TRANSPORT AND TOXICITY 0F
SULFUR. DIOXiDE EN THE YEAST
SACCHAROMYCES CEREViSIAE VAR.
ELLEPSOIDEUS

_ Thesis for the Degree of Ph. D.
MlCHIGAN STATE UNIVERSITY
BASIL MACRIS
21972

 

‘Iliiii'il‘flmit]Iiiiiiiflililmifliflifiu “ mu“
3 1293 01591 4199 Michigan State
University

 

This is to certify that the

thesis entitled

TRANSPORT AND TOXICITY OF SULFUR DIOXIDE IN THE
YEAST SACCRAROMYCES CEREVISIAE VAR. _E_I_.ELIPSOIDEUS

presented by

Basil Macris

has been accepted towards fulfillment
of the requirements for

Ph.D. Food Science and

Human Nutrition

M W «

Dr . Pericles Markakis

 

 

degree in

 

Major professor

Date August 2, 1972

 

0-7639

    
      

   

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800K BWUERY NC.
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A B S T R A C T

TRANSPORT AND TOXICITY OF SULFUR DIOXIDE
IN THE YEAST
SACCHAROMYCES CEREVISIAE VAR. ELLIPSOIDEUS
By

Basil Macris

Investigations were carried out on the tranSport and
toxicity of sulfur dioxide in the yeast §. cerevisiae var.
ellipsoideus.

It was found that 302 was transported inside the cell
by a mediated tranSport system.

The transported SO2 was not all retained inside the
cell and a certain portion of it could be removed by washing.
The amount of sulfur dioxide taken up by the cells as well
as that which could not be washed out was increased as the
pH value of the incubation mixture was decreased.

The $02 transport system operated very rapidly. The
802 reached its maximum level in about two minutes. There
were two equilibria involved in this transport. The first
was a chemical equilibrium which regulated the availability
of substrate (molecular 802) to this transport. The second
was a tranSport equilibrium which regulated the inward and

outward fluxes of 802 across the cell membrane.

B8811 Macris

Kinetic data showed that SO tranSport was saturable

2
and conforms to the MichaeliS-Menten type kinetics. Also
the transport System was apparently temperature dependent
and it was irreversibly heat inactivated.

Molecular 802 is the only transported form. The H805
and SO? ions are not taken up by this yeast.

In regard to $02 toxicity, it was found that the sup-
pression of cell growth was dependent upon the amount of
molecular 802 and the time during which this form remained
in contact with the cell. The curve which relates cell
viability and time to 802 exposure is linear on a semilog
plot.

A preliminary study on the localization of 358 in cells
exposed to 35802 showed that sulfur dioxide was not actively
metabolized. Practically all 802 which reacted inside the
cell. was found in the small molecule fraction. Most likely.
SO2 reacts with compounds containing carbonyl groups as well

as with other molecules inhibiting various steps which causes

termination of cell growth.

TRANSPORT AND TOXICITY OF SULFUR DIOXIDE
IN THE YEAST

SACCHAROMYCES CEREVISIAE VAR. ELLIPSOIDEUS

 

By

W"
Basil Macris

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

1972

{51 ACKN O'w'L EDG EN EN T5

The author is greatly indebted and appreciative
of his major professor. Professor P. Markakis, for guidance,
encouragement and constructive criticism during his
graduate work. He also wishes to eXpress his appreciation
to Professors C. L. Bedford. L. L. Bieber, T. Corner and
R. McFeeters for their help. advice and critical reading

of this manuscript.

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . .
LIST OF FIGURES . . . . . . . . . . . . . . .
CHAPTER

I. INTRODUCTION 0 . .

II. LITERATURE REVIEW . . . . . . . . . .

III. MATERIALS AND METHODS

organi Sm o o o o o o o o o o o o o 0
Synthetic Growth Media . . . . . . .
Preparation of Radioactive 302 . . .

Molecular Species of 802 in Aqueous
Solutions

Analytical Determination of 802. . .

Preparation of the Samples . . . . .
IV. RESULTS AND DISCUSSION . . . . . . . .

Characterization of 802 Uptake by
yeaStCellsoooooooocooo

Measurement of Sulfur Dioxide Pool .

SO2 TranSport as a Function of Time

Identification of SO2 TranSport System

Transport of Molecular 802, H503, and SO3

802 TranSport and Toxicity . . . . .

Localization of 802 in the Yeast Cell

iv

vi

vii

10
10
10
11
12

17
22

)0

3O
31
BL;
37
50
57
60

CHAPTER

V.

SUMMARY AND CONCLUSIONS

LITERATURE CITED . . . . .

APPENDIX 0 O O O O 0 O O O

Table

l.

7.

LIST OF TABLES

Percent distribution of the three SO molecular
species in aqueous solutions as a fuRction of pH

Determination of pellet intercellular Space and
cell volume of S. cerevisiae var. ellipsoideus .

Comparison between direct and indirect method
of SO tranSport in measuring SO transport
in S. cerevisiae var. ellipsoideus . . . . . . .

35
Fate of SO upon incubation with S. cerevisiae
ValueIIILSoneuS.o.............

Effect of washing of cells of S. cerevisiae var.
ellipsoideus after incubation with 55302 . . . .

Effect of metabolic inhibitors on SO tranSport
in S. cerevisiae var. ellipsoideus .2. . . . . .

Distribution of 35$ in various fractions of the
yeast S. cerevisiae var. ellipsoideus . . . . .

vi

Page

19

26

29 '

32

33

48

63

9.

10.

11.

12.

LIST OF FIGURES

Page

Percent distribution of molecular SO .

bisulfite (H805) and sulfite (80:)

forms as a function of pH in 802 aqueous

SOlution O O O 0 I I O O O O O O O 0 O O O O O O 18

Packing rate of S. cerevisiae var.
€111p501deu3 Cells 0 o o o o o c o o o o o o o o 25

TranSport of SO in S. cerevisiae var.
ellipsoideus asza function of time . . . . . . . 35

 

Time course of SO tranSport in the yeast
S. cerevisiae var. ellipsoideus . . . . . . . . 36

Sulfur dioxide tranSport in S. cerevisiae var.
ellipsoideus as a function of 802 concentration. 39

 

Lineweaver-Burk plot of SO tranSport in
S. cerevisiae var. ellipso deus . . . . . . . . 40

Effect of temperature on SO transport in
S. cerevisiae var. ellipsoi eus . . . . . . . . 42

Thermal inactivation of the SO transporting
system in S. cerevisiae var. eIlipsoideus . . . 44

Time course of SO tranSport in S. cerevisiae
var. ellipsoideus as a function of pH . . . . . 51

Relationship between rate of SO tranSport in
S. cerevisiae var. ellipsoideus . . . . . . . . 53

Relationship between rate of SO2 tranSport in
S. cerevisiae var. ellipsoideus and amount of
mOJ-ecular 502 I O I I O O O I O O I O O O I O 0 51+

Percent survival of S. cerevisiae var. ellipsoideus
incubated with different SO concentrations

and times 0 O O O O O O O 20 O O O O O O O O O 0 59

vii

Figure Page

13. Percent survival of S. cerevisiae var.
ellipsoideus incubated at various total SO2
concentrations and pH levels . . . . . . . . . . . 61

viii

CHAPTER I
INTRODUCTION

The problem of finding satisfactory methods for pre-
serving foods has been a challenge to mankind for centuries
and our evolution to an industrial society has not lessened
this problem.

Sulfur dioxide. bisulfite and sulfite salts are used
extensively in a wide variety of foods and food products for
the purpose of preservation from microbial deterioration.
Literally hundreds of millions of people have consumed sulfur
dioxide in the low concentrations permitted in foods without
apparent harm. It is one of the most common beverage pre-
servatives (Jacobs. 1944; Braverman. 1963). In the fermen-
tation industries SO2 is used as a selective inhibitor of
undesirable Organisms (kean and Mash. 1956). A rather unique
application of sulfur dioxide involves the bleaching of Spe-
cialty products such as Maraschino cherries (Bullis and Wiegand.
1931) and the decolorizing of sugar-cane and sugar-beet Juices
in the sugar industry. Treatment of grapes with sulfur dioxide
reduces molding and repels insects (Joslyn. 1952). In the
case of flour it modifies the baking characteristics of flour

by breaking disulfide bonds in the gluten molecule. Sulfur
dioxide. bisulfite and sulfite salts are also used to prevent
enzymatic and non-enzymatic browning and preserve the color
of various food products (Vas and Ingram. 1999; Joslyn and
Braverman. 1959; Kyzline et al.. 1961). Various pharmaceu-
tical formulations use 802 as an antioxidant (Jenkin et al..
1951; Remington, 1961).

Contrary to its wide commercial uses sulfur dioxide is

one of the most harmful air pollutants (West. 1970); it
affects both living organisms and inanimate structures.
Flue gases from smelters or power stations may contain suf-
ficient sulfur dioxide to cause serious damage to vegetation.
It is also toxic to the human and animal reSpiratory system
(Fairhall. 1957) and it has been implicated in eye irritation
(Nhittenberger and Frank. 1963). In the food field the main
disadvantage of sulfur dioxide are its unpleasant flavor and
its destructive action on thiamine (Thomas and Berryman. 1999).

DeSpite the importance of $02 as a food preservative the

mechanism of transport of SO inside the cell. as well as its

2
metabolism in the living cell are not well understood. The
purpose of this study is to clarify some of the mechanisms
relative to tranSport, toxicity and metabolism of sulfur

dioxide in yeast cells.

CHAPTER II
LITERATURE REVIEW

For many centuries SO2 has been used for protection of
foods from microbial deterioration caused by yeasts. molds
and bacteria. .According to Joslyn and Braverman (1959).
fumes of burning sulfur were used as a sanitizing agent in
wine-making by the ancient Egyptians and Romans. However.
the literature on the mechanism of its antimicrobial action
is very limited. Although much knowledge has been obtained
on the inhibition of various metabolic processes in microbial
cells by certain food preservatives. the factor which deter-
mines the antimicrobial effect is not clear (Hyss. 1948;
Bosund. 1962). Lipid solubility and surface activity were
correlated to antimicrobial actions of various series of
compounds (Meyer. 1937; Cavill and Vincent. 1949; Cavill
et al.. l9h9; Danielli, 1950; Hirai. 1957).

Wyss (1998). pointed out that the preserving effect
of $02 solutions is derived from their inhibiting influence
on enZymes of the living cells. EnZymeS containing 8-8
bonds may be inactivated by reducing substances. e.g. by
sulfur dioxide which breaks S-S bonds and reduces them to
SH-groups. Furthermore, the formation of sulphonates of
sulfur dioxide with carbonyls. e.g. acetaldehyde. is well

known.

Oka (1960 a-e; Oka, 1962 a—f) studied the antimicro-
bial effect of various food preservatives and classified
them into two groups. according to the manner by which
this effect is determined. In the first group belong the
acid preservatives, salicylic. benzoic, sorbic and dehydro-
acetic acids, their esters and phenols; the second group
includes quinones and nitrofurane. Sulfur dioxide aqueous
solutions belong to the first group. The acid antiseptics
affect all stages of microbial growth. The lag phase is
extended, the specific growth rate during logarithmic phase
decreases and the total yield also decreases (Huntington,
1995; Nomoto et al.. 1955). The antimicrobial effect of
acid antiseptics is strongly pH-dependent in that they are
powerful inhibitors of microbial growth under acidic conditions
and ineffective at neutrality. This appears to be dependent
on the amount of unionized molecular fraction which increases
as the pH value decreases. .At any pH. equal concentrations
of unionized molecules of these compounds show similar anti-
microbial effects (Hahn and Conn. 1949). In the case of
$02 solutions Bosund (1962) explained this by assuming that
the unionized form penetrates the cell wall of the micro-
organisms more rapidly than do the ionic forms (H803. 50;).
Consequently greated amounts can enter the cell and interfere

with its life processes.

