The effect of ball-milling upon certain properties of proteins

The Effect of Ball-Milling upon Certain Properties of Proteins Carl Monderl From the Cornell University

and P. E. Ramstad School of Nutrition,

Received April

Ithaca, New York

13, 1953

The ,purpose of the work reported here was to explore some of the changes ‘produced in proteins by severe mechanical treatment in a laboratory ball mill. The in vitro digestibility of wool by trypsin and pepsin can be increased. Keratins increase in solubility during ball-milling (l-4). Cohen (5, 6) found the solubility of ovalbumiq’casein, gelatin, hemoglobin, sheep wool, and proteose-peptone increased. Boissevain (3, 7) suggested that ball-milling caused hydrolytic changes in tubercle bacilli and gelatin. Stahl et al. (8), Edwards and Routh (9), and Routh (4) concluded that ball-milling may cause oxidative changes in keratins since their studies showed a decrease in cystine. Stahl et al. (10) later suggested that the changes in digestibility during ball-milling were due to the breaking of disulfide bonds in the keratin molecule. In the current inve&igation, two proteinaceous materials, dried egg albumen and sheep’s wool, have been subjected to ball-milling and sampling at intervals to follow the extent and rate of change in several properties likely to be important from the standpoint of in wivoutilization of protein. These two materials were selected because of the great difference between them. Egg albumen is readily soluble in water and is very susceptible to enzymatic hydrolysis. Wool is fibrous, insoluble and resistant to enzymatic hydrolysis. 1 Charles J. Quillman, Jr. Fellow, 1950-52. Present address : Central Research Laboratories, General Foods Corporation, Hoboken, New Jersey. 2 Present address: Oscar Mayer & Co., Madison 1, Wisconsin. 376

BALL-MILLING

OF PROTEINS

377

MATERIALS AND METHODS The egg albumen3 had been ferment)ed to removesugarbefore drying. Its moisture content was 9.0%. The wool was scoured with three baths of 0.025ojo detergent? at a pH of 6.8 and at 54”C., and rinsed three times at the same temperature. It was carefully picked free of foreign matter and brown discolorations and ground in a Wiley mill using a 20-mesh sieve. The wool was extracted with 95% ethyl alcohol and ethyl ether, then dried.b One-gallon porcelain jars revolving at 3840 r.p.m. were used in a three-jar vertical roller mill. The balls were “Borundum “6 cylinders, 2 cm. long and 2 cm. in diameter. An 800-g. charge and 225 “balls” were placed in each jar in alternate layers. When samples were withdrawn during grinding, some of the balls were removed to maintain a constant ratio of balls to charge. The jars were never less than half full. Nitrogen was determined by the Kjeldahl method, using copper sulfate as the catalyst. Total sulfur was determined by oxidation in a Parr bomb and gravimetric determination of sulfate. Sulfhydryl groups were determined by the o-iodosobenzoic acid method of Hellerman et al. (11). Cystine was estimated by the Sullivan-Hess-Howard procedure (12) and by the Block and Bolling (13) modification of the Winterstein-Folin method on hydrolyzates prepared by autoclaving l-g. samples in 5 ml. of 18% hydrochloric acid in sealed tubes for 8 hr. at 15 lb. pressure and 120°C. The amino acids methionine, histidine, tryptophan, tyrosine, and phenylalanine were determined by the microbiological assay methods described by Williams (14). Tryptic digestibilities were determined using the potentiometric form01 titration (15,16) modified as follows: Ineach serieaof experiments, 60 ml. of 3% protein suspension was incubated with 5 ml. of 3% trypsin solution (1:300) at pH 8.18.2 and 37 f l.O”C. Each 5-ml. aliquot was diluted to 20 ml. with distilled water and adjusted to pH 6.0. Five milliliters of formaldehyde (37%) adjusted to pH 7.0 and containing a small amount of phenolphthalein indicator was added. Standard sodium hydroxide was added quickly from a buret until a light-pink color was obtained, then slowly to pH 9.2, stirring the solution with a stream of nitrogen. Ash was determined in egg albumen by the official AOAC method (17), and in wool by aching in a mu5e furnace at 550°C. overnight. Moisture was determined by heating 2-g. samples in a vacuum oven at 85°C. for 6 hr. Solubility of ball-milled wool and egg albumin was measured by suspending 2-g. samples in 25 ml. of water and stirring the samples at high speed with an electric stirrer for 15 min. The suspensions were centrifuged and the supernatant and washings were combined and diluted to 100 ml. The micro-Kjeldahl procedure was used to determine the soluble nitrogen. 8 Nutritional Biochemicals Co., Cleveland, Ohio. 4 Igepal Ca Extra from General Dyestuffs, New York, N. Y. 6 The authors are indebted to Kenwood Mills, Rensselaer, N. Y. for their assistance in cleaning the wool. 6 U. S. Stoneware Co., Akron, Ohio.

