Use of gel filtration in the study of human amylase

CLINICA

918

USE OF GEL

FILTRATION

CHIMICA

IN THE

ACTA

STUDY

OF HUMAN

AMYLASE

P. WILDING Department

of Biochemistry, The General Hospital, Birmingham (Great Britain) (Received

January 7th, 1963)

SUMMARY Serum

and urine

were

G. IOO) and the presence tion

of the

amylase

technique

with

iso-enzymes

other

described serum

of amylase.

as an unbound

moiety.

by

The

gel filtration

results

investigated,

obtained

in respect

gel (Sephadex

determined

by a modifica-

possible

from

that

amylase

human

serum,

all have an electrophoretic

further

to molecular

indicate

association

of

existence

of

also the possible

indicate

that the amylases results

on dextran

fractions

LOON et al .4. The

VAN was

of pig pancreas

to y-globulin.

sources are similar

The

by

in the eluate

proteins

Also

juice, urine and extract similar

fractionated

of amylase

that

exists saliva,

pancreatic

mobility

the amylases

in serum (on paper)

from

all these

weight.

INTRODUCTION The

methods

are generally obtained

by

An

properties

for the stationary ases of human human

serum

serum

proteins

a starch with

the

serum. though

by paper

activity total

In the present of

to study or

activity work,

the amylase serum.

More

was

serum dextran

recently

solutions amounts

activity

with

acetone

fractions

separation

found

always

BAKER

size

found

MCGEACHIN from

by a modification

in the

and urine were fractionated ge16. Gel

filtration

acetone

separated

at

saline

In

in the

using

associated using

paper

and assayed

of the method

fraction.

fraction

affinity

of amylase

in all fractions

albumin

of the y-globulin

are

the amyl-

the amylase with

low

the albumin

AND LEWIS~

the paper

of amylase

and

with

accompanies

the presence

at

on several

to fractionate

AND PELLEGRINO~ they

sera

examined

depends

molecular

attempted

and showed

fractions

varying

the

in human

of the serum proteins

precipitation

solubility,

have

By this method

in the saline

“Sephadex”

point,

isoenzymes

is that

electrophoretic

electrophoresis

the protein

found

by

methods

the

in fractions

AND B.~SSANI~ used precipitation

in horse

fraction.

activity

workers

that

agar plate.

a rise in the amylase

columns

Several

CATTANEO not

eluted

of the

these

example

and claimed

et ~1.~.They

portion noted

used

such as isoelectric

y-globulin

the amylase LOON

For

impregnated

electrophoresis

to

medium.

low temperatures

been

separation

objection

heterogeneous.

physical

have

on the assay of enzyme

electrophoretic

temperatures. usually

which

based

of VAN

but the major addition

they

in cases of pancreatitis. by gel filtration

in combination

with

through ion

ex-

(‘En. Chirn. Acta, 8 (1963) 918-92t

GEL FILTRATION IN THE STUDY OF HUMAN AMYLASE

919

change by the technique of thin layer chromatography has previously been used for studying the isoenzymes of lactic dehydrogenase B. These gels act as molecular sieves and separate proteins chiefly according to molecular size (and shape). They are obtainable in various grades, differing in their degree of cross linkage and hence the range of protein molecular weights which can be separated. An additional advantage of this method is that after separation other physical and chemical properties of the isolated fractions can be determined, such as enzyme activity and electrophoretic mobility. In the course of the work it was found necessary to adapt VAN LOON’S method for determining amylase activity to the Technicon Autoanalyzer. Using this method of protein separation the amylases in several abnormal sera were studied.

normal

and

METHODS Separation of serum and urine proteins into fractions of decreasing molecular weight was carried out using a column of dextran gel (Sephadex G. IOO from Pharmacia Ltd., Uppsala, Sweden) 60 cm x z cm. The Sephadex G. IOO was equilibrated

50 El&ion

100 150 volume (ml)

Fig. I. Calibration curve for Sephadex de&ran gel column. The elution pattern of a mixture of 7s y-globulin and 3.5s albumin added to a Sephadex G. IOOcolumn (60 x 2 cm) prewashed and eluted with 0.85% saline. Peak I is the y-globulin and Peak II the albumin. in 0.85O/~ sodium

chloride and elution of the samples placed on the column was also carried out with this solution. The method of preparation of the column was the same as that described by FLODIN 5. The void volume of the column was about 40 ml.