Studies on the effect of SO2 solutions on the growth

of different microorganisms at various pH levels showed

that not only the unionized but also the ionized forms
(H805. so?) had an appreciable antiseptic effect on those
microorganisms. This work indicated that the unionized

form was greated than 1.000-fold more active against
Escherichia coli than the H803 ions. In the case of Sacchar-
omyces cerevisiae the unionized form was only 100 to 500-
fold greated than the H80; ions; the effect of the unionized
form against ASpergillus niger is only lOO-fold greater

than that of the ionized forms (Rehm and Uittmann, 1962).
However, the concluSions of this work are dubious because
an inaccurate pK2 value was used in calculating the distri-
bution of the three molecular forms of 802 in Solution as

a function of pH.

Yeast cells suSpended in solutions of acid antiseptics
caused a decrease in the concentration of these compounds.
The rate of this decrease was very high and the concentration
attained an equilibrium state in a very short time. The
transported quantity of these acid antiseptics was considered
to be in quilibrium with the concentration of unionized mole-
cules of these compounds in the medium (Bosund. 1960).
Studies on the distribution of the acid preservatives
between yeast cells and the surrounding medium have shown
that except for dehydroacetic acid. these preservatives

were absorbed on the solid phase in equivalent quantities

under equilibrium conditionS. In this case the quantities

dissolved in the water and in the lipid phase of the cells
varied greatly according to the kind of preservatives (Oka.
1960 b-e). Essentially the same results have been obtained
with S. 22;; and Staphylococcus aureus (Oka, 1960 a). Oka
(196A) concluded that the antimicrobial effect of the acid
preservatives depended upon the adsorption of the compound
on the solid phase of microbial cells. Since the food
products to which the acid preservatives are applied consist
also of water. lipid and solid phases. the adsorption on the
solid phase of microbial cells is in equilibrium with the
preservative dissolved in the water phase of the food and
not the average concentration in the food.

Rehm (1969) investigated the antimicrobial effect of
SO2 and found that it decreased due to the formation of
hydroxy-sulphonates with different carbonyl compounds.
However. the magnitude of this decrease did not correSpond.
in all cases. to the extent of the binding of SO2 with the
carbonyl. This difference was due to the antimicrobial
action of hydroXy-sulphonates which were formed by reaction
of $02 with acetaldehyde, pyrurate, a-keto-glutarate and
acetone and which had significant inhibiting effects on the
respiration of S. cerevisiae.

Sulfur dioxide affects both reSpiration and fermentation.

As mentioned in the beginning, SO is a potent inhibitor of

2
SH-group bearing enzymes. The high sensitivity of different

SH-enzymes to sulfites is caused by a primary inhibiting
action against nicotinamide adenine dinucleotide (NAD).
according to Pfleiderer et a1. (1956). Sulfur dioxide
forms an addition product with NAD as demonstrated by
spectrophotometric methods (Meyerhof et al.. 1938). SO2
can also destroy thiamine and Split different disulfides.
e.g. cystine to cysteine (Tehn, 1964).

Rehm (1964) investigated the effect of 80 on fermenting

2
cells of S. cerevisiae and S. coli. He first examined the
following NAD dependent steps in glycolysis:

l. The step from 3-phOSpho-glycera1dehyde to 1,3
diphOSphoglycerate.

2. The reaction from pyrurate to lactate.
3. The reaction from acetaldehyde to ethanol.
In the case of reaction (1). simultaneous addition of

3-phOSphoglycerate and ATP to SO -inhibited cultures of S.

2
cerevisiae restored fermentation.

 

 

Step (2) is strongly inhibited by 802 in S. coli. This
inhibition was due to the formation of an addition product
between SO2 and NAD. The back reaction, that is from lactate
to pyrurate. was also inhibited by sulfur dioxide.

Yeast ethanol dehydrogenase which may catalyze Step (3)
is slightly inhibited 3g 222:2 (Pfleiderer et al.. 1956).
Rehm has found that. in equilibrium. the fermentation was

inhibited almost to the same extent as in the case when

free SO2 bound acetaldehyde. Most likely, yeast ethanol
dehydrogenase is not inhibited directly. since fermentation
of ethanol in this step is not possible as free SO2 is
bound by acetaldehyde.

Rehm (1964) also investigated the effect of 802 on
reSpiration. ReSpiring cells ofIS..gg;; are strongly
inhibited by sulfur dioxide lg_z$£gg. The step from malate
to oxaloacetate was strongly inhibited by $02 in S. 231;.

In the Krebs cycle and from oxalosuccinate to a-ketoglutarate
there is a pyridoxal-dependent decarboxylating reaction

which is inhibited by SO Another step of reSpiration

2.

which is inhibited by SO is that from a-ketoglytarate to

2
succinate through succinyl-CoA. Between succinyl-COA and
succinate there is a thiamine-dependent decarboxylation
which is always strongly inhibited by $02. Addition of
succinate activated the reSpiration of cells of SN ggli.
Besides these significant inhibitions of SO2 on fermentation
and reSpiration. an inhibition by formation of addition
products must be considered. The formation of these addition
products begins at the level of glucose and may also include
3-phOSpho-glycera1dehyde. dihydroXy-acetonphOSphate. pyrurate.
acetaldehyde, oxalacetate and a-ketoglutarate.

Shapiro (1970) in a preliminary SQ 11332 experiment
showed that SO may cause point mutationSin nucleic acids

2
by converting cytosine to uracil under mild conditions.

The same investigator. in an unpublished work. had found
that H803 can change up to 90 percent of the cytosine resi-
dues of yeast RNA into uracil residues. In another preliminary

experiment. also unpublished. Makai (1970). has succeeded

in producing bacterial mutations with HaHSOB.

CHAPTER III
METHODS AND MATERIALS

Organism

The microorganism used in this study was the yeast

Saccharomyces cerevisiae var. ellipsoideus ATCC 1&824.

Synthetic growth media
a. Solid medium. The synthetic medium used for the stock
culture was the same used for the survival eXperiments.
This is the medium which Etchells et a1. (1953) called
Synthetic Agar B and prepared (Wickerham, 1951) as follows:
Difco yeast nitrogen base (DYNB) solution, to which glucose
was added to reach a concentration of hi. and a 3% agar
solution were separately heat-sterilized (121°C for 15 min)
and mixed at equal volumes Just prior to plating. The ready-
to-use medium had a concentration of 2% glucose. 1.5% agar
and a pH of 5.
b. Liquid medium. This medium was Similar to that used by

Schultz and McManus (1950). One liter of this medium contained:

10

11

I. Sugar and Salts

 

Sucrose 12.52 g
KH POL. 0.57 g
M3012 0.13 8
NH 0 1.57 8
Fe 1 0.01 g
NaSOR 0.04 g
II. Buffer (McIlvaine's Citrate buffer).
Citric acid 12.823 g
NaHP04.7H20 22.196 g
III. Growth factors
Inositol 16.30 mg
Calcium Pantothenate 10.50 mg
Biotin 0.05 mg
Thiamine 0.05 mg
Pyridoxin 0.05 mg
Nicotinic acid 0.05 mg

The liquid medium was prepared as follows: The sugar.
the salts and the buffer walte were dissolved in 900 ml
distilled water. The growth factors, dissolved in 25 ml
water were added to the above solution and the volume was
made up to 1 liter with distilled water. The pH of this

medium was 4.2. The medium was then filter sterilized.

Preparation of radioactive SO9
Sulfur dioxide is a readily condensable. colorless
gas. It was prepared by the reduction of hot concentrated

sulfuric acid by means of copper.

3580 + 2H 0 +-3580
2 2

35
Cu-y-ZH sob, _______.. Cu u

2

12

A flask provided with a safety tube and exit tube was
filled about one third with copper turnings which were

covered with labelled sulfuric acid. Specific activity

111C1 per ml.

Sulfur dioxide was evolved when the flask was heated

and it was trapped in 0.1 N NaOH.

3530 1- NaOH—-——> NaH35so

2 3

The NaHBSSO3 was redistilled by reacting with moder-

ately concentrated sulfuric acid in order to further purify

the labelled 302.

*35‘ + ‘ ' S -+ + 35S
NaH 503 H250u_____¢.NaH O)+ H20 0

Again the evolved 3530

2
was trapped in cold 0.1 N NaOH
35

2

and kept as sodium disulfite (NaH 803) under atmoSphere of

nitrogen in order to avoid oxidation during storage in the

freezer.
Molecular Species of 802 in aqueous solutions
Sulfur dioxide is soluble in water providing sulfurous

acid.

+Ho-—---‘ HSO

SO2 2v---23

The solubility of 802 in H 0 depends upon temperature.

2
At room temperature (20°C) and atmOSpheric pressure it is 11.3%
(w/w) (Handbook of Chemistry and Physics. 1959).

Ley and Koning (1938) have Questioned the existence of
the compound sulfurous acid in aqueous solutions. Ultra-

violet absorption Spectra of aqueous solution of sulfur

dioxide suggested the following equilibrium.

3 _ +
so2 + H20‘____. Hso3 + H

Infrared Spectral studies (Falk and Giguere, 1958:

13

Jones and McLaren. 1958) have demonstrated that molecular
sulfurous acid (H2803) is not present in aqueous solutions
of sulfur dioxide. Falk and Giguere (1958) inferred that
sulfur dioxide is dissolved in the molecular state since
no stable H2803 molecules were found in aqueous solution.
Eigen et a1. (1961) studied the rate of hydrolysis of sulfur
dioxide by sound absoprtion technique and concluded that
sulfurous acid is not an intermediate product. The hydra—
tion of sulfur dioxide appears to be one of the most rapid
hydrolytic reactions known (DeMeyer and Kustin, 1963).

Raman Spectra (Simon and Waldmann. 1956) of aqueous
solutions of sulfur dioxide of concentrations greater than
1M contain lines attributable to pyrosulfite (8205). Dilute

solutions contain lines assigned to 80 and H805 (Simon

2
and Kriegsmann. 1956). Raman Spectra of dilute aqueous
solutions of alkali bisulfites (NaHSO3) show the presence
of H803 while more concentrated solutions contain increased
concentration of pyrosulfite (Simon and Waldmann. 1956).
Ionization constants of sulfurous acids have been
reported by Tartat and Barretson (1991). The values they
found appear to be reliable in Spite of the fact that modern
evidence indicates that aqueous solutions of sulfur dioxide
contain no detectable sulfurous acid molecules. The unionized
sulfur dioxide Species in aqueous solutions of sulfur dioxide

is almost entirely free 802 molecules. The first disso-

ciation of Sulfur dioxide in aqueous solutions at 25°C is;

1h

 

SO + H O .122: H” + H80-
2 2 3
+ _ -‘ _
K =3 M93 = [H ] [H3031YHEY P1503 = 1.514 X 10-2
1 8802 [502] YSOZ

and the second dissociation at 25°C

8

K2: [H ] [so3l*r H+ ‘r 803 _ 1.02 x 10- -7
[H803] Y H303

Applying the Henderson-Hassebalch equation to 802

dissociations the following equations are obtained:

_ [HSO' ]
pH _ pK14- log [80%]
pH = pK2 + 108 L333.

