231
t /° o+. . . .
1 /
25 o 259, 878-883
20 ° o
15 o 5Umbreit, W W, Burris, R H and Stauffer, J F (1959) 'Manometric
1 1 5 10 ~s~(mM-1 )
Km
Figure 1 Lineweaver-Burk plot of acid phosphatase without
addition of phosphate and in the presence of different phosphate
concentrations (indicated on the figure)
students) collect the ceils from one of the six flasks by low speed
centrifugation. After washing the cells with 0.1 M acetate buffer,
pH 3.8 (especially important for cells grown with 100 mM
phosphate, since even a small amount of phosphate remaining
could cause inhibition of the enzyme during activity measure-
ment), cells suspensions are made in the same buffer giving
absorbances at 600 nm in the range 0.2-0.4. After recording the
absorbance at 600 nm, 1 ml of this suspension is used for
measuring the enzyme activity in a standard reaction mixture (it
is important to mix the yeast suspension vigorously on a
vibromixer before the 1 ml aliquot is taken). After the reaction
has been stopped by the addition of NaOH, the suspension is
centrifuged and the absorbance (A400) of the supernatant is
measured. Specific enzyme activity in terms A400/A60o is plotted
against phosphate concentration in the growth medium. The
results (Fig 2) clearly show that with increasing phosphate
Ill|YIIi
ACTIVITy
I A~-~-~ I e
o i 2 a 4
Figure 2 Acid phosphatase activity of yeast cells grown in media
with different concentration of inorganic phosphate
concentration the acid phosphatase activity of the cells de-
creases, due to the repression of enzyme synthesis. At 2 mM
phosphate, nearly total repression of the repressible isoenzyme
is achieved and even at fifty-fold higher concentration (100 raM)
enzyme activity does not significantly decrease further. This low
but significant activity, representing about 20% of the total
activity in the phosphate depleted medium, is due to the
presence of constitutive isoenzyme, synthesis of which is not
influenced by phosphate.
BIOCHEMICAL EDUCATION 16(4) 1988
References
1Suomalainen, H, Linko, M and Oura, E (1960) Biochim Biophys Acta
37, 482-490
2Toh-e, A, Ueda, Y, Kakimoto, S J and Oshima, Y (1973) J Bact 113,
727-738
3Barbari6, S, Kozuli6, B, Ries, B and Mildner, P (1984) J Biol Chem
4Mildner, P, Ries, B, Golubi6, Z and Poto, E (1972) J Gen Microbio172,
o [,J:o
403-405
Techniques', Second edition, pp 268-281, Burgess Publishing Co,
Minneapolis
T h e Partial Purification and Characterization of
Lactate D e h y d r o g e n a s e
EDWARD C WOLF
Department of Chemistry
University of Nevada
Las Vegas, Nevada 89154, USA
Introduction
Lactate dehydrogenase (LDH; EC 1.1.1.27) offers several
advantages over other possibilities as the enzyme of choice for a
student's first exposure to a purification scheme. These advan-
tages include its relative abundance in an easily obtainable
source, high specific activity, stability, ease of assay, well-
understood kinetics, and the availability of a good competitive
inhibitor. Also, when checking for purity using gel electrophor-
esis, one tube can be stained with a general protein stain while a
second tube can be stained with a dye system specific for LDH to
show which protein band contains LDH.1 This can be compared
to an L D H control to identify the isozyme of L D H that was
purified.
The simple, inexpensive procedure reported here uses equip-
ment and materials normally found in biochemistry laboratories
and yet incorporates several important biochemical techniques
including spectrophotometry, chromatography, centrifugation,
and electrophoresis. The procedure presented is a modification
of one reported by Reeves. 2
Materials
All chemicals were obtained from common sources and were
reagent grade or its equivalent. The porcine heart L D H control
was obtained from Sigma Chemical Company. Sephadex and
Sepharose were obtained from Pharmacia, Inc. Hearts from
freshly slaughtered pigs were obtained from a local slaughter-
house. For the purification described here one pig heart is a
o-.1~ convenient starting mass (300 g). An equivalent mass of beef or
rabbit heart may be substituted. If desired the experiment can be
proportionally scaled down.
