PTOC., 9, 205 (1950). 3. Prusoff, W. H., Meacham, G. C., Heinle, R. W., and Welch, A. D., Abstracts, American Chemical Society, 118th meeting, Chicago, Sept. 38, 27A (1950). 4. Shaw, G. E., Biochem. J., 47, p. xxxv (1950). 5. Skeggs, H. R., Huff, J. W., Wright, L. D., and Bosshardt, D. K., J. Biol. Chem., (1948).

10 THE VITAMIN B 12 BINDING POWER OF PROTEINS O. D. Bird and Barbara Hoevet J. Biol. Chem. 1951, 190: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#reflist1