378

CARL MONDER AND RESULTS

P.

E. RAMSTAD

AND DISCUSSION

The total nitrogen content of the wool was 15.7 y0 and remained unchanged after 2064 hours of ball-milling. The nitrogen content of the egg albumen remained 14.7 Y0 for over 2200 hours of ball-milling. These observations confirm and extend those of Routh (4) and Cohen (6) for wool, and those of Cohen (5) for egg albumen. Denaturation of egg albumen is characterized by an increase in detectable sulfhydryl groups and a decrease in solubility of the protein 7

2 ii ‘0 0.16 h

0” -

. - SH GROUPS

:

0

E

HOURS

FIG. 1. Changes in tit&able during ball-milling.

SOLUBILITY

-

2200

-2000

5 m E L q

WILLED

sulfhydryl

groups and solubility

of egg albumen

(18, 19). To determine whether ball-milling caused similar changes, the detectable sulfhydryl content and water solubility of the egg albumen were determined. Figure 1 shows that a decrease in solubility and an increase in titratable -SH groups are concurrent; the greatest insolubility coincided with the greatest number of -SH groups. Though the solubility of egg albumen decreased rapidly during the first 600 hr. of ball-milling, at no time during the process did the egg albumen become completely insoluble. After 600 hr., the solubility of the egg albumen increased slowly, then more rapidly. After the maximum sulfhydryl value was attained, a slow decrease occurred, probably caused by oxidation. That the denatured fraction of the albumen was degraded during the

BALL-MILLING

OF

379

PROTEINS

first 600 hr. is indicated by the observation that at no time was the albumen ever completely insoluble and that, though the maximum number of -SH groups in heat-denatured albumen was about 0.15 mequiv./ g., the maximum value for ball-milled albumen was 0.14. During this time, there may have been oxidation and breaking of secondary linkages in the molecule. After 1800 hr., because primary bonds were being made more “available” to the grinding action, more extensive splitting of primary bonds and the formation of small, soluble peptides may have occurred.

5i 5i ifE

I

0L

L

5

0.30

-

1 0 - SH GROUPS SH o

-2500

g

-2000

m’ *

-500

g

SOLUBILITY

a

0

500

1000 HOURS

FIG. 2. Changes in titratable ball-milling.

l!NO Iso0

2000 2000

MILL20

sulfhydryl

groups and solubility

of wool during

In wool, the number of detectable -SH groups increased throughout the grinding treatment, as shown in Fig. 2. Solubility increased slowly for the first 1300 hr., and more rapidly thereafter. There was no significant change in the percentage of methionine, tyrosine, and phenylalanine in the albumen. A slight decrease of questionable significance was observed in the levels of histidine and tryptophan. The largest change was in cystine which decreased from 2.9 to 2.7 g. per 16 g. nitrogen. The changes in the amino acid content of wool during ball-milling are summarized in Table I. There was a large decrease in each of the amino acids determined. This began to be evident at about 800 hr. of

380

CARL

MONDER

AND

P.

E.

RAMSTAD

grinding. The period of rapid increase in solubility of wool (Fig. ‘2) and that of most rapid destruction of amino acids appear to correspond rather closely. Destruction of amino acids played a minor role in the action of the ball mill on egg albumen. This might be expected on the basis of the close, compact nature of the molecule. On the other hand, the amino acids in a long, relatively exposed wool keratin molecule might be expected to be more readily destroyed. Evidence for degradation of the protein molecules into smaller units during ball-milling was obtained from form01 titration results which showed during the full grinding period an increase of 1.0 X 10” carboxyl groups/mg. albumen and 1.8 X lOl’/mg. wool.

The

Time milled hr.