To calibrate the column (i.e. determine its elution pattern) z ml of a solution containing 0.24 g 7 S y-globulin and 0.24 g 3.5 S albumin per IOO ml was placed on the column and eluted with 0.85% sodium chloride. The eluate was collected in sixty 2 ml fractions after discarding the first 40 ml (void volume). The protein content of the fractions was determined by ultraviolet spectrophotometry at 280 rnp. The

results are shown in Fig.

I.

Random checks made during test runs on serum showed the remarkable ability of the column to maintain an elution pattern identical with the calibration curve for at least 6 weeks. After this time there was a tendency for a more rapid elution of the protein fractions. Columns of similar dimensions were found to give almost identical elution patterns. Runs were made by placing 2 ml of serum, urine or other samples on the column and eluting with saline. After discarding the first 40 ml of eluate at least sixty 2 ml fractions were collected. Protein was determined as above. Amylase was determined in the eluate fractions by an adaptation of the method C&z. Chim.

Ada,

8 (1963)c)IB-gz4

P. WILDING

920

of VAN LOON et aL4 to the Technicon Autoanalyzer (Fig. 2). Eluate was incubated at 37’ with starch-buffer (0.0125~/~ starch in 0.05 M phosphate buffer at PH 7.0) for 12 min, 0.001 N iodine solution (VAN LOON’S working iodine solution diluted I in IO) added and the optical density of the blue colour measured at 660 m,u. It was necessary to carry out a control run (Fig. 2) omitting incubation to determine the amount of diminution of the final starch-iodine colour due to factors other than amylase in the fractions. These factors were found to be particularly concentrated in the fractions containing the 3.5 S protein (mostly albumin). Protein has a decolourising effect on the starch iodine colour which is dependent upon time and temperature. In order accurately to determine this effect it was found necessary to ensure that the time and temperature of mixing the iodine with the starch-sample line was the same during both -Test 0.32

ml/min

1.20 ml/min

Sample Air

2,9Oml/min

Starch-

2.9Omml/min

Iodine

Buffer

L-A Recorder

Fig. 2. Amylase estimation on the Technicon Autoanalyzer test and control runs. If the amylase activity of the sample placed on the column was known to be above 300 units per IOO ml then the concentration of starch in the substrate line was doubled (i.e. 0.025~&). No attempt has been made to develop a method which gives absolute values of amylase activity per unit volume of eluate. The method is merely a sensitive and accurate way of detecting and comparing amylase activity in individual fractions. Whole serum, urine and those eluate fractions which contained the highest levels of amylase activity were subjected to paper electrophoresis in 0.05 M phosphate buffer at pH 7.0, duplicate strips being run in each case. 80 ,~l portions of the samples were spotted on strips of Whatman 3MM paper (5 cm x 34 cm). Eight IO ~1 spots were placed 2.5 mm apart and 12 cm from the anode. A current of 2 mA per strip was then applied for 15 h. In some cases where the amylase activity was known to be high, smaller amounts of sample were spotted. One of each pair of the electrophoretic strips was taken directly from the electrophoresis bath and laid on a starch impregnated agar plate. The remaining electrophoretic strips were stained for proteins with bromphenol blue for 3 min. The plates were prepared by adding 0.1 g starch and 0.8 g New Zealand agar-agar to IOO ml of 0.05 M phosphate buffer at pH 7.0 and boiling. When the starch and agar had dissolved the solution was allowed to cool slightly. It was then poured evenly over a glass plate (20 cm x 20 cm) and allowed to set. Clin.

Chits.