[H803]

The sum of all three forms adds to 100% and since

pK = 1.81 and pK = 6.91 (Handbook of Chemistry and Physics.

1 2
1970-1971)
pH = 1. 81.+ log [H803] (a)
[ 802
+ [so=]
pH = 6.91 108 2 (b)
[H803]

[803 + [HSO§]+ [80;] = 100 (c)

This system has 3 equations and 4 unknowns. namely pH.

[$02] . [H803] and [303'] .

15

Solving (a) and (b) for [H803] and [803] as a function

of 80 and substituting those values to (c) the follOWing

2
equation is obtained:

(pH-1.81) (2pH-8.72)
1 + 10 + 10 = 13g 1 (I)
2
In the same way:
- - , 100
1 _ lo(8.72 2pH) _ lo(pH 6 91) = H803 (II)
(8.72-2pHO (6.91-pH) 100
1 - 10 — 10 = g6?“ (III)

Equations (I). (II) and (III) provide a direct relationship
between pH and each form of 802. On the other hand these
three equations can not be Solved when any one of these
three 802 forms gets 0 or 100% value. The latter is proved
mathematically as follows:

If [802] = 0, then substitution to (I) gives:

(pH-1.81) (ZPH-8.72)
[802] [10 + 10 + l] = 100

(pH-1.81) (ZpH-8.72)
or [0] [10 +-10 +1J = 100

or 0 = 100. This is impOSSible.

In the same way [H805] and [80; ]cannot have a zero

value. Therefore. all three 80 forms are present in all

2
pH values and none of them disappears completely. This also
means that none of those forms can have a 100% value. The

latter is mathematically proved as follows:

16

Substitution to (I) for [802] = 100 gives:
1-+ 10 + 10 = TDD = 1
pH 1 .+ 10pH
or 10 lol.8| 108.72 = 0 (d)

Equation (d) has the following solutions:
pH
10 a O

or 1 + 10pH
. 2
1.81 108 7

10
8.72
or lOpH = - 1.9.73... (r)
101. 1

Both (e) and (f) equations are mathematically impossible
and therefore 802 never assumes the value of 100%.
In the same way H803 and 80; do not become 100%
Therefore none of the three 802 forms gets a value
of 100% and none of them disappears at any pH value. The
amount each of those forms takes at pH values between 0
and 14 appears in Table 1. and Figure 1 shows the distri-
bution of these forms at the same pH values. Molecular
SO2 predominates at low pH values. it has its maximum value
at pH = 0 and approaches assymptotically to zero as pH gets
closer and closer to pH = 14. Bisulfite (H803) form has
its maximum value at about pH 4.4 and decreases as pH

value goes above or below its maximum. approaching zero

assymptotically as pH approaches zero or 14. Finally

17

sulfite 80; gets its maximum value at pH = 14 and approaches

assymptotically zero as pH value approaches zero.

Analytical Determination of 802

The importance of SO in many fields has been responsible

2
for the development of a number of analytical methods.

The following methods were used in this study:

a. Iodometric method. This determination is based upon
oxidation of sulfurous to sulfuric acid by iodine (Treadwell

and Hall. 1946):

+ + 2 8
802 ZHZO IZ-————-—+ 4H + I '1’ On

The iodine solution was previously standardized by
Na28203. The latter compound was in turn standardized
against KIOB' which served as a primary standard.

1 liter of 0.1 N iodine = 802 = 3.203 g 302
:0

b. Spectrophotometric method. This method was described

by ScOSSins (1970). The sulfur dioxide solution. containing
not more than 0.08 mole 802, is transferred into a 50-ml
volumetric flask and diluted to 40 ml with water. Then

5 ml of a 5M sulfuric acid solution is added to the flask.
the volume iS made up to 50 ml with H20 and the absorbence

of the solution is measured at 276 nm XE a reagent blank.

The 802 concentration is calculated from a standard curve.

.mOm mo wcofiusaom msomscm CH :a mo coauocsm m mm manm

 

 

AWOmv muwwasw new AMOmzv munHSmHL.NOm awasomHoE mo mcowuscwuumfiv wwmucoouom .H ouswwm
In
e_m_~___o_om~omem~_
llflfiflflm n N II.IIII||)I-IH.-IT
I \\
/ x

18

2

cm
cc

8
2

cm

205 ‘lViOi :IO %

-mom m // \\ New
-Om: (l 223322...

 

19

Table 1. Percent distribution of the three 800 molecular
species in aqueous solution as a funEtion of pH.

 

--.- .—

 

 

 

 

 

pN Molecular Bisulfite Sulfite
SO2 (H803) (803)
0.2 97.60 2.39 0.00
0.4 96.26 3.73 0.00
0.6 94.19 5.80 0.00
0.8 91.10 8.89 0.00
1.0 86.59 13.40 0.00
1.2 80.29 19.70 0.00
1.4 71.99 28.00 0.00
1.6 61.86 38.13 0.00
1.8 50.58 49.41 0.00
2.0 39.23 60.76 0.00
2.2 28.95 71.04 0.00
2.4 20.45 79.54 0.00
2.6 13.95 86.04 0.00
2.8 9.28 90.71 0.00
3.0 6.06 93.92 0.01
3.2 3.91 96.07 0.01
3.4 2.50 97.46 0.03
3.6 1.59 98.36 0.04
3.8 1.01 98.91 0.07
4.0 0.64 99.24 0.11
4.2 0.40 99.40 0.19
4.4 0.25 99.44 0.30
4.6 0.16 99.35 0.48

 

 

 

20

 

 

 

pH Molecular Bisulfite
SO2 (H803)
4.8 0.10 99.13
5.0 0.06 98.72
5.2 0.04 98.05
5.4 0.02 96.98
5.6 0.01 95.32
5.8 0.00 89.04
6.0 0.00 83.68
6.2 0.00 76.39
6.4 0.00 67.12
6.6 0.00 56.30
6.8 0.00 44.83
7.0 0.00 33.89
7.2 0.00 24.45
7.4 0.00 16.96
7.6 0.00 11.40
7.8 0.00 7.51
8.0 0.00 4.87
8.2 0.00 3.13
8.4 0.00 2.00
8.6 0.00 1.27
8.8 0.00 0.80
9.0 0.00 0.51
9.2 0.00 0.32

 

 

Sulfite

(803)

 

 

0.76

1.21

1.90

2.99

4.66

10.95

16.31

23.60

32.87

43.69

55.16

66.10

75.54

83.03

88.59

92.48

91.12

96.86

97.99

98.72

99.19

99.48

99.67

-.¢-- -—--—.

21

 

 

pH Molecular Bisulfite Sulfite
802 (11803) (503)

9.4 0.00 0.20 99.79
9.6 0.00 0.12 99.87
9.8 0.00 0.08 99.91
10.0 0.00 0.05 99.94
10.2 0.00 0.03 99.96
10.4 0.00 0.02 99.97
10.6 0.00 0.02 99.97
10.8 0.00 0.01 99.98
11.0 0.00 0.00 99.99
11.2 0.00 0.00 99.99
11.4 0.00 0.00 99.99
11.6 0.00 0.00 99.99
11.8 0.00 0.00 99.99
12.0 0.00 0.00 99.99
12.2 0.00 0.00 99.99
12.4 0.00 0.00 99.99
12.6 0.00 0.00 99.99
12.8 0.00 0.00 99.99
13.0 0.00 0.00 99.99
13.2 0.00 0.00 99.99
13.4 0.00 0.00 99.99
13.6 0.00 0.00 99.99
13.8 0.00 0.00 99.99

 

 

 

22
Preparation of the Samples

The culture received from the American Type Culture
Collection was transferred to DYNB-glucose-agar Slants and
served as a stock culture.

Cells from the stock culture were used to inoculate
the 500 ml liquid medium contained in 1 liter Erlenmeyer
flasks. The cells were grown aerobically by shaking on
a gyratory shaker (300 rpm) for about 36 hours at constant
temperature (25°C). The cells were then centrifuged (10,000
xg for 3 min) and washed 3 times with demineralized water.

A simple eXperimental procedure for studying 802

tranSport in a microbial system would be to incubate the

cells with labelled 80 for a certain period of time. inter-

2
rupt the incubation either by filtration or centrifugation.
wash the cells and measure the radioactivity of those cells.
This procedure was tried but gave inconsistent results.

The reason for the inconsistency was radioactivity losses
during washing of the cells after incubation. The following
two methods proved to be more satisfactory for the study

of the $02 transport in this yeast:

a. Indirect method. 0.4 ml of a dense cell suSpenSion

containing about 4.8 x 108

cells of §. cerevisiae var.
ellipsoideus were incubated with 0.4 ml of a solution con-
taining the desired concentration of labelled 802 and 0.2 ml

McIlvain's buffer (0.25 M citric acid and 0.5 M disodium

23

phOSphate). The incubation was terminated by filtering
the cell suSpension through 0.6 11Millipore filter, 1.4
cm in diameter. With this technique approximately 0.3
ml of clear filtrate could be obtained in 2 to 4 sec.

The radioactivity present in the filtrate was mea-
sured by placing 0.2 ml of the filtrate in a polyethylene
vial. containing 15 m1 of a dioxane base scintillation
liquid. The scintillation liquid was prepared by diluting
in 2.7 liters 1.4-dioxane, 300 ml of absolute ethanol.

21 g'of 2.5-diphenyloxazol. 750 mg 1.4 bis-2-(5 phenyl-
oxazolyl) benzene and 375 g of naphthalene. The vials
were counted in a Packard Tri - Carb liquid scintillation
counting system. model 3310.

In the indirect method it was necessary to determine
the volume occupied by the cells and the intercellular Space.
In determining cell volume and intercellular Space a pro-
cedure similar to that followed by Black and Gerhardt (1961)
was employed. Water-suSpended yeast cells were distributed
into four tared 35-m1 round-bottom glass centrifuge tubes
and packed at 15,000 15 for 30 minutes in a clinical cen-
trifuge at 5°C. The results of packing are illustrated
in Figure 2. Detailed values are presented in Appendix.
Table 1.

This curve determines the plateau region between time
and centrifuge force. This plateau region was used for all

cell packing experiments carried out in this study. Four

24

ml of water-suSpended yeast cells. representing an average
dry weight of 200 mg, were packed at 15.000 Xg for 30 min
at 5°C. Four replicates were used. After centrifugation
the supernatant water was decanted and the inside of tubes
were wiped free of water with absorbent tissue. Weighed
to the nearest 0.0001 gr, each tube contained about 1 gr
of packed yeast cells. Following packing. the cells were
resuspended in 3 ml of a 3% water solution of dextran
(M.W. 500,000). This type of dextran does not penetrate
the yeast cell wall. because of its high molecular weight.
The cells. after being suSpended in the dextran solution,
were agitated vigorously with the aid of a LAB-LINE
supermixer for 1 minute at 5°C in order to allow equilibration
and finally repacked at 15.000 Xg for 30 minutes. The
supernatant fluids were decanted into centrifuge tubes.
clarified by additional centrifugation and assayed for dex-
tran concentration. This assay was carried out by measuring
the refeactive index in a Bausch & Lomb refractometer at
constant temperature (27°C). The degree of dilution of
dextran solution due to intercellular water was calculated
by the means of a standard curve of dextran concentration
XE refractive index. The results appear in Table 2. Detailed
results appear in Appendix. Table 2.