Methods
Enzyme Assay The assay mixture contains 2.7 ml of 0.1 M
Tris, pH 7.4, 0.1 ml of 22.7 mM sodium pyruvate and 0.1 ml of
5.0 mM N A D H (prepared fresh). The reaction is initiated by the
addition of 0.1 ml of diluted enzyme and the decrease in
absorbance at 340 nm as a function of time is monitored. The
enzyme is diluted in Tris buffer until a reasonable rate is
obtained. The reference and control contain all the components
except the N A D H and pyruvate respectively. The total number
of units for each purification step is calculated from:
Units = (A/mins - A/minc) x (1 unit) x (DF) x (Vol)
2.0733 A/min 0.1 ml
232

where A/min is the initial slope of the absorbance versus time Table 1 Purification of lactate dehydrogenase from pig heart
graph, subscript s refers to the sample and c to the control,
2.0733 is the change in absorbance expected for the disappear- Volume Protein Specific Yield
ance of one micromole of N A D H in 3.0 ml; DF is the dilution Procedure (ml) (rag) Units~ Activity Purification (%)
factor of the enzyme, 'Vol' is the total volume in ml of enzyme
for a given purification step and 0.1 ml is the volume of enzyme Crude
Extract 500 8 496 42 710 5.03 -- --
assayed. 40-60%
Preparation of Crude Extract The heart, freed of fat, vessels Salt Cut 34 1 406 18 730 13.3 2.6 44
and valves, is cut into strips and processed through a meat DEAE Ion
grinder. The ground heart is homogenized in a Waring Blender Exchange 27 150 16 920 113 22.5 40
with two volumes of cold buffer (0.1 M Tris, pH 7.4) for one
minute. The extract is centrifuged at 3000xg for 15 min and the aAll enzyme assays were performed in triplicate
supernatant filtered through Miracloth.
Salt Fractionation The extract is brought to 40% saturation by

8__11
iO
slowly adding solid (NH4)2SO 4 at 4°C (22.6 g per 100 ml of
solution). The suspension is stirred for an additional 30 min and
then centrifuged at 14 500×g for 15 rain. The supernatant is 6
brought to 60% saturation with (NH4)2SO4 (12.0 g per 100 ml of
4
solution), stirred for 30 min, and centrifuged. The pellet is
resuspended in 20 ml of cold buffer, and the suspension clarified 2
by centrifugation at 3000xg and desalted on a Sephadex G-25 0 :::::==:::: ::=:
column (2.5 × 17.5 cm). The protein is eluted with 0.1 M Tris,
pH 7.4. o ""i0 ~ 6 ~ 6 i0" ~6" "~6 %" "~0
Anion Exchange Chromatography The desalted solution (30-
m:
35 ml) is applied to a DEAE-Sepharose CL-6B column (2.5 × t--
0.8 B
17.5 cm) at 4°C previously equilibrated with 0.1 M Tris, pH 7.4,
and the protein eluted with a linear salt gradient (starting buffer o.6
250 ml 0.1 M Tris, pH 7.4; final buffer 250 ml 0.1 M Tris, pH
~_ 0 . 4
7.4 containing 0.4 M NaC1). Five ml fractions are collected and
aliquots assayed for L D H activity and their protein concen- nr- O.2

tration measured. The fractions with the highest L D H activity
are pooled.
Protein Determination Protein concentration may be deter-
mined by the Lowry m e t h o d ) The standards range from
0.03 mg to 0.30 mg bovine serum albumin per ml of 0.1 M Tris,
pH 7.4. A preliminary estimation of the protein concentration
may be obtained by the 280/260 method 4 in order to ascertain an
appropriate dilution factor for each of the three samples.
