Tubidimetric Method for Determination of Yeast Mannan and Glycogen

Irwin J. Goldstein , Harry C. Winter , Ronald D. Poretz. 1997 ... Irwin J. Goldstein , Colleen E. Hayes. 1978 ... S.A. Barker , A.G. Giblin , C.J. Gra...
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V O L U M E 27, N O . 10, O C T O B E R 1 9 5 5 Table I. HIO in Sample Taken, Mg.

29.8 32.2 33.1 38.6 39.7 40.1 41.0 42.6 43.5 4F.4 50.2 51.5 98.G

1639 Solution B is best standardized by titrating a known weight of a stable hydrate (4)and should he checked daily.

Titration of Water

Water Found, hlg.

Difference

29.7 32.4 32.9

3 8 , .5

39.8 40.0 41.2 42 5

L70

-0.1 f0.2 -0.2 -0.1 f0.1

-0.34 +O.G2 -0.60 -0.Zi f0.25 -0.25 fO.49 -0.24 -0.23 -0.43 0.00 +0.78 -0.51

-0.1 f0.2 -0.1 -0.1 -0.2 0.0 f0.4 -0 5

43.4 46.2 50.2 51.9 98 1

RESULTS

~-

Mg.

The precision attainable is evident from Table I. ACKNOWLEDGMENT

The authors wish to thank J. R. H. van Nouhuys, director of the Fibre Research Institute, T.N.O., for permission to publish the results of this investigation, and are indebted to Nicolaas de Hart of this institute for his assistance with the analytical work. LITERATURE CITED

(1) Almy, E. G., Griffin, W.C., and Wileox, C. 9 . . 1x1.ENG.CEmLf,, ANAL. ED., 12, 392 (1940). (2) Faulk, C. W.t and Rawden, d. T.. J . .4m. C'heru. ,Cot.. 48, 2045

of anhydrous methanol and 500 ml. of anhydrous pvridine. Solution B: dissolve 50 grams of resublimed iodine in 1 liter of anhydrous methanol.

Procedure. Place 30 to 50 ml. of Solution A in the titration vessel, start the stirrer, and rapidly titrate with Solution B until a sharp inflection is noted on the vacuum tube voltmeter. Continue the titration dropwise until the increased voltage remains constant for a t least 1 minute. Add the sample to be tested and repeat the titration with Solution B, recording the volume used. This is equivalent to the moisture in the sample taken.

(1926). (3) Fischer, K., Angew. Chem., 48, 394 (1935). (4) Frediani, H. A , , et al., ANAL. CHEM.,23, 1332 (1961). (5) Heinemans, B. J., J . Dairy Sei., 28, 845 (1945). (6) Johansson, A, Saensk Papperstidn., 50, 11B, 124 ( 1 9 4 i ) .

(7) hIitehell, J., Jr., and Smith, D. &I.,"Aquametry, .4pplication of the Karl Fischer Reagent to Quantitative Analyses Involving

Water." Interscience, London-New York, 1948. (8) Seaman, W. hI., McComas, W. H., and Allen, G . Ai.,.4iv.a. CHEM.,21, 510 (1949). (9) Willard, H. H., and Fenwick, F., J . Am. Chem. Sock, 44, 2504, 2516 (1922).

RECEIYIDD for review March 15, 1954. Accepted M a y 23, 1955,

Turbidimetric Method for the Determination of Yeast Mannan and Glycogen J. A. ClFONELLl

and

F.

SMITH

Department o f Agricultural Biochemistry, University o f Minnesota, St. Paul, M i n n .

The interaction of concanavalin-.& a globulin extracted from jack bean meal, with glycogen and with yeast mannan has been utilized for the determination of these two polysaccharides. It is suggested that the reaction between concanavalin-.4 and glycogen could be used for the determination of alpha-amylase activity.