The amount of SO tranSported was calculated from the

2
equations

CELL PACK WEIGHT, (0)

 

25

 

 

2.55 T-

250 '
14.

2.45 5'
2.40 J l J
0 I0 20 30

TIME, MIN

Figure 2. Packing rate of S. cerevisiae var. ellipsoideus cells.

Water suspended cells were packed by centrifugation at

15,000xg at different times.

Table 2.

volume of §. cerevisiae var. ellipsoideus.

26S

 

Determination of pellet intercellular space and cell

Cells

suspended in 3% dextran (M.w. 500,000 solution were

packed at 15,000xg at 5°C.

 

 

Weight of cell pack

 

Intercellular volume

~v—.—-—.--—--. - ---.

 

 

(8) of cell pack Weight
Wet Dry (cm3) (g)
1.0107 0.1926 0.3211 0.6896

 

 

 

—.—.~

Cells

Pao-Qa— .-..._‘ ~. ~-

Volume

(cm3)

~. ‘ “.“*_—,.-.

0.626

 

 

- n“...-

. —-—.—.-

 

Number

4.8)(109

 

---—.—.— ‘---

b‘w—

 

27

%=£-‘—§—§—x100
where A represents the amount of radioactivity present
in the control and B represents the amount of the radio-
activity present in the reaction mixture filtrate.
b. Direct method. In order to prove that cells do take
up $0 the following direct measurement of the tranSported

2

80 into the cells was employed. The cells were incubated

2
as in the indirect method with the difference that the

cell population was 3 or 4 times smaller than that in the
indirect method in order to separate the cells from the
incubation mixture in less than 4 seconds. [After incubation
the radioactivity of the cells plus the filter plus the inter-
cellular liquid was measured by dissolving the mixture

in 1 m1 of Soluenggloo. a commercial solubilizer and using

the scintillation liquid described for the indirect method.
The amount of radioactive SO2 tranSported in the direct

method was calculated from the equation:

T a (C - Y) x F »+ (C - Y) x V + Y

k_______«,_____J \__—_—_“v—"“’J

radioactivity radioactivity

present in the present in the

filter intercellular
Space

where T represents the amount of radioactivity present in
the filter plus cells plus intercellular Space, C the amount
of radioactivity present in the control (incubation medium
Without cells). V the intercellular volume of the yeast

cells after filtration, Y the radioactivity present in

28

cells and F the precentage of radioactivity retained by

the fiber. Solving equation (I) for Y gives:

CF+CV-T
F4V-l

From this equation the radioactivity present in the cells

Y:

was calculated.

In order to compare the direct and indirect method.
cells were incubated with SO2 at pH=3.8 for 5 and 10 min
With each method. The results appear in Table 3. and show
that the difference between the direct and indirect method
is not significant. Detailed data appear in Appendix,
Table 3. However. the indirect method was used in sub-
sequent eXperiments for the following reasons:

1. Simplicity. The only parameters to be measured
are the volume of cells and the radioactivity of filtrate.
whereas in the direct method the cell and filter solubili-
zation step complicates the procedure.

2. Precision and accuracy. The possibility of errors
caused by differences in the radioactivity retained by the
filter and the intercellular space and the losses due to
the adhesion of cells on the walls of the container and
filtering apparatus decrease the precision and accuracy

of the direct method.

29

Table 3. Comparison between direct and indirect method in
measuring transport in_§._gergvisiag.var._ellips idgug.
Cells representing a dry weight of 20 mg (4.8x10 cells)
were incubated in 1 ml of 10"3 M 802 at pH=3.80 and

 

 

 

 

 

20°C.
I

Method Incubation time Counts per minute Uptake
(min) Control Expt Z

Direct 5 3,417 751 21.97
10 3,417 779 22.79

Indirect 5 4,475 1,041 23.26
10 4,475 979 21.87

 

 

 

 

 

 

 

CHAPTER IV
RESULTS AND DISCUSSION

Characterization of SO2 Uptake by Yeast Cells

Oka (1964) considered that the uptake of acid pre-
servatives was the result of adsorption of those antisep-
tics on the cell surface. Other investigators, however,
have demonstrated that $02 inhibits microbial growth at the
level of reSpiration and fermentation. Therefore it should
enter the cell (Meyerhof et al.. 1938; Pfleiderer et al..
1956; Rehm, 1964). In order to determine whether 302
enters the cell or absorbs on the cell surface. yeast
cells representing 200 mg dry weight were incubated for
5 minutes with 10.5 moles 802 at pH=4.11. After incubation
the cells were washed with distilled water five times until
the recovered radioactivity in the supernatant approached
background radiation. The cells. after being washed. were
mixed with 20 grams of glass beads (0.5 mm diam.) into a
previously chilled metal cylinder and shaken for 60 seconds
in a mechanical cell homogenizer. Microscopic examination
revealed a Small portion of broken cells. The homogenate

was centrifuged at 3.000 xg for 3 minutes to separate the

30

31

beads. Further centrifugation at 15.000 Xg for 5 minutes
yielded a supernatant free of membrane which was assayed

for radioactivity. The results appear in Table 4. Detailed
results are presented in Appendix. Table 3. The recovered

radioactivity in the supernatant is a direct proof that

SO is transported inside the cells.

2

Indirect evidence that SO2 is not adsorbed on the cell

surface is that metabolic inhibitors, not reacting chemically

2. If 802 was taken up

by adsorption. the use of metabolic inhibitors should not

with 802. inhibit the uptake of SO
interfere with the adsorption process.

Measurement of Sulfur Dioxide Pool

In studying a transport system it is important to
determine whether the substrate leaves the cell, and to
what extent. after its entry into the cell. The phenomenon
of counterflow was predicted by Widdas (1952) and verified
by Rosenberg and Wilbrandt (1957) in their studies of
sugar movement across erythrocyte membranes. Dreyfuss
(1963) has also described an outward movememt of sulfate
upon washing of Salmonella typhimurium cells. loaded with
soff.

A certain portion of the radioactivity acquired by

3550

cells eXposed to 2 can be removed by washing (Table 5).

32

Table 4. Fate of 3580 upon incubation with S, cerevisiae var.

ellipsoideus.

2

 

cells (dry weight) suspended in 10 m1 of 10'-5
tion at pH=4.1l.

ground radiation in the supernatant.

 

then partly fractionated in a cell homogenizer and

after centrifugation (15,000 xg; 5 min), the radio—

activity in the supernatant was assayed.

The reaction mixture contained 200 mg

Repeated washing resulted in back-

The cells were

M solu—

 

 

Counts per

 

Operation 10 min Z

3SSO2 removed in the original incubation 240,544 56.74
5S recovered in:

First wash 57,973 24.10

Second wash 11,598 4.82

Third wash 2,022 1.36

Fourth wash 83 0.03

Fifth wash 81 0.03

Supernatant of broken cells 2,778 1.53

 

 

33

Table 5. Effect of washing of cells of S. cerevisiae var. ellipsoi-

35

deus after incubation with SO The reaction mixture

2.
contained 250 mg of cells (dry weight) suspended in 10 m1

of 10-5 M SO2 solution. Incubation time 10 min. The

volume of cells was about 0.6 m1 and the volume of “20

used in each washing was 10 m1.

 

 

 

Type of operation pH=3.19 ph=3.32 pH=4.ll
. 35 . . . . ,

A $07 removed in the original incubation 68.01 51.48 16.24
35

SO (as X of that removed in the original
incubation) recovered in:

First wash 15.77 15.59 17.65
Second wash 8.97 6.55 0.90
Third wash 6.91 2.11 0.22
Fourth wash 1.72 0.15 0.22
Fifth wash 0.48 0.09 0.29
Sixth wash 0.09 0.08 0.19
Seventh wash 0.06 0.08 0.25
Eighth wash 0.05 0.08 0.21
Ninth wash 0.05 0.09 0.20
Tenth wash 0.06 0.08 0.25
Total 35802 recovered 34.16 24.82 20.38

,3-

m 35. . . .
iotal SO7 remaining in cells after ten

°/

washes (as N of that removed in the

original incubrti L) 44.78 38.71 12.93

 

34

Qualitative analysis by the method of Scoggins (1970)
revealed the presence of SO2 in the cell washes. The

amount of SO2 recovered in 10 washes as well as the amount

of residual 80 remaining in the cell after these washes

2
depended upon the pH of the incubation medium. In Appendix.

Table 4, detailed values are presented.

802 TranSport as a Function of Time

Yeast cells suspended in solution of acid antiseptics
eSpecially benzoic acid caused decreases in the concentra-
tions of these antiseptics in the solution (Oka, 1964).

In the case of benzoic acid the rate of decrease was very
high and the concentration attained an equilibrium stage
in less than 5 min. Figure 3 shows the tranSport of SO2
as a function of time. Detailed values are presented in

Appendix. Table 5. The amount of SO transported in 5,

2
10. 20. 40. and 60 minutes was practically the same. The
yeast reached their maximum load of 802 in less than 5
minutes. The pattern of transport was dimilar to that
found by Oka (1964) for benzoic acid.

Cells were incubated with labeled SO for 10. 20,

2
40. 60, 300 and 600 seconds. The initial rate of uptake
was very rapid and seemed to follow a linear course up to
about 10 seconds (Fig. 4). Beyond this time the uptake

started leveling off and reached maximum value in about 2

 

35

 

 

es

7
I)

a
b

 

I l l l I I

 

25r-
N
CD
(I)
._l
E
.215—
LL
CD
32
{Blot
uJ
ii
<E
[.—
De
:3
5 .
0
Figure 3.

10 20 3O 40 50 60
INCUBATION TIME, MIN

Transport of SO in S. cerevisiae var. ellipsoideus as a

2

function of time. The reaction mixture contained 20 mg

 

of cells (dry weight) or 4.8x108 cells suspended in 1 m1

of 10-3 M SO2 solution adjusted at pH = 3.80 and 200C.

 

 

36

 

| 1 I l l I 1 1 I l I I l l J

 

30 1"
N

CD
00

2" 20
|—'
CD
I—'
LL
CD
as
‘2

10
LLI
>4
<E
[.—
CL
:3

0

Figure 4.

2 4 6 8 10
INCUBATION TIME, MIN.

Time course of SO

transport in the yeast S, cerevisiae
var. ellipsoideus.

f Cells representing a dry weight of
20 mg (4.8x10b cells) were incubated in 1 m1 of 10‘3H SO
solution at pH=3.80 and 20°C. The direct (dotted line)

and indirect (solid line) methods were used in measuring
uptake.

 

37

minutes. After maximum load was attained the 802 pool
remained at a constant level. In Appendix. Table 6. de-
tailed results are presented.

Identification of SO TranSport System

2

Compounds in solution may undergo a net movement from
outside to inside the cell and vice versa by either non-
mediated or mediated processes. The non-mediated move-
ment has two characteristics derived from the phenomenon
of simple diffusion.

a. The tranSported compound does not change throughout

the tranSport process.

b. The rate of tranSport is linearly dependent upon

the concentration of the compound being tranSported.

The mediated transport has a number of identifying
characteristics. Those characteristics are often used
as experimental criteria and can be studied by measurements
of the kinetics of transport through the cell membrane.