Kinetics The assay mixture contains 1.8 ml of 0.1 M Tris,
pH 7.4; 0.1 ml of 5.0 mM N A D H ; and 0.1 to 1.0 ml of 1.5 mM
sodium pyruvate. The difference in volume is made up by
appropriate addition of water. The reaction is initiated by the
addition of 0.1 ml of diluted enzyme from the anion exchange
chromatography step. The enzyme is diluted such that the assay
containing 1.0 ml of pyruvate results in a reasonably fast rate.
This same dilution factor is maintained in all the kinetic assays.
For the inhibition study 0.1 ml of 3.0 mM sodium oxamate is
substituted for an equivalent volume of Tris.
Disc Gel Electrophoresis Fifty txg of protein from the anion
exchange chromatography step are analyzed by polyacrylamide
disc gel electrophoresis at 4°C using a modified procedure of
Cooper. 5 Ten to 20 ~g of purified porcine heart L D H are
electrophoresed as a control. Tube gels are prepared with a
7.5% acrylamide running gel in Tris, pH 8.6, and a stacking gel
in Tris-phosphate, pH 6.6. The upper reservoir buffer is Tris-
glycine, pH 8.3 and the lower reservoir buffer is Tris pH 8.6.
Bromophenol blue and glycerol are added to the protein solution
and the tubes are electrophoresed at 1 mA/gel. The tubes were
either stained for protein using Coomassie brilliant blue G-250
or stained for lactate dehydrogenase usin~ the phenazine
methosulfate/p-nitroblue tetrazolium system. 1'~
Results
Table 1 shows the results of the partial purification of lactate
dehydrogenase from pig heart. The (NH4)2SO4 cut results in a
two-and-a-half-fold increase in specific activity with a con-
comitant loss of over half the original activity. The high loss is
easily tolerated by the large initial activity. The D E A E ion-
exchange step results in about a ten-fold reduction in total
rr~ 0.(I
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . j
0 t0 20 30 40 50 60 70 80
FRACTION NUMBER
Figure 1 DEAE anion exchange chromatography. A 40-60%
(NH4)2SO 4 cut of the crude extract was desalted, applied to a
DEAE-Sepharose CL-6B column and eluted with a linear salt
gradient as described in Methods. The fractions collected were
assayed for L D H activity (Figure 1A) and total protein (Figure
117)
protein with a minimal loss of enzyme activity. The success of the
ion-exchange step is illustrated in Figure 1. The bulk of the
protein is eluted very early from the column while the enzymatic
activity elutes with a much smaller protein peak half way through
the elution profile. Disc gel electrophoresis of an aliquot of this
smaller peak shows a number of protein bands present after
staining with Coomassie brilliant blue (Figure 2A). An alternate
gel, loaded with an aliquot of the smaller peak and stained with
the phenazine methosulfate/p-nitroblue tetrazolium system
(PMS/pNBT), shows which of these bands contain LDH (Figure
2B). Purified porcine heart L D H was applied to lanes C and D as
controls. Lane C was stained with Coomassie brilliant blue and
lane D with PMS/pNBT. The major heart LDH isozyme
migrates at the same rate as the band identified from the D E A E
peak as LDH. The very sensitive PMS/pNBT stain also picked
up two minor L D H isozymes in the control.
Aliquots of the peak from the D E A E ion-exchange step
containing the L D H activity were also used in a kinetic study of
the partially purified LDH. A Lineweaver-Burk plot of the data
is shown in Fig 3. The Km and Vmaxfor pyruvate calculated from
the plot are 0.14mM and 100.6 txmoi/min, respectively.
Oxamate is shown to be a competitive inhibitor with a Ki of
0.031 mM.