Acta, 8 (1~63) 918-924

GEL FILTRATION IN THE STUDY OF HUMANAMYLASE

921

The plate with the paper strips lying on it, was incubated for one hour at 37” after which the paper strips were removed, taking care that the point where the origin on the paper was in contact with the starch agar had been marked on the plate. The agar plate was then sprayed with 0.01 N iodine. The amylase activity in the various electrophoretic fractions was seen as a clear zone against a blue background. The decolourising effect of protein on the starch-iodine blue colour increases with time. Therefore the plates were read immediately after staining with iodine, that is before there was any appreciable “protein effect”. Immunological assay of proteins was also carried out on the fractions containing amylase activity in order to determine which serum proteins, if any, were being eluted with the amylase. The techniques used were gel diffusion and immuno-electrophoresis using specific anti-sera. The normal sera used in this experiment were taken from healthy members of the hospital staff, while abnormal sera came from patients having raised serum amylase. RESULTS The adaptation to the autoanalyzer of VAN LOON’S method for estimating amylase was prompted by the large number of samples to be analyzed. However, it was soon apparent that the instrument gave reproducible results, a factor not always obtained with the standard method. Six normal sera were fractionated on the Sephadex G. IOOcolumn and the eluate fractions analyzed for amylase activity. The total serum amylase in all the normal sera was below 170 units per IOO ml and in each case the amylase activity was eluted from the column well after the elution of detectable major serum proteins. All the normal sera examined gave identical elution patterns. In Fig. 3 a typical Autoanalyzer trace obtained during the analysis of a normal serum is shown with its “blank” trace superimposed below it. The trace emphasises the necessity for accurate blanks. The first two peaks (I and z) coincide with the elution of globulins and albumin from the column and are due entirely to the high protein concentration. Fig. 4 gives a trace typical of those obtained when analysing serum from patients with acute pancreatitis. It again shows that all the amylase present was eluted as one band well after the elution of the major serum proteins. Serum from a patient with mumps gave an identical pattern. Several samples of normal urine, saliva and pancreatic juice were also fractionated on the Sephadex column. In each case only one band of amylase activity was demonstrated, its point of elution being identical to that obtained with serum. Three sera from patients who had undergone total pancreatectomy and yet who still had normal serum amylase levels were also analysed. In both cases the results were similar to those obtained with normal sera. It is of interest that when a commercial extract of pig pancreas (Pancreatin B.P.) was examined, it also gave an identical elution pattern. In every case already mentioned the amylase was eluted from the column at a point which suggests that amylase has an apparent molecular weight of less than 20.000. However, the results obtained using the extract from pig pancreas would indicate that the molecular weight of the amylase isolated from human serum and urine, is the same as that of pig pancreatic amylase, namely 45,000 (Ref. 7). FLODIN~ Clilz.CJzim.Ada, 8 (1963)91%924

922

P. WILDING

x

AMYLASE

120 UNITS 2

%

3

2

1

Fig. 3. Autoanalyzer trace obtained when determining amylase activity in fractions of eluate collected following fractionation of normal serum on Sephadex G. roe. Top trace is “Test” and lower trace “Blank”. Peaks I and 2 are due to decolourisation of starch-iodine colour by protein. Peak 3 is amylase activity.

AMYLASE

~Oi~ME

of ELUATE

7150 UNITS

%

lmll

Fig. 4. Autoanalyzer trace obtained during analysis of serum from patient with acute pancreatitis. Top trace is “Test” and lower trace “Blank”. Peaks I and 2 are due to dtcolourisation of starchiodine colour by protein. Peak 3 is amylase activity.

has suggested that this anomaly is due to interaction between the enzyme and the gel matrix. He claims this is a reasonable hypothesis in view of the similarity in structure of starch and dextran. Following paper elctrophoresis, serum, urine and eluate fractions containing amylase were examined by the modified technique of BAKER AND PELLEGRINO. The results obtained with samples having high amylase activity showed marked activity Clin.Chisa.

Acta, R (rg63)

9x8-924

GEL FILTRATION in the slow y-globulin possible

The technique paper

region.

to be certain

described

of protein

able amylase paper

was found

was examined albumin

serum

some

factors

in view

activity

it was im-

the amylase

due allowance

on the starch-iodine

high

was found

923

levels

for eluting

When

fractions

of the small

containing

low amylase

AND LEWIS

in any of the protein

was not unexpected

(20 ,ul). When

with

AMYLASE

in any of the bands.

was also investigated.

and other possible

activity

serum. This

was present

by MCGEACHIN

strips

OF HUMAN

In the case of serum

if amylase

electrophoretic

the effect

IN THE STUDY

amylase

of serum

activity

in the y-globulin

colour,

separated

volume

for

no measurfrom normal

applied

(800 units

fraction,

from

was made

to the

per IOO ml)

but none

in the

fraction.

Attempts

were

demonstrate

the

containing

the enzyme

were also carried efficiency ability

also

with

made,

presence

using

gel diffusion

of proteins

other

activity.

The

results

out in parallel

with

paper

respect

to the separation

of the column

to carry

and

than were

immuno

amylase always

electrophoresis,

in the

negative.

electrophoresis was found

to

fractions

These

to determine

of 7 S and 3.5 S proteins.

out this separation

eluate

techniques the column

In all instances

the

to be good.