In identifying the SO2 tranSport in the yeast S. 2333-
visiae var. ellipsoideus the following criteria were used:
A. Kinetic characteristics:

1. §QZ transport as a function of SO2 concentration. While
in the case of non-mediated tranSport the rate of transport
is linearly dependent upon the concentration of the compound
being tranSported, mediated tranSport shows saturation kine-

tics.

38

Sulfur dioxide transport in this yeast displayed
saturation kinetics, that is the SO2 transport sites
were saturated Just as enzymes became saturated with their
substrates (Fig. 5). Detailed values are presented in
Appendix. Table 7. Plotting of the initial (or close to
the initial) rate of SO2 transport XE 802 concentration
showed a hyperbolic curve which approached a maximum and
was similar to the Michaelis - Menten curve of an enzyme.
The rate of tranSport between 0 and 10'2M 80 concentration

2
seemed to be proportional to 80 concentration and it was

2
of first order. Beyond this concentration the rate of
tranSport started leveling off up to the point where it
became zero order with reSpect to SO2 concentration. The
pattern of this tranSport suggested the presence of carriers
containing Specific sites to which 802 bound.

A double reciprocal plot according to the method of
Lineweaver - Burk (Fig. 6) gave a straight line with an
apparent Km - 2.65 * 0.40 x 10-2M and Vmax = 6.95 t 0.39
x 10’3 M/ 10 sec. The meaning of these two constants in
the process of SO2 tranSport through the plasma membrane
is the following:

The Vmax expresses the maximum rate of tranSport

that the yeast cell can demonstrate toward SO and depends

2
upon the number and the physiological state of these cells.

The Km is independent of the number of cells present

(V) MICROMOLES OF TOTAL S02 TAKEN UP PER 10 SEC

 

Figure 5.

 

39

 

I J

L I I I 1

IL J
12345678910

so2 CONCENTRATION, M x 10'2 (S)

Sulfur dioxide transport in S. cerevisiae var.
ellipsoideus as a function of SO concentration.

 

 

The reaction mixture c ntained 20 mg of cells

(dry weight) or 4.8x10U cells suspended in 1 m1

SO solution adjusted to ph=3.80. Incubation time:
10 sec. at 20°C.

40

. (M'1x1o3) (10 sec)

 

l

 

1- (M'1x102)
s

Figure 6. Lineweaver—Burk plot of SO transport in _S_.

-g—o-.--._.-‘

41

and eXpresses the SO2 concentration at which the rate of
tranSport is one-half of maximum rate of transport (vmax)'

2. Effect of temperature on 802 tranSport. .Another kinetic
characteristic of SO2 tranSport is the effect of temperature.
In the non-mediated tranSport process the temperature co-
efficient is usually that of physical difquion, namely

1.4 per 10°C rise in temperature (Lehniger. 1970).

The pattern of SO tranSport is affected by temperature

2

as it is shown in Figure 7. Detailed results are presented

in Appendix. Table 8. It is clear that the temperature

at which the tranSport system operates has a very pronounced

effect on the rate of 802 tranSport in this yeast. Between

0 and 20°C there is no significant difference in the rate

of tranSport. The rate increases sharply with temperature

in the range 20-50°C with a maximum temperature coefficient

of 2.4. Above the optimum temperature. 50°C. the rate of

tranSport decreases rapidly. At 70°C the tranSport process

is very slow. The effect of temperature on 802 tranSport

displays a pattern similar to that observed in enZyme kinetics.
The inactivation of SO2 tranSport system is not rever-

sible. Cells were subjected to different temperatursSfor

two minutes and after cooling at 20°C. the $02 transport

at that temperature was measured. The results appear in

Figure 8 and show that the pattern of the SO tranSport

2
inactivation by temperature, besides being irreversible.

50

40

30

UPTAKE AS '70 OF TOTAL 502 IN 30 SEC

 

42

 

.I I 11 I I I ll
10 20 30 40 50 60 70
TEMPERATURE, ° C
Figure 7. Effect of temperature on SO transport in §._gggggisigg Egg.

e11ipsoideus._
in 1 m1 of 10
temperatures.

 

Cells, 4.8x1 8, 20 mg dry weight were incubated

M SO

2

at pH=3.80 for 30 sec at different

43

appears to be similar to the pattern of protein denatu-
ration by temperature. In Appendix. Table 9 detailed
results are presented.

B. Specificity of tranSport system.

The Specificity of the tranSport system for the com-
pound transported constitutes the second criterion of
mediated tranSport. The degree of specificity of mediated
tranSport systems starts from structural Specificity to
stereOSpecificity. It has been postulated that mediated
tranSport systems reside in membranes and contain binding
sites complementary to the compound tranSported. This
process resembles in its Specificity the active site of
enzyme molecules.

The 802 tranSport system in the yeast S. cerevisiae
Egg. gllipsoideus diSplays a degree of structural Specifi-
city tranSporting from a mixture of bisulfite (H503).

sulfite (SO?) and the molecular 80 only the latter form.

2
The mechanism and results on the tranSport of molecular

SO . H805 and 803 will be discussed later on.

2 3
C. Inhibition of tranSport system.

This is the third criterion of a mediated tranSport
system. In addition to competitive and non-competitive
inhibition of tranSport Systems. inhibitors that affect

energy metabolism abolish the accumulation of the transported

compound inside the cell (Winkler and Wilson, 1966).

UPTAKE AS % OF TOTAL 502 IN 2 MIN

30

10

 

44’

 

 

 

I)
I l I I I 111
10 20 30 40 50 60 70
TEMPERATURE, ° C
Figure 8. Thermal inactivation of the SO transporting system

in S. cerevisiae var. ellipsoi‘egg. Cells representing

a dry weight of 20 mg (4. 8x10° cells) were subjected

to different temperatures for 2 minutes and then cooled

at room temperature (200C). The SO transport was studied
by incubating these cells in 1 m1 of 10 3 M SO at

pH=3. 19, 200 C, for 2 min. 2

 

45

Inhibitors that block the formation or utilization of
high energy phOSphate bonds inhibit tranSport. Details
on this inhibition will be discussed later.

All the above criteria characterize $02 tranSport
as a mediated tranSport system. These systems may be
divided into the following two types:

Facilitated diffusion. which does not require energy
since it merely catalyzes equilibration of solute across
the membrane.

Energy-coupled permeability or active transport. which
requires energy since it proceeds against the concentration
gradient.

In order to determine whether the SO tranSport is

2
of the facilitated or energy-coupled type (active) of

transport, the following criteria were used:

1. The first criterion refers to whether or not SO2
tranSport proceeds against the concentration gradient.
This can be easily determined from Figure 4. The trans-

ported SO is much higher than that justified by the

2

cell volume. Therefore the transport of SO proceeds

2
against the concentration gradient in this yeast and this
is a criterion of active tranSport. However, this criterion
requires not only precise knowledge of the SO2 concentration
inside and outside the cell but also an assurance that the

tranSported substance eXists in the same molecular Species

inside and outside the cell. Since the latter requirement

46

is very difficult to establish from the analytical measure-
ments alone, the criterion of concentration gradient is not
always useful for proving the existence of active tranSport.
In the case of $02 transport this is even more difficult
because of the complications due to differences in pH
inside and outside the cell.

2. The second criterion to characterize whether or not
the mediated tranSport is facilitated diffusion or active
tranSport can be eXpressed in two ways:

a. The inhibition can produce its effect by acting
on the metabolic system involved in the supply of energy
for tranSport or the maintenance of the membrane. Because
active tranSport proceeds against the concentration gradient
it must account for thermodynamic work and therefore it
should be hooked into some energy producing systems such
as the respiration or glycolysis. Inhibitors that block
those sources of metabolic energy must also affect active
transport.

b. The inhibitor can act on the membrane or tranSport
system directly. Inhibitors reacting with SH groups can
inhibit tranSport by acting on membranes, the protein portion
of which contain SH groups. probably important in maintaining
the integrity of membranes.

Active transport processes may. for convenience. be

considered as consisting of two parts:

47

I. Exergonic reactions providing the energy required.
usually oxidation coupled with.ATP production.

II. Endergonic reactions by which the substrate is
transported.and the energy is utilized (the functional
tranSport system itself).

Inhibitors that affect tranSport by reducing ATP
production (e.g. 2.4-dinitrophenol. sodium azide. etc.)
can be considered as exerting a rather nonSpecific effect
with reSpect to transport. Since any cell function requiring
energy will be depressed eventually.

Inhibitors that affect the endergonic system may be
more selective, blocking tranSport and leaving other cell
functions intact.

Four metabolic inhibitors, at three different con-
centrations, were used in the study of SO2 tranSport in
S. cerevisiae gag. ellipsoideus. These inhibitors were
found not to react with SO2 chemically. The results appear
in Table 6. In Appendix. Table 10. detailed values are
presented.

2,4-Dinitrophenol is one of the best known uncoupling
agents of oxidative phOSphorylation inhibiting production
of ATP from.ADP (Lehmniger, 1970). Sodium azide has mul-
tiple effects. acting as inhibitor of electron tranSport.
as an uncoupling agent like 2,4-dinitrophenol. and as a

phOSphorylation blocking agent like oligomycin (Robertson

1+8

Table 6. Effect of metabolic inhibitors on 807 transport in S.

Cells of 20 mg dry weight

, -3 .
were suspended in 1 m1 of 10 N 809 containing the

quantities of the inhibitor appearing in this Table.

The ph was 3.19, 200C.

 

9....-- w“-- - -v-O—p.-..--.—-.——“—-

 

 

Inhibitor Inhib. concentration (M) Z Inhibition
5 x 10’4 18.54
2,4—Dinitrophenol 10—3 30.48
5 x 10"3 31.28
10‘3 66.86
~3
HgCl2 5 x 10 , 103.51
—2
10 104.72
10’2 1.69
—2
Iodoacetamide 5 x 10 1.41
10'2 1.66
«2
10 40.04
—2
Sodium azide 5 x 10 64.85
10'1 72 29

 

 

 

 

1+9

and Boyer. 1955). Both 2,4-dinitrophenol and sodium

azide inhibit the production of ATP. Both have a signi-

2
the inhibitor concentration.

ficant effect on SO tranSport. This effect depends upon

Iodoacetamide and mercuric chloride are enzyme inhi-
bitors that react with SH groups. Iodoacetamide frequently
does not react with SH as readily as mercuric chloride does
(Webb. 1966). .AS far as energy production is concerned.
iodoacetamide blocks glycolysis at the level of 3-ph08pho-
glyceraldehyde oxidation and prevents glycolytic generation
of ATP.

In essence. iodoacetamide did not inhibit SO2 tranSport.
Since this inhibitor reacts selectively with SH groups
(Webb. 1966) the possible eXplanation concerning its inability
to affect SO2 tranSport is that it does not react with SH
groups present in the tranSport system, therefore, leaving
the endergonic System intact. On the other hand. since
the yeast cells were grown aerobically, the glycolytic
generation of ATP was low in comparison to the ATP coming
from the reSpiratory mechanism (Pasteur effect) and there-
fore one should expect that the presence of iodoacetamide
would not have any effect.

Mercuric chloride inhibited SO2 transport completely.

Since it was found that mercuric chloride is a very strong

enzyme inhibitor (Webb. 1966) one would assume that mercuric

50

ions affect the endergonic system of SO2 transport by
reacting with SH groups.