Discussion
There are three major techniques at the disposal of the
enzymologist for protein purification that utilize equipment

BIOCHEMICAL EDUCATION 16(4) 1988


A B C D
| Of course, additional steps could be added to increase the purity
Figure 2 Polyacrylamide disc gel electrophoresis. Fifty tLg of
protein from the D E A E ion exchange purification step was
applied to Lanes A and B. Twenty l~g of purified porcine heart
L D H was applied to Lane C and 10 I~g was applied to Lane D as
controls. Lanes A and C were stained with Coomassie brilliant
blue and Lanes B and D were stained with the L D H visualization
system as described in Methods
standard to an undergraduate biochemistry laboratory. These
techniques are differential solubility, chromatography, and
electrophoresis. Centrifugation, as a technique for protein
purification, requires an ultracentrifuge which is not always
available in an undergraduate laboratory. It was this author's
intent to devise an enzyme purification scheme that would
illustrate one variation of each of the three major techniques.
After considering twenty-five enzymes, representing the major
pathways of intermediary metabolism and selected pathways of
secondary metabolism, lactate dehydrogenase emerged as the
enzyme of choice. Among others, L D H offers the advantages of
stability (a high priority for a student purification), inexpensive
assay, interesting electrophoresis, and unambiguous kinetics.
The purification scheme reported here was not primarily
designed to yield a preparation of the highest specific activity
with the maximum percent yield since others have succeeded
admirably in this respect. 2'6 Instead, the intent was how best to
illustrate the major techniques in a project-oriented approach
utilizing a single enzyme. The two purification steps chosen,
ammonium sulfate cut and ion exchange chromatography, are
certainly widely employed by enzymologists. As shown in Table
1, the ammonium sulfate cut chosen sacrifices a large percent of
the activity. In any purification step there is a trade-off between
purity and yield. Whereas a broader cut could be used to
increase the yield, it was decided to favor purity since this is only
a two-step purification. Because each student starts with one
heart, even this large loss of activity will result in excess LDI-I for
the subsequent studies. On the other hand, Table 1 and Figure 1
show that the ion exchange step was particularly effective in
increasing the specific activity while minimizing the loss in yield.
BIOCHEMICAL EDUCATION 16(4) 1988
233
0.1~
E
O.IC
o
m
o
L.
(.2
m
0.0
-'0t 0 ' ' ' t'o' ' '
1/S ImM)
Figure 3 Kinetic analysis of partially purified LDH. Aliquots of
enzyme from the D E A E ion exchange purification step were
assayed from 0.05 mM to 0.50 mM pyruvate in the absence (A)
and presence (11) of O.1 mM oxamate as described in Methods
of the enzyme as time permits, but those described here are
suitable for a half-semester project (50 hours) that includes time
for an electrophoretic analysis and a kinetic study.
In the electrophoretic analysis of the L D H from the ion-
exchange step the appearance of a large number of protein
bands, while expected, is disappointing to the students. This
disappointment can be partially offset by identifying which of the
protein bands is LDH. In Figure 2, protein from the ion
exchange step was applied to both Lanes A and B. Lane A,
which was stained for protein, shows the many expected bands,
but Lane B, which was stained with the visualization system
specific for LDH, immediately identifies which of the protein
bands contains LDH. Heart tissue, while primarily containing
the H4 isozyme of LDH, also contains progressively smaller
amounts of the other four isozymes H3M , H2M2, HM3 and M4.
The higher the proportion of the H subunit, the faster the
electrophoretic mobility. 7 To identify the isozyme present in
Lane B, purified porcine heart L D H was applied to Lanes C and
D as a control. The protein stain (Lane C) could only detect the
most abundant isozyme (ie H4) which migrated at the same rate
as the band in Lane B. The more sensitive L D H stain (Lane D)
demonstrated that of the minor L D H isozymes detected, both
migrated more slowly. Therefore, Lanes C and D identified the
LDH band found in Lane B as the more abundant, faster
migrating H4 isozyme.
Even though the LDH at this stage of purity is a mixture of
isozymes it is still suitable to illustrate a kinetic study since the
more abundant H4 dominates the analysis. In fact, the KM
calculated from the Lineweaver-Burk plot in Fig 3 is identical to
that reported for purified H4 LDH. 8 A direct comparison of the
Vmax between this partially purified preparation and a purified
preparation is not appropriate since it contains less L D H per mg
of protein. The Vmax calculated from Fig 3 is, as would be
expected, less than the reported value. 8 Oxamate is a known
competitive inhibitor of LDH. 9 The use of oxamate in the
inhibition study illustrated in Fig 3 unambiguously demonstrates
competitive inhibition.