D

URING a study of the purification of yeast invertase it n as desirable to have a method which would permit the estimation of small concentrations of yeast mannar (yeast gum). Although yeast mannan was generally detected by means of its precipitation with alkaline Fehling solution ( 7 ) , this method lacked the sensitivity necessary for the determination of w a l l amounts. Sumner and O'Kane (9) had reported that a jack bean globulin, concanavalin-A, was effective in precipitating not only invertase but also yeast mannan. This observation was utilized t o develop a simple method for the determination of yeast gum. The method was also found to he suitable for the determination of glycogen. METHODS

Preparation of Concanavalin-A Solution. A partially purified concanavalin-A solution suitable for determining yeast mannan and glycogen was prepared as follows (6, 9). Twenty grams of jack bean meal (Arlington Chemical Co., Yonkers, N. Y.) was stirred n;ith dilute sodium chloride (2%, 200 ml.) for about 10 minutes and then centrifuged for 10 minutes a t 2000 r.p.m. The turbid supernatant solution was treated with 2 ml. of 2M acetate, p H 4.2, and after standing for an hour was centrifuged. The solution was warmed to 50" to 55" C. with

continual stirring, and after standing a t room temperature for Generally, the solution was clear a t this point, but occasionally it was slightly turbid, in which case 2 or 3 ml. of 0.1% glycogen in water was added, and after standing in the refrigerator overnight the solution was filtered or centrifuged. The clear solution was treated with 15 ml. of a 7% aqueous solution (15 ml.) of poly(viny1 alcohol) (Elvanol, Grade 71-24, Du Pont Co.) and the reagent kept in the refrigerator until ready for use. The solution (pH 6.0) is stable for several months when refrigerated. It is possible to re-use the concanavalin-h reagent several times ; it should be clarified before being re-used. With some solutions little change in absorbance was noted when they were treated with a standard glycogen solution even after the reagent had been used several times. Generally the reagent develops a slight sediment on standing longer than several days, hut this is much less in solutions prepared by acetate and heat treatment than in those prepared under neutral conditions. Lyophilization of the acetate- and heat-treated preparations gives products which are stable a t room temperature and which dissolve completely in 1% saline to yield solutions which are stable v hen refrigerated. Preparation of Yeast Mannan and Standard Liver Glycogens. The yeast polysaccharide was obtained from baker's yeast autolyzate (4)b y precipitation with alkaline Benedict solution and subsequent purification according to the method dexribed bv Haworth, Hirst, and Isherwood ( 5 ) . The product was dried in vacuo a t 56" C. and n-as slightly green. The color, presumabljdue to bound copper, could not be removed by dialysis against distilled water or against 0.0LV acetic acid, but was removed by passage over a cation exchange resin (Amberlite IR-120, Rohm & Haas Co.). The addition of concanavalin-A reagent to a standard weight of the yeast mannan before and after passage through Bmberlite IR-120 produced the same absorbance. Hence traces of copper do not influence the analytical results. The specific rotation of the yeast mannan was +88' in water ( r , 1.0). in agreement with the value reported previously ( 5 ) .

0.5 hour was centrifuged.

ANALYTICAL CHEMISTRY

1640 The sample of human liver glycogen used as a standard in this study was purified according to the procedure previously described (Z), and the rabbit liver glycogen was fractionated n-ith alcohol ( S ) , after a preliminary purification, as for the human liver glycogen. Both samples had a specific rotation of [a17 +195' in water (c, 0.5). Turbidimetric Procedure. One milliliter of an aqueous solution of yeast mannan containing 10 to 100 y or of glycogen containing 100 to 1000 y is mixed a t once with 9 ml. of concanavalinA solution a t room temperature. -4fter standing for 10 minutes, the absorbance of the solution is determined in an Evelyn colorimeter using a No. 420 filter. A blank is prepared by using water in place of the polysaccharide solution.