Cyst&

0

646 1008 1680 2064

12.16 11.03 9.10 8.23 6.82

TABLE I Amino Acid Composition of Ball-Milled Data in grams/l6.0 g. N. Histidine

0.95 0.94 0.87 0.66 0.66

Methionine

0.50 0.47 0.43 0.36 0.31

%Xk 2.93 2.64 2.43 2.23 1.95

Wool

Tryptophan

0.55 0.42 0.31 0.23 0.12

Tyrosine

4.34 3.83 3.34 3.05 2.27

The small increase in free amino acid groups might result from little hydrolysis during ball-milling, or extensive hydrolysis followed by destruction of amino-carboxyl groups which could not be detected by the procedures used. The former is more probable since solubility increased only slightly during prolonged milling. Table II contains a summary of the contributions of cystine and methionine to the total sulfur content of egg albumen and wool during ball-milling. The total sulfur of egg albumen did not change during ballmilling. However, the proportion of organic sulfur (methionine plus cystine) dropped during 2000 hr. of ball-milling from 93 Y0to about 78 y0 of the total sulfur. The methionine and cyst&e content of wool decreased during ballmilling as shown in Table I. The contributions of cystine plus methionine to the total sulfur content of wool decreased in 2000 hr. from 94 to 55 %. The total sulfur content of wool decreased from 3.58 to 3.46 %, but

BALL-MILLING

OF

381

PROTEINS

no HB was detected by sense of smell or by use of the lead acetate test, contrary to the experience of Stahl e2 al. (8). Previous experiments by Routh and Lewis (1) and Stahl et al. (10) have demonstrated that digestibility of wool by trypsin was increased during ball-milling. Haurowitz et al. (20) and Cohn and White (21) found that tryptic digestibility and extent of denaturation of egg white were apparently linearly related. Experiments were undertaken in order to determine the rate of and the extent to which the digestibility of wool

Contributions

of

TABLE II Cystine and Methionine to the Total and Wool

Time

milled

TOtdldfW

%

hr.

During

Sulfur

Content

of Egg Albumen

Ball-Milling

cy&

s

%

III Methionine

S

%

Egg albumen 0 96 240

670 831 1464 2160

1.67 1.60 1.69 1.65 1.67 1.71 1.70

0.79 0.76 0.83 0.79 0.72 0.71 0.72

0.77 0.77 0.76 0.75 0.76 0.72 0.62

93.4 95.6

0.12 0.09 0.12 0.08

93.8 92.6

94.1 93.3 88.6 83.6 78.2

Wool 0

168 353 2064

3.58 3.59 3.70 3.46

3.25 3.23 3.09 1.82

86.8 54.9

changes during ball-milling. An attempt was also made to determine whether the digestibility of egg albumen by trypsin was altered during ball-milling. Figure 3 depicts the increased tryptic digestibility of both proteins resulting from ball-milling. The digestibility of wool increased most during the hrst 909 hr. ; further grinding produced little additional change. Ball-milling increased the tryptic digestibility of the egg albumen throughout the grinding period. The next question was how the increase in digestibility of albumen by ball-milling compared with that caused by other treatments. Acidcoagulated albumen was prepared according to the method of Hendrix and Wilson (22). Heat-denatured, uncoagulated egg albumen was pre-

382

CARL MONDER AND P. E. RAMSTAD

pared by Rosner’s method (23), heating the albumen solution for 30 min. to insure complete denaturation. Albumen was heat-denatured at pH 9.0. The tryptic digestion of these preparations was compared with that of untreated albumen and with albumen that had been milled for 2160 hr.

l

EQG

ALBUMIN

0 WOOL

SO0

IO00 HOURS

moo

2000

MILLED

FIG. 3. Effect of ball-milling upon the susceptibility to tyrptic digestion (4-hr. digestion p&iods).

of egg albumen and wool

All digestions were performed at a substrate level of 3 %. Figure 4 shows that the increase in digestibility is greater for ball-milled egg albumen (2160 hr.) than for the heat-denatured product. The digestibility of ball-milled wool increased up to 800 hr. of milling. After this time, no increase in digestibility could be obtained, although the number of sulfhydryl groups increased throughout the period of milling. Geiger and Harris (24) and Goddard and Michaelis (25, 26) using

BALL-MILLING

OF

PROTEINS

333

disulfide groups as the type secondary bond, postulated that the digestibility of wool is dependent on the arrangement of secondary linkages in the molecule. Apparently, the first effect of ball-milling was to break secondary linkages, gradually exposing peptide groups to the action of the mill. After a sufficient number of primary bonds had been exposed,