Whereas each instructor of an undergraduate biochemistry
laboratory will have a favored enzyme,1 0 ' 11 •12 LDH certainly
should not be overlooked. In a single project, L D H allows many
opportunities that were previously achieved only by separate
proteins in unrelated experiments.
Acknowledgements
This research was partially supported by grants from the UNLV
Foundation and the University Research Council, University of Nevada,
Las Vegas.
234
References
1Gabriel, O (1971) Methods Enzymol 22, 578-604
2Reeves, W and Fimognari, G (1966) Methods Enzymol 9, 288-294
3Lowry, O et al (1951) J Biol Chem 193, 265-275
4Kalckar, H (1947) J Biol Chem 167,461-475
5Cooper, T (1977) 'The Tools of Biochemistry', J Wiley, 228-231
6LeJohn, H and Stevenson, R (1975) Methods Enzymol 41,293-298
7Tietz, N (1982) 'Fundamentals of Clinical Chemistry' W B Saunders, p
655 and 599
SWiner, A and Schwert, G (1958) J Biol Chem 231, 1065-1083
9Novoa, Wet al (1959) J Biol Chem 234, 1143-1148
l°Busquets, M and Franco, R (1986) Biochem Educ 14, 84-86
llStrang, R (1984) Biochern Educ 12, 57-59
12Bonomi, F (1981) Biochem Educ 9, 12-14
A Visual-electrophoretic Method for Following the
Purification of Ribulose-l,5-bisphosphate Carboxyl-
ase Oxygenase
L PElqARRUBIA, J MORENO and P CARRASCO
Departament de Bioquimica i Biologia Molecular
Universitat de Valencia
A v Dr Moliner 50, Burjassot
46100 Valbncia, Spain
Introduction
The principles of protein purification are taught in most
undergraduate biochemistry courses. However, it is more
difficult to introduce the students to appropriate practical work
since the techniques required are often too complex, laborious
and expensive to be used in laboratory classes especially in
departments with many students and an equipment shortage.
Ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco)
(EC 4.1.1.39) accounts for approximately 50% of the total
protein in the leaves of higher plants, and is probably the most
abundant protein in the world. ~ This abundance, together with
its large size, makes it possible to extract and purify the protein
in few steps, making it ideal for introducing students to some of
the basic procedures used in protein purification. We describe
here an inexpensive isolation method, which is easy to perform
and that does not require sophisticated laboratory equipment.
Background
Rubisco is one of the plant proteins that is currently being
studied intensively due to some of its remarkable features. The
enzyme catalyzes both the carboxylation and the oxygenation of
ribulose-l,5-bisphosphate thereby starting both the Calvin cycle
and the photorespiratory pathway, 2 two apparently 'opposite'
metabolic routes. The protein of higher plants is an oligomer
with eight large (50-55 kDa) and eight small (12-16 kDa)
subunits. Genetic regulation of the enzyme is mediated by
nuclear and chloroplastic g~enomes, which code for small and
large subunits respectively. ~ Besides its enzymatic activity, the
protein may play an important role as a storage protein in view
of its selective degradation during leaf senescence. 4
Rubisco has been purified and characterized from a great
variety of photosynthetic organisms, 5 and different methods
have been developed to obtain the pure protein. 6 The purifi-
cation procedure proposed here is based on that given in Ref 7
with unpublished modifications and additions. It includes
fractionation with polyethylenglycol (PEG) 3350, and ion-
exchange chromatography on DEAE-cellulose. Sodium dodecyl
sulphate (SDS) polyacrylamide gel electrophoresis is carried out
to visualize the protein patterns of the different steps of the
BIOCHEMICAL EDUCATION 16(4) 1988
purification procedure. Due to its abundance and well-estab-
lished mol wt, the Rubisco subunits can easily be identified, even
in crude extracts (see Fig 3) with the aid of mol wt markers. The
accepted way of determining the degree of purification of an
enzyme is to assay its activity. However, methods to assay
Rubisco are based on the rate of formation of phosphoglycer-
aldehyde measured in a continuous spectrophotometric assay
employing coupled enzymes or measuring the uptake of 14CO2 in
a radioactive assay, both of which are too expensive and
complicated to be used by the students. In contrast, the
electrophoretic method we propose is easy to perform and allows
the purification process to be visualized in an instructive manner
for the students.