0

YEAST MANNAN ( Y ) 25 50 75

0

250

I00

U

m b:

0

500

750

protein than the use of either condition alone. Thus, protein precipitation occurred when the acetate-treated extracts were heated to 71" C., and also when scetate w-as added to the heattreated extracts. To obtain reaction nlixtures which give consistent readings turbidimetrically, it is necessary to operate under conditions which do not cause rapid precipitation. The most convenient range for glycogen was found to be between 0.1 and 1.0 mg. and for yeast mannan, 10 to 100 -/ per ml. (The sensitivity of the concanavalin-A reagent toward glycogen and yeast mannan may be increased by the use of a more concentrated concanavalin-h reagent.) Even a t these concentrations it is advisable to add a stabilizer such as poly(viny1 alcohol) to prevent precipitation during the test. Concentrations of polysaccharides highei than those indicated should not be used; otherwise precipitation will take place even in the presence of the stabilizer. $n excess of polysaccharide acts as a protective colloid (see Figure 2), and at a yeast mannan concentration of 20 mg. per ml., the reaction niixture with concanavalin-h remains clear for approximately 30 minutes. Thereafter, the mixture shows an opalescence which increases sloi~ly,but even after standing overnight the mixture remains only moderately opalescent and shows no suspension or precipitate.

1000

GLYCOGEN

Figure 1. Interaction of concanavalin-A with glycogen and w-ith yeast mannan

A standard curve for comparing the polysaccharide concentration of the unknowns is prepared with standard yeast mannan or with standard normal human or rabbit liver glycogen, using the concentration ranges noted above (see Figure 1).

i

RESULTS AND DISCUSSION

The preparation of crystalline concanavalin-A was first described by Sumner (8). More recently a simplified method has been reported ( 6 ) . While the precipitation of both glycogen and yeast gum was effected (9), presumably by the use of crystalline concanavalin-& in this study it was found that a relatively crude solution of concanavalin-A was suitable for the determination of these polysaccharides. The procedure as finally developed makes use of both heat and acetate for the removal of inert protein. Heating the crude jack bean meal extract to 70" to 72" C. left much of the concanavalin-A in an "active" form, though the decrease in activity of the concanavalin solution was related to the temperature t o which it was heated. The use of acetate buffer of pH 4.1 to 4.5 (the value is not critical) in conjunction with heating produced a solution with greater precipitating power than the use of either acetate or heat alone (see Table I). The use of both heat and acetate apparently removes more inert

Table I.

Effect of Acetate and Heating on Preparation of Concanavalin-A Extract"

Vol. 2.11 Acetate ( p H 4.1) Added, M1.

Temp., O C.

S o h . after Centrif. b

Absorbance after Adding 1 M I . of 1% Glycogen Soln.

0 Volume of extract in each case was 50 ml. and contained 2 ml. of 5% poly(viny1 alcohol). Extracts were prepared by treating Jack Bean meal with 10 parts by weight of 2% saline solution and after mixing for 10 minutes. centrifuging a n d then treating t h e centrifugate with 5 % poly(viny1 alcohol) solution a s indicated above. b 10 minutes a t 2500 r.p.m.

I

0

2

4

6 MG

Figure 2.

8 YEAST

IO

12

14

16

18

MANNAN/ML

Interaction of yeast mannan with concanavalin-A

Absorbance measured after 10 minutes

Glycogen behaves somewhat differently, for a t a concentration of 60 mg. per ml. it gives a precipitate after shaking for a short while with concanavalin-A. On the other hand, the addition of glycogen to 201, yeast mannan does not result in the formation of a precipitate with concanavalin-A. Moreover, dilution of a clear mixture of 2% yeast mannan-concanavalin-A with saline does not promote precipitation whereas the addition of one volume of concanavalin-A solution results in the formation of a turbidity within a minute or two and precipitation within several minutes. The control of pH is important in the reaction between the two polysaccharides and concanavalin-A. A pH of 5 to 6 is optimal, whereas pH values either loner or higher result in decreasing absorbances of the glycogen-concanavalin-A reaction mixtures. Under alkaline conditions the concanavalin--& reagent produces little or no turbidity with glycogen. Similarly, no turbidity occurs a t pH values of 2 to 3 when a concanavalin-A reagent prepared by acetate and heat treatment is used, but when the reagent is prepared under neutral conditions and a t room temperature, bringing the pH to 4 to 5 results in the precipitation of much inert protein. Thus, it is best to prepare the reagent by the use of acetate to eliminate the possibility of erroneous results from this source of inert protein precipitation. It appears that proteins do not influence the concanavalin-polysaccharide reaction. The addition of an equal part or more of blood-serum protein to a solution of glycogen does not interfere with the reaction of the latter with the concanavalin-A reagent.