Of these 2 criteria for an active tranSport system.
the movement of SO2 against a concentration gradient is
difficult to defend. since no definite information exists
regarding the pH on the cell Side of the cell membrane.

If the pH inside the cell is high (6 or 7) the cytoplaSm
could act as a sink for the transported 502 by converting
it to disulfite and sulfite. The effect of inhibitors does
point to an active tranSport although 2,4-dinitrophenol
should have inhibited the SO2 tranSport to a greater extent
at the concentrations used. At any rate. the pOSSibility

of Simple difquion is ex01uded on the basis of the above

findings.

Transport of Molecular SOZ._HSO§ and so;
J

Once it was established that SO was transported inSide

2
the yeast cell the question was raised whether all three
forms of 802 in solution participate in the tranSport or
only one or two.

It has been known that the molecular form of SO2 is
the most toxic of all three forms and predominates at low
pH values. Therefore, it was deemed desirable to study
it. first from the tranSport vieWpoint. Figure 9 shows the

tranSport of 802 at four pH values and clearly demonstrates

UPTAKE AS "I. OF TOTAL so2

60r-

8

8

 

 

Figure 9.

 

51

 

 

 

INCUBATION TIME, MIN

Time course of 802 transport in S. cerevigiag vag.

ellipsoideus as a function of pH. The reaction mix—

 

ture contained 20 mg cells (dry weight) or 4.8xl06

cells suspended in 1 m1 of 10_3 M 802 and adjusted

at different pH values. Incubation time: 10 sec.

at room temperature (20°C).

52

that lower pH values increase the 802 tranSport. Detailed
values are presented in Appendix, Table 11. The amount
of molecular SO2 present at pH 3.19. 3.50, 3.80 and 4.80
were 4, 2. l and 0.1%, reSpectively.

The effect of pH on tranSport may be due to the

following:

a. The 80 tranSport system is affected by pH directly.

2
thereby influencing the tranSport itself.

b. The SO tranSport System is not affected by pH

2
directly: instead, the pH dependent distribution of SO2
forms in solution is responsible for the change in 802
transport.

In order to determine whether the effect of pH is
direct or indirect two experiments were carried out as
follows:

1. Yeast cells were incubated with labeled 502 for
10 sec at five pH values and the rate of tranSported total
802 was plotted against the amount of molecular SO2 present
at: those pH values. The results appear in Figure 10.
Detailed data appear in Appendix, Table 12.

2. The cells were incubated at the same pH values

as above but the total SO concentration was adjusted so

2
that the same concentration of molecular SO2 at each pH
value was obtained. The amount of tranSported total 802
in 10 sec was measured and the results appear in Figure 11.

In.Appendix. Table 13 detailed values are presented.

% OF TOTAL S02 TAKEN UP IN 10 SEC

 

Figure 10.

53

 

I I I I

l 2 3 4

MOLECULAR 502 AS % OF ALL 3 FORMS
PRESENT IN 10 3 M AQUEOUS SOLUTION

Relationship between rate of SO transport in_§..gerevisi§e
var. ellipsoideus and am unt of molecular SO . Cells of

20 mg_§ry weight (4.8x10 cells) were incubaged in 1 m1

of 10 80,, for 10 sec. and at pH 4.11, 3.80, 3.50,

3.32 and 3519. The amount of molecular 809 ag those pH
values was 0.5, 1.0, 2.0, and 3.0 and 4.0x107‘ M,

respectively.

 

 

B 8
I T

|—l
CD
I

”la OF TOTAL 502 TAKEN UP IN 10 SEC

 

Figure 11.

3.|9 3.50
l I

3. 32 P”

I I I I I

2 4 6 8
TOTAL SO2 CONCENTRATION, MxIO‘3

Relationship between rate of SO transport in S.
cerevisiae var. ellipsoideus an amount of molecular
800. Cells of 20 mg dry weight (4.8x108 cells) were
inEubated in 1 m1 of a solution containing 1.0, 1.33,
2.0, 4.0, and 8.0 10‘3 a so, at pH 3.19, 3.32. 3.50,
3.80 and 4.11, respectively? The amount of molecular
803 at those ph values was the same in all sarpigw

”13:11!

?".. >' . .. ‘ ,_.a_.' \. :.',,. n4 ‘1' ‘._ .u "3"“7 ‘
.LIILJ .LIILLIIJTALLKIII. LLI .g: liens) .L.) bLC, (II... ...\J (J.

55

The results from both SXperimentS prove that:
A. The effect of pH on the SO2 transport system is
not direct at those pH levels because: (1) As pH decreases
the transport rate increases being linearly proportional to mole-

cular SO2 concentration. (2) When the pH is adjusted so

that the molecular 802 concentration is the same in all

samples the rate of tranSport remains constant.

is the only SO form tranSported

B. The molecular 502 2
inside the cell. The bisulfite (H805) and sulfite (80;)
are not transported at all, since only the molecular SO2
Species forms a linear relationship with the amount of total
SO2 transported in 10 sec (Table 7). The H803 and so? are
not related at all with the observed rate of tranSport.

The fact that molecular SO2 is the only tranSported
species aids in further elucidation of tranSport mechanism.
The results appearing in Figure 9. Show that the amounts
of tranSported molecular SO2 at each pH value are much larger
than those originally present in the incubation medium.

For example at pH 3.8. 1% of the total SO is present as

2
molecular $02. After 2 minutes of incubation with the
cells at the same pH about 23% of total 802 was tranSported
into the cell. Since only molecular SO2 is tranSported

the amount of this form which disappeared from the solution

in 2 minutes Should be about 23 times more than the ori-

ginal amount of molecular 802. In order for this to happen

56

and Since the pH of the incubation mixture remains con-
Stant, sulfite (80;) and bisulfite (H303) must be converted
to molecular 802 as follows:

H+ + 80‘..__..“"“J H803
3

+ _ __.i
H + H503 v—— 1120+ 802

As the amount of molecular 802 tranSported inSide the cell
increases the concentration of the same form outSide the

cell decreases and the curve describing 802 tranSport as

a function of time starts levelling off. This chemical
equilibrium aSpect is supported by the fact that concentration
saturation kinetics (Figure 5) Showed that in order to
saturate the tranSport System sites a concentration of

2

2Km=4.70 0.80 x 10' M total 802 was necessary. This value

is very high compared to the amount of total 80 used.

2
for the same pH (3.80). in the eXperiment shown in Figure 9.
However, molecular SO2 exhaustion can not itself eXplain
the pattern of SO2 tranSport because. if this were the

case. the tranSport curve after reaching a maximum should
start gradually going down. Since this was not observed
but. instead. a constant tranSport level was attained. the
latter could be understood as a consequence of a tranSport
equilibrium in which equilization of the rate of inward
flow to outward flow is obtained. This is supported by

the following:

57

(a) Washing of cells resulted in 502 losses (Table 5).
The 802 that was not washed out reacted inside the cell,
whereas the non-reacted SO portion was the one that should

2
be in equilibrium with 80 in the incubation mixture.

2
When this equilibrium was attained the rate at which 502
entered the cell was equal to the rate at which the same
compound left the cell. The mechanism of outward movement

of $0 is not a diffusion process since the amount of SO2

2
released in each cell washing is less than that justified
by simple difquion. as the data of Table 5 show. (b)

Cell fractionation data appeared below suggest that $02

is not metabolized inside the cell. Therefore SO2 uptake
should not increase continuously with time since the $02
transported inside the cell was not used up. (0) Oka (1964)

described the same pattern of tranSport equilibrium for

benzoic acid.

SO2 Transport and Toxicity

Sulfur dioxide is toxic to cells. The inability of
yeasts. molds and bacteria to reproduce and form colonies in
the presence of this agent has been used as a measure of
lethal damage by this chemical.

The toxicity results obtained in this study were ex-
pressed as precent survival or percent outgrowth of organism.

Number of colonies after exposure to $02

% survival 3 x 100
Number of colonies before exposure to 802

 

58

In studying the relationship between tranSport and
toxicity the following factors were examined.
a. The relationship between the amount of SO2 and
toxicity.
b. The dependence of toxicity upon the time during
which SO2 remained in contact with the cells.
Cells representing a dry weight of 4.2 mg (107 cells)
were incubated with 2.5. 5.0 or 7.5 x 10'3 M total 802
concentration for 5. 10, 15. 20 and 30 minutes. The effect
of SO on the yeast cells was terminated by diluting 0.1

2
ml of the incubation mixture to 400 ml sterile water. This

resulted in that:
a. The SO2 concentration was diluted 4.000 fold.

b. The level of SO into the cell pool was loWered

2
significantly due to the outward flow of SO2 upon

dilution.

The results appear in Figure 12 and Show that the
toxicity of 802 on the yeast cells depends upon both 802
concentration and time during which SO2 remains in contact
with the cells. On the other hand, the relationship between
inhibition of growth and time of exposure to 802 is eXpo-
nential. The calculated 0 values or the time required for
90% growth inhibition under the conditions of this experiment,
were 01:83 min. 02:25 min and 03:19 min for total SO2

3

concentrations 2.5. 5.0 and 7.5 x 10' M respectively.

Detailed data appear in Appendix, Table 14.

% SURVIVAL

 

59

 

100
90 _
80 ..
70 - 9
60 -- ‘ _3
‘ 2.5x10 M SO7
50 '- e
C)
m —
30 P ‘
r —3
5.0x10 M 809
20 .—
7.5x10"3 M 80; ‘
t C)
10 I I I I I 111
O 10 20 30
TIME, MIN
Figure 12. Percent survival of S. cerevisiae var.

ellipsoideus
incubated with different 80 concentrations and
Cells representing a dry weight 4.2mg (107

 

tlJQCS.
cells) were

incubated I19801 ml 80 solution containing 2.5, 5.0

and 7. 5 at p 3.19. Incubation was interrupted
by diluting 0. 01 m1 of cell suspension in 400 m1 sterile
water.

Aliquots were plated on g1ucose~yeast nitrogen
base-agar medium.

60

An eXperiment was carried out in order to determine

the relationship between the three 80 forms and toxicity.

2
Since the molecular SO2 was the only form transported one
should expect that this would be the toxic one. Therefore.
if molecular 802 were the only toxic form. then by changing
the pH so that the same amount of molecular form would be
present at different total 802 concentrations. the same toxic
effect would be obtained. The results appearing in Figure

13 Show that SO had a very pronounced toxic effect when

2
the concentration of this preservative increased while the

pH value was kept constant (pH=3.19). On the other hand.

the toxicity remained the same when the pH was adjusted

so that the same amount of molecular SO2 was present although
the concentration of total 302 was increased 40 fold.
Detailed data appear in Appendix. Table 15 and 16. Therefore
it is the molecular form only that exerts a significant

suppression of growth of S. cerevisiae var. ellipsoideus.

Localization of $02 in the Yeast Cell

An attempt was made to determine the fate of 802 after
it was tranSported inside the cell. .A fractionation was
carried out similar to that described by Chaloupka and
Babicky (1958). Yeast cells were incubated with labeled

802 for 20 minutes at room temperature. The incubation

was terminated by centrifugation and the cells were washed

% SURVIVAL

B 3 8 8

61

 

B
I

 

100

 

   
 

3. .19 3. .19

pH
3. 19
I

 

Figure 13.