Materials
Leaves from orange trees were used, but leaves from any other
plant can be used as source of enzyme.
Media and solutions Extraction buffer: 100 mM Tris-H2SO4,
pH 8.0, containing 10 mM MgSO4 and 20 mM 2-mercapto-
ethanol. PEG 3350 precipitation solution: 60% (w/v) PEG in
extraction buffer. DEAE-cellulose equilibration buffer: 10 mM
Tris-H2SO4, pH 7.8. DEAE-cellulose gradient solution: 0.15 M
(NH4)2504 in equilibration buffer.
Solutions for electrophoresis were as in Ref 8 except that
N,N,N',N'-tetramethyldiamine (TEMED) was included in Tris
buffer solutions B and C. (A) 30% (w/v) acrylamide, 0.8% (w/v)
bis-acrylamide; (B) 0.5 M Tris-HC1, pH 8.0, 0.4% SDS, 185 Ixl
TEMED; (C) 1.5 M Tris-HCl, pH 8.8, 0.4% SDS, 400 ixl
TEMED, and (D) 10% ammonium persulphate (freshly pre-
pared). Electrophoresis buffer: 14.4 g Tris, 3.07 g glycine, 0.5 g
SDS in 500 ml H20. Samples were prepared by adding 42.8 Ixl of
a solution containing 0.27 M Tris-HCl, pH 8.0, 2.0 M 2-mer-
captoethanol, 6.2% (w/v) SDS, 31.1% (w/v) glycerol and
0.0075% bromophenol blue to 100 ixl of sample and boiling for 4
min. Fixing solution: 12.5% trichloroacetic acid (w/v), 25%
isopropanol (v/v). Staining solution 0.1% aqueous Coomassie
brilliant blue R-250. Reagents such as Tris, PEG 3350, and
DEAE-cellulose, were all purchased from Sigma Chemical Co.
The cost of the reagents is about $5 per group of students.
Laboratory equipment: Preparative centrifuge, UV-spectro-
photometer, fraction collector, and electrophoresis power
supply are needed.
Experimental Procedure
An outline of the purification scheme of Rubisco is shown in
Fig 1.
Extraction Cut leaves (about 20 g) into small pieces with
scissors and extract proteins in a blender with the addition of
4 ml extraction buffer per g fresh weight (about 80 ml). Filter
through muslin, add polyvinylpolypyrrolidone 2% (w/v) (1.6 g)
and stir for 10 min. Centrifuge the extract at 3300 g for 10 min
and save the supernatant to precipitate with PEG 3350.
Fractionation with PEG 3350 Make the supernatant 10% (w/v)
PEG 3350 by adding 20 ml PEG 3350 50% (w/v) (Ref 8) per
80 ml extract. Stir for 30 rain and centrifuge at 12000 × g for 20
min. Take the supernatant and make it 20% (w/v) PEG 3350 by
adding 33 ml PEG 50%. Stir and centrifuge as before and
discard the supernatant. The pellet is used in the next step.
These first two steps must be carried out at 4°C.
DEAE-cellulose column chromatography Dissolve the pellet in
about 25 ml equilibration buffer. Load the sample onto a
DEAE-cellulose column (2 × 12 cm) equilibrated with the same
buffer. Wash the sample with 15 ml buffer and elute with
0-0.15 M (NH4)2SO4 linear gradient. Collect 3 ml fractions and
determine the absorbances at 280 and 260 nm.