DISCUSSION The introduction gation

an excellent It was found

and from

results indicate to molecular amylase.

normal

of the amylase and urine

that the amylases weight,

present

mobility

colour,

amylase

in the albumin

activity

were

studied,

of normal

When

results

marked

activity

“test”

and

were

determined was only

the findings

was

“blank”

when only

amylase

the

effect”

presence

high levels

VAN

used a total

in optical

LOON’S

of

of amylase

in the y-globulin difference

using

of

the abnormal

region. density

method

is at

of 2 ~1 for the assay

bands. containing

examined

by electrophoresis

by the method detected

one

differing

for “protein

indicate

demonstrated

AND LEWIS

than

with the y-globulin.

sera having

fractions

of BAKER

not

the

while

care to allow

to give an adequate

of more

that

to albumin,

did

These

with respect

properties.

electrophoretically taking

pig pan-

of these amylases

demonstrated

when

juice,

for the presence

obtained

although

in all 5 electrophoretic

and urine

the amylase

the

MCGEACHIN

the eluate

moves

G. IOO. identical

or mumps.

sources are identical

similar

was repeated,

fraction,

the

IO ,ul. However,

of amylase

with

serum

at a point

pancreatic

or chemical

to have

on Sephadex

pancreatitis

the possibility

mobility

serum required

at 660 rnp between

amylase

claimed

investi-

times to be

proteins.

urine,

with

was found

many

as one band,

saliva,

patients

or in other physical

in pancreatitis

starch-iodine

individual

from these different

when their technique

The volume

from

this does not exclude

AND LEWIS~

in biochemical

by gel filtration

was eluted

from

and no evidence

Nevertheless

However,

serum

amylase

serum has an electrophoretic

amylase

least

serum

method

it has been demonstrated

of isolating

point

MCGEACHIN

on the

method

serum

in electrophoretic normal

as an analytical

However,

serum was fractionated

that normal

the elution

creas,

recent.

and reproducible

In this study with

of gel filtration

is comparatively

amylase

from

the gel

on paper

of BAKER AND PELLEGRINO

in the y-globulin

AND PELLEGRINO,

region.

on starch-agar

For normal

but not with

filtration

column,

and the mobility

of the plates,

serum this agrees

those of MCGEACHIN

AND

LEWIS. Clin.

Chim.Acta, 8

(1963) gI8-924

P. WILDING

924

The analyses of the sera from patients who had undergone total pancreatectomy were essentially the same as those for normal sera. This is in agreement with the findings of ROE et a1.8 and MCGEACHIN et aks who have both shown that the serum amylase levels in rats are unaffected by pancreatectomy. The identical behaviour on the gel filtration column of amylase from serum and urine suggests that the ready excretion of amylase by normal kidneys is a simple process. There is no evidence for the carriage of amylase in serum by other proteins. Certainly the amylase isolated in these experiments was never shown to be associated with any other Provided a large number study of other

known protein. that suitable methods are available for measuring enzyme activities in of samples, the technique of gel filtration might well be applied to the serum enzymes, especially those already known to exist in iso-enzyme

form. ACKNOWLEDGEMENTS The author wishes to thank Dr. R. GADDIE for constant help and encouragement, Dr. W. T. COOKE and Dr. B. E. NORTHAM for advice and useful criticism, Dr. J. F. SOOTHILL for carrying out the immunological assays and Mr. J. CHATTERLEY for technical

assistance. REFERENCES

1 C. CATTANEO AND B. BASSANI, Boll. SOL. Ital. Biol. Sper., 13 (1938) 424. 2 R. W. R. BAKER AND C. PELLEGRINO, Stand. J. Clin. Lab. Invest., 6 (1954) 94. * R. L. MCGEACHIN AND J. P. LEWIS, J. Biol. Chew., 234 (1959) 795. 4 E. J. VAN LOON, M. R. LIKINS AND A. J. SEGER, Am. J. Clin. Pathol., 22 (1952) 1134. 6 P. FLODIN, D&ran gels and their apfilications in gel filtration, Pharmacia Ltd., Uppsala, ~962. B TH. WIELAND AND H. DETERMANN, Experientia, 18 (1962) q?r. ’ C. E. DANIELSSON, Nature, 160 (1947) Sgg. 8 J. H. ROE, B. W. SMITH AND C. R. TREADWELL, Proc. Sot. Exptl. Biol. Med., 87 (1~54) 79. O R. L. MCGEACHIN, J. R. GLEASON AND M. R. ADAMS, Arch. Biochem. Biophys., 75 (1958) 403. Cl%&.Chim. ii&,

8 (1963) 918-924