0

BALL-MILLED

0

HEAT

X

UNTREATED

2278

HOURS

DENATURED

ALBUMIN

ALBUMIN

5 HOURS

FIG. 4. Tryptic

digestibility

IO DIGESTION

of ball-milled

versus heat-denatured

egg albumen.

the amino acids were,in a position of greatest availability to the digestive action of trypsin. The apparent relationship between the increase in sulfhydryl groups, decreasein solubility, and increase in tryptic digestibility of egg albumen during the first 800 hr. of milling was due largely to the denaturation process. After this time, the molecule exposed ever more primary linkages, causing a continuous increase in susceptibility to proteolytic attack beyond that which could be explained by denaturation.

384

CARL

MONDER

AND

P. E.

RAMSTAD

SUMMARY

Grinding in a ball mill caused the solubility of egg albumen to decrease and then to increase, while free sulfhydryl groups reached a maximum and then decreased. Similar treatment of wool caused a continual increase in both aqueous solubility and titratable sulfhydryl groups. Both were made more susceptible to tryptic digestion. REFERENCES 1. ROUTA,

2. ALSBERQ,

J. I., AND LEWIS, H. B., J. Biol. C. L., AND GRIFFITH, E. P.,

(1925). BOISSEVAIN, ROUTH, J. COHEN, H. COHEN, H. BOISSEVAIN, 8. STAHL, W. 9. EDWARDS, 10. STAHL, W.

3. 4. 5. 6. 7.

11. 12.

13. 14. 15.

16. 17. 18. 19. 20. 21. 22.

23. 24.

25. 26.

Chem.

134, 725 (1933).

Proc. Sot. Exptl. Biol. Med. 23, 142

C. H., Am. Rev. Tuberc. 31, 547 (1935). I., J. Biol. Chem. 136, 175 (1940). R., Arch. Biochem. 2, 1, 345, 353, 359 (1943). R., Arch. Biochem. 4, 145, 151 (1944). C. H., Am. Rev. Tuberc. 31, 542 (1935). H., MEQUE, B., AND SIU, R. G. H., J. Biol. Chem. 177, 69 (1949). B., AND ROUTH, J. I., J. Biol. Chem. 154, 593 (1949). H., MEQUE, B., MANDELS, G. R., AND SIU, R. G. H., Terfile Research J. 20, 570 (1950). HELLERMAN, L., CHINARD, F. P., AND RAMSDALL, P. A., J. Am. Chem. Sot. 32, 2551 (1941). SULLIVAN, M. X., HESS, W. C., AND HOWARD, W. H., J. Biol. Chem. 146, 621 (1942). BLOCK, R. J., AND BOLLINO, D., The Determination of the Amino Acids. Burgess Publ. Co., Minneapolis, Minn. 1949; idem, The Amino Acid Composition of Protein and Foods. C. C. Thomas, Springfield, Ill., 1951. WILLIANJ, H. H., Proceedings 1950 Cornell Nutrition Conference. LEVY, M., J. Biol. Chem. 99, 767 (1932). LEVY, M., J. Biol. Chem. 106: 157 (1934). ASSOCIATION OF OFFICIAL AGRICULTURAL CHEMISTS. Official Methods of Analysis, 7th Ed., p. 476. Washington, 1950. ANSON, M. L., Advances in Protein Chem. 2,261 (1945). MIRSXY, A. E., J. Gen. Physiol. 24, 709 (194641); ibid. 725 (1940-41). HAUROWITZ, F., TUNCA, M., SCHWERIN, P., AND G~KSU, V., J. Biol. Chem. 167, 621 (1945). COHN, E. W., AND WHITE, A., J. Biol. Chem. 199, 169 (1935). HENDRIX, B. M., AND WILSON, V., J. Biol. Chem. 79, 389 (1923). ROSNER, L., J. Biol. Chem. 132, 657 (1940). GEIGER, W. B., AND HARRIS, M., J. Research Null. Bur. Siandards 29, 271 (1942). GODDARD, D. R., AND MICHAELIS, L., J. BioZ. Chem. 108, 605 (1934). GODDARD, D. R., AND MICHAELIS, L., J. Biol. Chem. 112, 361 (1935).