V O L U M E 27, NO. 10, O C T O B E R 1 9 5 5 Furthermore, it was possible to determine the amount of yeast mannan in an invertme preparation which contained 85% protein with an accuracy equivalent to that involving acid hydrolysis of the mannan followed by determination of the reducing sugar liberated. It is thus possible to determine the concentrations of glycogen or yeast mannan in the presence of protein and consequently the determinations are greatly simplified, since isolation of the polysaccharides is unnecessary. Determinations of yeast mannan in the presence of glycogen niay be accomplished simply by adding diluted saliva to the mixture of polysaccharides. (Such a mivture is obtained when yeast is extracted by dilute alkali.) lt7hen the glycogen has been destroyed, the addition of concariavalin-.4 reagent then measures the yeast mannan which ic: not affected by the salivary a-amylase. Pretreatment of the glycogen-yeast mannan mixture with salivary a-amylase before addition of the concanavalin-A reagent is not necessary, a3 a-ani) lsie is active even in the presence of the reagent. The precipitating ability of glycogen is generally destroyed within a few minutes, and only the turbidito caused by the interaction of 4 east mannan nith concanavalin-.$ remains. By use of the above method, it is possible also to determine the amount of glycogen in admixture with yeast mannan. The turbidity produced by the mixture with the concanavalin-A reagent is determined first, and then that produced by the aamylase treated reaction mixture. The difference between the two results gives the glycogen concentration. It will be apparent that the concanavalin-h reagent coultl be utilized for the detection and determination of a-amylase activity.

1641 The reaction of concanavalin-A with glycogen or yeast iiiannan requires an intact molecule, since when either polysaccharide is oxidized with periodate. the products no longer react lyith the concanavalin-h reagent ( 3 ) . Reduction of these periodatetreated polysaccharides produces polyalcohols (1) which likewise show no reaction with concanavalin--4. .U1 glycogen specimens tested thus far including those from plant, vertebrate, and invertebrate sources give precipitates while the varioiis starches and mannans, other than yeast mannan, which were examined, failed to give precipitates with the concaii:iv:tlin-A reagent (3). LITERATURE CITED (1) A4btiel-Akher,AT., Hamilton, J. K., Jlontgomery, R . , and Smith, F., ,I. Am. Chem. Soe., 74,4970 (1962). (2) A4bdel-Akher,AI., and Smith, F., Ibid., 73, 994 (1951). ( 3 ) Cifonelli, J. d.,Montgomery, R., and Smith, F.. Ibid., in press. (4) Cifonellj. J. A., and Smith. F., Ibid., in press. (5) Haworth, W. N.. Tlirst, E. I,., and Isherwood. F. h., J . Chem. Soc.. 1937,784. ( 6 ) Howell, 8. F., Federation PTOC., 12, 220 (1953). ( 7 ) Salkon-ski, E., 2. physiol. Chem., 34, 162 (1901). (8) Sumner, J. B., and Howell, S. F., J . Biol. Chem., 115, 583 (1936). (9) Sumner. ,J. B.. and O’Kane. D. J., Enzymologia, 12, 251 (1948).