 

I—‘
5 10 15 20 100
TOTAL SO2 CONCENTRATION, M x 10‘3

Percent survival 9f.§-.EQEQKLQEQQIZQE-.ElliREQEQSBi-
incubated at various total SO9 concentrations and pH
levels. Upper Eggye. Cells fepresenting a dry weight
of 4.2 mg (10T cells) were igcubated in 1 ml containing
2.5, 5.0, 10, 20 and 100x10’ u so for 5 min at on
3.19, 3.50, 3.80, 4.11 and 4.80. he incubation

period was interrupted by diluting 0.1 m1 of cell
suspension to 400 m1 sterile H O and then plating on

a glucose—yeast nitrogen—agar medium. nggrflcgryg.

Same procedure as above with the difference that the
ph of the incubation mixture was 3.19.

62

six times until practically no radioactivity was recovered
in the wash. Trichloroacetic acid (TCA). 10% solution.
was added to the washed yeast pellet and the suSpension
left for 30 minutes at 5°C. It was then centrifuged and
the extraction of the yeast pellet was repeated with 5%
TCA. The joined extracts contained inorganic ions. nucleotides,
free aminoacids and other Small molecules. The residue after
TCA extraction was suSpended twice for 60 minutes in 95%
ethanol and once in a mixture of ethanol and ethyl ether
(1:1). The extracts were separated from the residue by
centrifugation and combined into one solution. This solution
contained the lipid sulfur. The residue again was extracted
twice for 5 minutes with 1N NaOH to obtain polyphosphate-
like compounds and ribonucleic acid and the remaining
sediment was finally hydrolyzed in 6N HCl in a glass tube
for 15 hours at 110°C. The hydrolyzate was centrifuged
and a sample of supernatant was assayed for radioactivity.
In addition to the above extracts further fractions were
obtained from the TCA extract by precipitation with barium
chloride at pH=3.8 (inorganic sulfate and sulfite). at
pH=8.2 (nucleotides) and further by cadmium chloride at
pH=6.5 (glutatathione).

The results of this fractionation appear in Table 8.
Detailed values appear in Appendix. Table 17. In view of
the broad Spectrum of reactions in which 802 can participate

inside the cell, as Rehm (1964) and other investigators

63

Table 7. Distribution of 358 in various fractions of the yeast_§.

cerevisigg var. ellipsoideus. The reaction mixture contained

 

1.2 g of cells (dry weight) suspended in 30 m1 of 1.2 M
SO2 at pH=3.19 for 20 min. Repeated washing resulted in
practically background radiation of the last wash. The
radioactivity present in the washed cells was designed

as control

 

Fraction Total activity % 35

(Counts per min/1.2 gr
dry weight of cells)

 

Control 15.222 100.00

1. Cold trichloroacetic acid

supernatant

a. Inorganic sulfate and sulfite 6 0.04

b. Nucleotide S. 2 0.01

c. Glutathione 3 0.02

d. Other small molecules 15.143 99.48
2. Lipid S. 21 0.14
3. Alkali-extractable S. 25 0.16

4. 6N—UC1 hydrolyzate S. 22 0.15

 

 

 

 

64

have demonstrated (Meyerhof et al.. 1938: Pfleiderer et
al.. 1956) the following observations can be derived from
this preliminary investigation:

1. 35s reacted with small molecules. the best known
of which are carbonyl containing compounds (sugars. pyruvate.
acetaldehyde.<1-ketoglutarate. etc.). forming hydroxy-
sulfonates.

2. Since practically no 353 was incorporated in glu-
tathione or in macromolecules like proteins, one might
say that $02 is not metabolized in this yeast. On the
contrary the sulfur of 3580: fed to the same yeast was found
to be present in all cell fractions. obviou81y because
it was actively metabolized (Kotyk. 1959). It is true that

SO“ is reduced to so; and then to sulfide (-SH2) prior to
incorporation into organic form in yeast. There is some
indication that the SO? generated remains bound to the
enzyme (Wilson and Bandurski. 1958: Wilson et al.. 1969:
Asahi et al.. 1961: Torrii and Bandurski. 1964) and follows

a pathway different from that of SO tranSported through

2
the cell membrane. The results of this fractionation are
in disagreement with those of Shultz and McManus (1950).
These investigators showed that 802 could be used as Sulfur
source in the yeast S. cerevisiae var. ellipsoideus but

their deductions are dubious, because they incubated the

cells with 802 overnight without taking into consideration

65

that 802 is oxidated rapidly to sulfate which is one of

the best sulfur sources for yeasts.

CHAPTER V

SUMMARY AND CONCLUSIONS

The tranSport, toxicity and localization of SO2 in
aqueous solution in the yeast S. cerevisiae var. ellipsoideus
were investigated in this study. The following conclusions
were derived:

A. The 802 tranSport system probably qualifies as
an active tranSport and has the following characteristics.
(a) The tranSport system operates very fast, reaching maxi-
mum 302 load in about 2 min and then remains at constant
level. This can be understood as a two equilibria concept.
The first in a chemical equilibrium and regulates the amount
of molecular SO2 being tranSported. The second one refers
to a dynamic state established between the inward and out-
ward flux of $02, maintaining the tranSport at a constant
level. (b) The 802 tranSport displays saturation kinetics
conforming to the Michaelis-Menten curve of saturation and
has a K = 2.65 t 0.40x10-2 M and Vmax = 6.95 t 0.93x10'"3
M/lO segonds. The pattern of this transport suggests the
presence of carriers containing Specific sites to which

302 binds. (c) The 30 transport system is apparently

2

66

67

temperature dependent and displays a pattern similar to
that observed in enZyme kinetics. It is also irreversibly
inactivated by heat and thus resembles protein denaturation.
(d) Metabolic energy is necessary for this transport to
proceed. Inhibitors that block the formation and utili-
zation of high energy phoSphate bonds abolish the accumu-
lation of SO2 insdie the cell. (e) Among the 802 forms
possible in aqueous solution, bisulfite (H803). sulfite

2, the latter is the only form trans—
ported. This underlines the high Specificity of this trans-

(SO?) and molecular SO

port system.
B. The toxicity of 802. eXpressed as the ability of
this chemical to suppress the growth of yeast cells is related

to 80 transport as follows. (a) The molecular form is

2
the only tranSported form and the only toxic one. (b)
SO2 toxicity depends upon the amount of the molecular 802
as well as the time during which this form remains in con—
tact with the cells. (c) There is an eXponential relationship
between cell viability and time of eXposure to 802.

C. The results obtained during fractionation suggested
that sulfur dioxide does not seem to be metaboliZed in the
yeast cell. Rather. it reacts with certain small molecules
depriving them of their metabolic role: the result is termination

of the growth of the cell.

 

Eur-u: :- 3..

68

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Fourth Intern. Symp. Food Microbiology. Goteborg,
Sweden. p. 3.

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I. Ubersicht uber einflussnehmende Faktoren auf die
antimicrobielle Hirkung der schwelfigen Saure. Z.
Lebensmittelunters. Forsch. 118. #13.

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Symp. Food Microbiology. Goteborg. Sweden. p. 105.

72

Remington. 1961. Practice of Pharmacy. 12th ed. Mark
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hunt. «a and

V“; V
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73

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Advances in Food Research I. 373.

APPENDIX

Table 1.

Packing rate of S. cerevisiae var._e

 

--

ipggidggg cells.

 

 

Centrifugation
time (min)

Weight of cell pack (8)

 

 

Expt I Expt II Expt III Expt IV Average
5 2.5112 2.4950 2.5180 2.5207 2.5112
10 2.4687 2.4592 2.4610 2.4625 2.4627
20 2.4675 2.4577 2.4377 2.4537 2.4527
30 2.4665 2.4585 2.4372 2.4527 2.4537

 

 

 

 

 

 

76

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

moaxw.q omo.o omwo.o Hmm.o Hm.~ Homm.a comm.H mommmAemmm.H cmmm.a wmmm.a noao.a oNoH.o .><
mao.o «quo.o mqm.o mo.~ comm.a quo.H onH.o c
omo.o wmwo.o mam.o mo.~ comm.a mmom.a nemH.o m
mmo.o Hoqm.o oq~.o nn.m Homm.a comm.a ommm.H mmm.a qmmm.H wmmm.a momm.o onH.o N
coo.o «000.0 mqm.o mo.~ comm.a qwmm.a nmmm.H mmm.H cmmm.a mmmm.H «moo.H HHmH.o H
So me So mafia”
m as .o
m m scooaooxooc on” 3.. o.N mg as o2 a z
umnasz .Ho> ucwfimz woman :H ace «0
.Hsaaou uxma#.uaasm smuuxma N Amy
mHHmo lumusH N menu um xovcH o>wuumummm xoma Hamo mo unwwoz maasmm
.msotfiomaaaam .um> mmfimw>muoo .m.sfi oaaao> Hamu paw woman amasflamoumusfi mo soauwswEumuoo .m canwa

77

 

 

 

 

 

 

 

 

 

 

mm.a mun.N mmm.N mmw.~ mHHmu soxoun
. mo usmumcumasm
mo.o aw mo 50 fimmz :ume
00.0 mm qq NNH cmms nuuaom
om.a NNo.N eao.~ Hso.H some cease
mm.e mom.HH cmm.aa aom.aa coma ocoomm
oa.e~ “mo.em aom.wm mmm.am coma betas

u G 0H0>OUQH

H b mmm

«n.0m eqm.oq~ mbH.qu ana.mm~ coaumnaocfi Hmcaw no

mnu cw 0m>oEwu 00
mm
oo.ooa emo.m~e New.oce oeo.eme Nemmm Hocamaeo

.um>< HH H .um><
N .umwm Houusou

Gas 0H Homimucsou cowumumao mo mamH

.msmbwommaaam .um> omfimw>oumo .w.:ufi3 cowumnsucfi son: Now we much .0 memH

mm

78

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

na.ee oa.en 00.0 no.0 no.0 00.0 00.0 no.0 NN.H no.0 s0.n as.na Ho.no N
an no on mm on 00H non no~.n oeo.n ooa.n non.nn .co><
mm on mm 0H mm was “on osn.n eon.~ Hea.n ona.nn n 0H.n
on on nn nn nn nos nnn ann.n ono.n ana.n ana.nn H
.Jmen_
non.nn onaine Hopscoo
aa.nn Nn.en no.0 00.0 no.0 no.0 no.0 00.0 nn.o HH.N nn.o 0n.nH ne.an N
an mm on on on on nn enn nno.a oon.n n0n.en .eo><
on na nn nH on on no one nno.a noe.n ean.nn n nn.n
ma on an no on nn an non en~.H nan.e onn.en a
limmmn
non.en onn4ne Hocucoo
n0.~a nn.0N n~.o on.o an.o n~.0 0H.o on.o nn.o NN.0 00.0 no.na en.oH N
on on as on ma nn nH nH an nnn.H onn.a .cc><
on 0a ea na na mm on na so non.a non.n n Ha.e
on on on mm es em on na nu ooe.H Hon.n a
nimnmw
onn.e ona.ne Hopscoo Ha.e
.nsocfiil‘
NHHUU an Hmuoe suoa sum cum fiuh sue sum fine bum ch umH .wHuo
we s we oaaaom ma
0 amnesz smmz an .>ou
Imp Om ucmumcuCQSm wzu an poum>oomu,Nwfi>HuumOfiwom Imp Emu
Hobos
.NOm suns cofiumnsucfi “mums msovwomaaaam .um> mmwmw>mumo .0 mo mHHou mo asfizmms mo uommmm .0 wficma

mm

79

Table 5. Transport of SO2 in_§. cerevisiae var. ellipsoideus as a

 

 

function of time.

