Charles E F Gersdorff

D Breese Jones

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California Avocado Association 1929 Yearbook 14:141-145 
 
Proteins of the Avocado (Persea Americana Mill) 
 
D. Breese Jones and Charles E. F. Gersdorff 
From the Protein and Nutrition Division, Bureau of Chemistry and Soils, United States 
Department of Agriculture, Washington 
 
Reprinted from The Journal of Biological Chemistry, March 1929, through courtesy of its 
editor & Dr. Jones.  (Received for publication, November 28, 1928.) 
 
In this paper are described the isolation and composition of some proteins of the 
avocado. Data are given concerning the proteins of a type of vegetable tissue that has 
been very little investigated. Aside from the work of Smith (1) on the orange there has 
been to our knowledge nothing published regarding the nature and properties of the 
proteins of fruits. 
In addition to the scientific aspects connected with a study of the proteins of the 
avocado, the growth of the avocado industry in the United States during recent years, 
and the increasing popularity of this fruit as an article of food lend further interest and 
importance to a consideration of the proteins from the standpoint of their biological 
value. 
Proteins of three different types have been isolated from the fresh avocado. One was 
removed by extracting the fresh fruit with 10 per cent sodium chloride solution. This 
protein was separated from the saline extract by three different methods; heat 
coagulation (Preparation I), precipitation with ammonium sulfate (Preparation II), and 
acidification with acetic acid Preparation III). The close agreement between these 
preparations obtained by different methods, with respect to their properties and 
composition (Table I), strongly indicates that they represent one and the same protein. 
This protein behaves in most respects like a globulin. It is soluble in salt solution, from 
which it can be precipitated by addition of ammonium sulfate to 67 per cent of 
saturation, or by addition of acetic acid to pH 3-9. On the other hand, it cannot be 
precipitated from a 5 per cent sodium chloride solution by dilution with 25 volumes of 
water, nor have we been able to separate it by dialysis. 
Another protein fraction was obtained which was insoluble in sodium chloride solution 
but soluble in an alcoholic solution of sodium hydroxide. By slightly acidifying the 
alkaline solution with acetic acid the protein separated as a flocculent precipitate 
(Preparations IV and V.) 
The slightly acid alcoholic filtrates from Preparations IV and V yielded on dilution with 
water another protein fraction (Preparation VI and VII). 
 
 
The method of preparing the fraction obtained by acidifying the alkaline alcoholic extract 
(Preparations IV and V) suggests a type of protein having somewhat the properties of 
glutelins. However, in view of the enzymic activity present in the pulped fruit of the 
avocado, it is possible that some of the salt-soluble protein originally present had 
become denatured, and thus escaped extraction with the salt solution, to be later 
dissolved from the residue by the alcoholic alkali extractant. It is also possible that this 
fraction represents a type of protein hitherto uncharacterized, and which does not fit into 
the generally accepted classification of proteins. 
It is of interest to note that Preparations VI and VII were separated from an alcoholic 
solution, although practically no protein could be extracted with alcohol from the fresh 
avocado. These preparations may represent a secondary product formed by the action 
of the alkali upon an original protein of more complex structure. 
 