RECEIJ-ED for review January 17, 1953. Accepted April 19, 1955. T h i s littper (30. 3296, Scientific Journal Series, X n n e s o t a Agricultural Experinreot Station) forms p a r t of a thesis presented by J. A. Cifonelli t o t h e graduate faculty of t h e University of Minnesota in partial fulfillment for t h e 1’h.D. degree, 1952.

Determination of Iron Pentacarbonyl in Commercial Carbon Monoxide JULIUS SENDROY, JR., HAROLD A. COLLISON, and HUBERT J. MARK Division of Chemistry, Naval Medical Research Institute, Bethesda, Md. In the course of experimental work in which carbon monoxide from steel containers was used, the presence in the gas of a substance which appeared on qualitative test to be iron pentacarbonyl [Fe(CO)J was noted. Further investigation led to the development of a specific, rapid, and accurate method for its determination, with particular application to its presence as an impurity in conimercial carbon monoxide. The gas sample to be analyzed, in volume up to 10 liters, is passed through traps in which the pentacarbonyl is condensed or absorbed, then measured spec trophoM in methanol solution. A n avertometrically at 235 m age accuracy within 1.370 is obtained in analyses of samples holding a minimum of 0.04 mg. of iron pentacarbonyl. The concentrations present in commercial carbon monoxide in steel cylinders have been found to range from 0.16 to 18 nig. per liter.

T

HE formation of carbon\l in carbon monoxide and other

technical gases held under pressure in metal cylinders, has been investigated by others (1, 9, 11, 1 4 ) . In extensive studies carried out by Pohland and Harlos ( I C ) , the commercial gases examined were air, carbon dioxide, nitrogen, oxygen, hydrogen, and carbon monoxide. Of these, only cylinders of the latter two were found to contain iron pentacarbonyl (in about 10 out of 50 cylinders). The conditions under which carbon monoxide reacts with iron to form the carbonyl, and the physical and chemical properties of the latter, were apparently first studied by Roscoe and Scudder ( 1 5 ) and Mond and Quincke ( I S ) , and subsequently by many others ( 2 , 4 , 6, 7 , 18, 19). An extensive bibliography compiising over 500 titles and abstracts on the metal

carbonyls has been compiled by Croxton ( 3 ) . However, very little of that literature is applicable to the treatment of the present paper based on more recent developments in instrumentation. Of the relatively few procedures described for the estimation of iron carbonyl compounds ( 1 , 3, &I$, 14), most have been based on the analysis of the iron component. For the determination of the carbonyl in concentrations likely to be found in gas cylinders, a rapid, precise, and specific method of analysis based on the ultraviolet absorption characteristics of iron pentacai bonyl was developed. METHOD

Apparatus. For determination of iron pentacarbonyl in pure carbon monoxide or in any gas mixture, the apparatus shown in Figure 1 is used. The gas to be analyzed is contained in a sample tube (tonometer), S, of known volume ( 1 or 2 liters). Water or saturated sodium chloride solution is used as displacement fluid. Tube S is attached to the 3-way T-bore stopcock, T, which provides connection with N , a cylinder of water-pumped nitrogen (free of organic matter), and with a trap. When the sample contains less than 2 mg. of iron pentacarbonyl per liter it is preferable to use a condensation cold trap consisting of the tube 1 immersed in a solid carbon dioxide-cellosolve mixture, C, held in a Dewar cylinder. The tube is a standard taper midget impinger vessel ( 5 ) of 30-ml. capacity (obtainable from H. s. Martin and Co., Evanston, Ill., M-14050). A narrow band of lubricant (high vacuum grease, Dow Corning) is applied to the up er portion of the ground glass joint, When larger volumes ( > 2 fters) of sample are taken, to obtain carbonyl in amount sufficient for accurate analysis, the gaa sample is passed from ita cylinder directly through a glass flow meter (Emil Greiner, Type G914C) connected to T. All connections (such as R ) are made with minimal exposure of the rubber tubing (pure gum, amber) to the gas. When the sample contains more than 2 mg. of iron pentacarbonyl per liter, the all-glass trap unit of the three collection