The three different proteins isolated are clearly differentiated from one another both by 
their elementary composition (Table I) and by the distribution of nitrogen (Table II). 
The avocados1 used in the work described in this article were grown in California and 
were mostly of the Fuerte variety. The edible portion contained about 71 per cent water, 
20 per cent oil, and 0.38 per cent nitrogen, equivalent to 2.37 per cent crude protein 
(Nx6.25). The proteins actually isolated amounted to 0.67 per cent of the fruit, and 
represented 28.5 percent of the total nitrogen.  In its content of nitrogen the avocado 
leads almost all fresh fruits. 
A meal prepared by drying the fresh fruit at room temperature, and extracting the oil 
with ether was found to be unsuitable for a study of the proteins on account of changes 
that had occurred in the proteins, caused apparently by the action of enzymes. It was 
therefore decided to abandon the idea of preparing a meal, and to work directly with the 
fresh fruit. 
It was found that three successive extractions of the fresh pulp with 10 per cent sodium 
chloride solution removed 39 per cent of the total nitrogen, and that 32 per cent of this 
nitrogen was contained in the coagulum formed by heating the salt extract. 
The residue that had been extracted with sodium chloride solution still contained 39 per 
cent of the original nitrogen of the meal that was extract-able by boiling 0.1 N, 60 per 
cent alcoholic solution of sodium hydroxide. By neutralizing the alkaline extract with 
acetic acid a precipitate formed which contained 26 per cent of the total nitrogen of the 
meal. 
By successive extractions of the fresh avocado with 10 per cent sodium chloride 
solution and alcoholic sodium hydroxide, 78 per cent of the total nitrogen was removed, 
58 per cent of which was protein nitrogen precipitable by tannic acid. 
Preparation of Proteins 
The edible portion of the fruit was finely comminuted and extracted with 10 per cent 
sodium chloride solution. The clearly filtered, light brown salt extracts were neutral to 
litmus, but on standing they gradually turned darker and became acid in reaction, with 
the separation of a small quantity of protein, which completely dissolved when the 
solution was neutralized with 0.1 N sodium hydroxide. When slowly heated the sodium 
chloride extracts yielded a flocculent precipitate at 68°. Continued heating of the extract 
up to boiling gave no further significant amount of coagulum. 
Heat-Coagulable Fraction. Preparation I—The salt extracts were heated to boiling, and 
the coagula were thoroughly washed, first with distilled water which had been slightly 
acidified with acetic acid, then with boiling distilled water, and finally with boiling 95 per 
cent alcohol These washings were repeated many times. The protein was then dried in 
the usual way with absolute alcohol and ether. The final product consisted of a light, 
cream-colored powder which weighed 22 gm. This quantity represented the yield 
obtained from 5 kilos of avocados. 
Fraction Precipitated by Ammonium Sulfate. Preparation II.—To the clear, filtered, 
sodium chloride extract of 3422 gm. of avocados was added enough ammonium sulfate 
to make the extract 67 per cent saturated with this salt. The precipitate which separated 
was dissolved by addition of water. It had been previously found that this protein could 
not be separated by dialysis. An attempt to separate the protein by heat coagulation 
gave a finely divided precipitate which could not be removed by filtration or 
centrifugalization. Addition of 5 volumes of 95 per cent alcohol, however, caused the 
separation of a flocculent precipitate. The thoroughly washed and dried product 
weighed 4 gm. 
Fraction Precipitated by Acidification of Sodium Chloride Extract with Acetic Acid. 
Preparation III.—Acidification of a 10 per cent sodium chloride extract of the avocado 
with acetic acid gave the maximum precipitation at pH 3-9. 
The washed and dried precipitate obtained in this way from 1618 gm. of avocado 
weighed 4 gm. 
Alcoholic Alkali-Soluble fraction. Preparations IV, V, VI, and VII.—The residue 
remaining after exhaustive extraction of 7 kilos of avocado pulp with 10 per cent sodium 
chloride solution was boiled for 10 minutes with a 0.1 n, 60 per cent alcoholic solution of 
sodium hydroxide. The alkaline extract was readily filtered through a folded filter paper, 
and the clear filtrate was acidified with acetic acid. The flocculent precipitate which 
separated was washed many times with distilled water which had been slightly acidified 
with acetic acid, and was finally washed with hot 50 per cent alcohol After drying in the 
usual way, 7 gm. of light material were obtained (Preparation IV). 
In a similar way, Preparation V (12 gm.) was obtained from 5040 gm. of avocado. 
The slightly acidified, 60 per cent alcoholic filtrate from Preparations IV and V still 
contained considerable protein which was precipitated by addition of several volumes of 
water. The dried and purified precipitates (Preparations VI and VII, respectively) 
consisted of light colored powders. Preparation VI weighed 4 gm. 
The elementary composition of the different protein fractions obtained as described is 
given in Table I. 
Preparations I and IV were obtained from a lot of avocados of the Taft variety. The other 
preparations were made from the Fuerte variety. 
Analyses of Proteins by Van Slyke Method.—The general properties and elementary 
composition of the six preparations described indicate that we were dealing with three 
different proteins. No essential difference was observed between Preparations I, II, and 
III, although prepared by different methods. Preparations IV and V were practically 
identical in their behavior and composition, but distinctly different from the preceding. 
Preparations VI and VII were alike, but differed from all the others. As representatives of 
the three proteins, Preparations I, V, and VI were analyzed by the Van Slyke method. 
The results are given in Table II. 
Determinations of Tyrosine, Tryptophane, and Cystine. 
The determination of tryptophane was carried out essentially by the colorimetric method 
of May and Rose (2). Tyrosine was determined colorimetrically by the method of Folin 
and Looney (3). In addition to the values found for cystine by the Van Slyke method, 
there are also included in Table II figures for this amino acid obtained colorimetrically. In 
connection with a comparative study of methods for the estimation of cystine, Dr. M. X. 
Sullivan of the Hygienic Laboratory, estimated the cystine in Preparations I and V 
according to the colorimetric method developed by him (4) which has been shown to be 
highly specific. In view of the well known uncertainty regarding values for cystine as 
determined by the Van Slyke method, and the high degree of specificity shown for the 
Sullivan method, the percentages obtained by the latter method are to be regarded as 
expressing more accurately the actual amount of cystine present in the proteins. This 
view is strongly supported by correlations observed in connection with the results of 
feeding experiments carried on in this laboratory with certain cystine-deficient proteins, 
and the cystine content of these proteins as determined by these two methods. 
Summary 
Three different types of protein have been isolated from ripe avocados, Persea 
americana Mill. By extraction of the edible portion of the fruit with 10 per cent sodium 
chloride solution a protein was obtained which behaved in most respects like a globulin. 
In the saline solution it coagulated at 68°, and was precipitated both by addition of 
acetic acid and by making the solution 67 per cent saturated with ammonium sulfate. It 
contained 15-31 per cent nitrogen. 
By slightly acidifying with acetic acid an alcoholic alkali extract of the residue remaining 
after the extraction with sodium chloride, a second protein containing 13-42 per cent 
nitrogen was obtained. Dilution of the slightly acid, alcoholic filtrate with water caused 
the separation of a third protein fraction. This protein contained 16.23 per cent nitrogen. 
Elementary composition and distribution of the nitrogen by the Van Slyke method in the 
proteins were determined. Cystine, tryptophane, and tyrosine were determined 
colorimetrically. 
Bibliography 
1. Smith, A. H., J. Biol. Chem., 63, 71 (1925). 
2. May, C. E., and Rose, E. R. J. Biol. Chem., 54, 213 (1922). 
3. Folin, O., and Looney, J. M., J. Biol. Chem., 51, 421 (1922). 
4. Sullivan, M. X., Pub. Health Rep., U. S. P. H. S., 41, 1030 (1926); J. Biol. Chem., 74, 
p.xiv (1927). 
 
1The avocados were furnished by the Calavo Growers of California, and were of the 
grade that are marketed under the trade name "Calavo." 


Proteins of the Avocado (Persea Americana Mill)

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Proteins of the Avocado (Persea Americana Mill)

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