 

 

 

 

Time Uptake (cpm/ZO mg dry weight)
min Expt I Expt II Expt III Average %
0 3,127 2,905 3,027 3,020 ---
5 618 620 601 613 20.3
10 621 611 628 620 20.5
20 680 639 619 646 21.4
40 566 561 604 577 19.1
60 713 633 610 652 21.6

 

 

 

 

 

 

Table 6.

Time course of SO

2

80

transport in the yeast.

 

 

Time

DIRECT METHOD

INDIRECT METHOD

 

Uptake (cpm/ZO mg d.w.)

Uptake (cpm/20 mg d.w.)

 

 

sec. 1 2 3 AV. 2 1 2 3 AV. Z

0 3,430 3,415 3,369 3,417 -—- 4,439 4,483 4,503 4,475 --—
10 260 238 263 254 7.43 412 490 500 467 10.43
20 278 319 347 315 9.21 567 431 641 546 12 20
40 458 433 424 438 12.81 689 856 679 741 16.55
60 558 592 585 578 16.91 820 889 819 843 1°.83
120 802 901 651 785 22.97 1,144 982 953 1,026 22.92
300 799 663 790 751 21.97 1,150 993 980 1,041 23.26
600 669 889 781 779 22.79 1,003 1,027 908 979 21.87

 

 

 

 

 

 

 

 

 

 

 

-

81

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

00.0 00.0 00.0 000.0 000.0 000.0 000.0 000.00 000.00 000.00 000.00 000
00.0 00.0 00.0 000.0 000.0 000.0 000.0 000.00 000.00 000.00 000.00 00
00.0 00.0 00.0 000.0 000.0 000.0 000.0 000.00 000.00 000.00 000.00 00
00.0 00.0 00.0 000.0 000.0 000.0 000.0 000.00 000.00 000.00 000.00 00
00.0 00.0 00.0 000.0 000.0 000.0 000.0 000.00 000.00 000.00 000.00 00
00.0 00.00 00.0 000.0 000.0 000.0 000.0 000.0 000.0 000.00 000.0 00
00.0 00.00 00.0 000.0 000.0 000.0 000.0 000.0 000.0 000.0 000.0 0
00.0 00.00 00.0 000.0 000.0 000.0 000.0 000.0 000.0 000.0 000.0 0
00.0 00.00 00.0 000 000 000 000 000.0 000.0 000.0 000.0 0
00.0 00.00 00.0 000 -u- 000 000 000.0 000.0 000.0 000.0 0
11¢ 20000
20000 .>< 000 00 0 .>0 000 00 0 .ceoo
IGOU
> 0 0
0. 0. com 00 on
\NOm .omm 00\.3.0 we 0N\an oxmua: Asaov Houuaov Hmuoa
.GOfiumHquUGOU Now MO COfiuUGDm m mm mdmfifiomafimHHm .Hm> mmfimfi>mumu .Ima CH uHOQmeHu mwfixowv HDMHDm .m. 9238

82

 

 

 

 

 

 

 

 

 

 

 

Table 8. Effect of temperature on 802 transport in §._gg§gvisi§g
var. ellipsoideus.
Temp. Counts per minute per 20 mg cells Z of total SO2
(dry weight) taken up in
30 seconds
Expt I Expt II Expt III Average
20 (Control) 11,806 ---- 11,694 11,750
0 (Expt) 1,365 1,843 1,372 1,525 12.98
10 (Expt) 1,204 1,295 1,834 1,444 11.28
20 (Expt) 1,386 1,687 1,512 1,528 13.00
30 (Expt) 3,075 3,108 3,122 3,102 26.40
40 (Expt) 4,810 4,693 4,816 4,773 40.62
50 (Expt) 5,487 5,339 5,194 5,340 45.44
60 (Expt) 5,308 5,025 4,863 5,065 43.10
70 (Expt) 1,867 1,872 1,866 1,868 14.98

 

83

 

 

 

 

 

Table 9. Thermal inactivation of the S02 transporting system in
§, cerevisiae var. ellipsoideus.
Temp. Counts per min:::sw2:;hi? mg cells Z of total 502
C taken up
Expt I Expt II Expt III Average in 2 min.
20 Contr. 2,982 2,992 3,072 2,985 ---
20 Expt. 1,746 1,601 1,651 1,666 55.81
40 Expt. 1,801 1,745 1,808 1,751 58.66
50 Expt. 1,606 1,591 1,623 1,606 53.80
55 Expt. 1,311 1,309 1,112 1,244 41.67
60 Expt. 879 865 815 853 28.57
65 Expt. 43 43 61 49 1.55
70 Expt. 44 44 62 50 1.67

 

 

 

 

 

 

84

 

 

 

 

 

 

 

 

 

 

Table 10. Effect of metabolic inhibitors on 802 transport in
S. cerevisiae var. ellipsoideus.
cpm per 20 mg (d.w.) X
Inhibitor Concentration Expt Expt Expt Aver Inhibition
I .1 II 111

Without —-- 3,240 3,343 3,141 3,241 O
10‘5M 3,134 3,258 3,163 3,186 1.69
Iodoacetamide 5x10-5M 3,138 3,154 3,244 3,195 1,41
10‘48 3,216 3,136 3,210 3,187 1.66
lO'SM 1,949 1,984 1,898 1,943 40.04
Sodium Azide 5x10’5M 1,196 1,071 1,152 1,139 64.85
10‘48 921 903 871 898 72.29
5x10‘7n 2,660 2,603 2,658 2,640 18.54
2,4-Dinitrophenol 10—6M 2,270 2,279 2,212 2,253 30.48
5x10‘6M 2,215 2,193 2,275 2,227 31.28
10’6M 1,299 —-— 849 1,704 66.86
HgClz 5x10‘6n -105 ~134 —103 —114 103.51
10’5h -186 —163 —111 -153 104.72

 

 

 

 

 

 

 

85

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

00.0 000 00.0 000 00.0 000 00.0 000 00.0 000 00.0 000 00.0 00 000.0 onoco><
-- -- -- -- -- -- -- 000.0 0
000 000 000 000 000 00 000 000.0 0 00.0
000 000 000 000 000 000 00 000.0 0
00.00 000 00.00 000 00.00 000 00.00 000 00.00 000 00.0 000 00.0 000 000.0 oneco><
000 000 000 000 000 000 000 000.0 0
000 000 000 000 000 000 000 000.0 0 00.0
000 000 000 000 000 000 000 000.0 0
00.00 000.0 00.00 000.0 00.00 000.0 00.00 000.0 00.00 000.0 00.00 000.0 00.00 000 000.0 onmco><
000.0 000.0 000.0 000.0 000.0 000.0 000 000.0 0
000.0 000.0 000.0 000.0 000.0 000.0 000 000.0 0 00.0
000.0 000.0 000.0 000.0 000.0 000.0 000 000.0 0
00.00 000.0 00.00 000.0 00.00 000.0 00.00 000.0 00.00 000.0 00.00 000.0 00.00 000.0 000.0 onmeo><
000.0 000.0 000.0 000.0 000.0 000.0 -- 000.0 0
000.0 000.0 000.0 000.0 000.0 000.0 000.0 000.0 0 00.0
000.0 000.0 000.0 000.0 000.0 000.0 000.0 000.0 0
m 808 0 Saw 0 895 m 83$ 0 808 0 895 0 .090 98
com 000 omm 00m 00w QNH own 00 awn 00 umm cm can OH .02 :0
A.3.U waamu wE 0N\anv mxmump HWMW mHaEmm
.:a mo cowuoasw m on mamvwommwaam .um> om0m0>mumu .w.:0 uuoamsmnu Now no mmusoo 6808 .00 magma

86

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.00 >0 000.0>< 000.0>< 0.00 >0 000.0>< 000.o0>< 0.00 >0 000.0>< 000.0><
0.00 000.0 0.00 000.0 0.00 000.0
00.00 0.00 000.0 0.00 000.0 0.00 000.0 0.0 0.0 0 00.0
0.00 000.0 0.00 000.0 0.00 000.0
0.00 >0 000 >0 00n.0>< 0.00 >0 0n0.0>< 000.00>< 0.00 >< 000 >0 000.0>0
0.00 000 000.0 0.00 000.0 0.00 000
00.00 0.00 000 000.0 0.00 000.0 0.00 000 0.0 0.0 0 00.0
0.00 000 000.0 0.00 000.0 0.00 000
0.00 >0 000 >0 000.0>< 0.00 >0 non.0>< 000.00>< 0.00 >< 000 >0 00n.0><
0.00 000 000.0 0.00 000.0 0.00 000
00.00 0.00 000 000.0 0.00 000.0 0.00 000 0.0 0.0 0 00.0
0.00 000 000.0 0.00 000.0 0.00 000
0.00 >0 000 >0 000.0>< 0.00 >< 000.0>< 000.00>< 0.0 >0 000 >0 00n.0><
0.00 000 000.0 0.0 000.0 0.0 000
00.0 0.00 000 000.0 0.0 000 0.0 000 0.0 0.0 0 00.0
0.0 000 000.0 0.00 000.0 0.0 000
I I I H.0 >< New >< 0mm.CH>< 0.0 >< NNH >< mmm.m>¢
- - - 0.0 000 -- 0.0 000 000.0
00.0 - - - 0.0 000 000.00 0.0 000 000.0 0.0 0.0 0 00.0
- - - 0.0 000 000.00 0.0 000 000.0
IIIMW N .umwm Hoaammwf!IIw .uQWm Hepucmu N .umwm Houucou 000x . moax
.uuamusoo 0 New
.um>< Auzwfims 000 .mHHoo 05 ON 000 anv meump mow amasomaoz 00009 :0
New unasumaos
mo u::0Em 0cm msmvfiowmwaam .um> om0m0>muum..m.s0 uuoamcwuu New mo musk smmsuon 003w:00umamm .NH manna

 

87

 

 

 

 

 

 

 

 

 

 

 

 

Table 13. Transport of SO in S. cerevisiae var. ellipsoideus
as a function 0 the molecular S02 concentration.
Total Molecular 802 Uptake (cpm/20mg d.w.)
SO Control
pH 33 Z Concentr. Expt.
x10 M x10-3M
6,075 1,761
4.11 8.0 0.5 4 6,166 1,960
6,227 2,027
Av. 6,289 Av. 1,916 Av. 30.46
1,044
3.80 4.0 1.0 4 1,046
Av. 3,144 Av. 1,045 Av. 33.23
542
3.50 2.0 2.0 4 552
549
Av. 1,572 Av. 558 Av. 34.19
344
3.32 1.33 3.0 4 278
316
Av. 1,025 Av. 313 Av. 30.53
254
3.19 1.0 4.0 4 229
245
Av. 786 Av. 243 Av. 30.91

 

 

 

 

 

 

 

8
8

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I I I I I I I I 0.00 0.0 0.00 0.00 N.om 0.0m m.wN N.om 0.0m N.mm 0.0m 0.mm N.0H m 000 m.m
I I I I m.NH 0.0a H.0 0.00 m.¢N 0.0N m.HN w.MN 0.00 0.00 0.Nm 0.00 0.N0 0.00 0.0m 0.00 w.NH N 0Nm o.m
w.m0 0.00 N.Hm 0.0m 0.0m 0.0m 0.N0 m.0m N.00 m.mm 0.00 n.00 0.00 0.00 0.N0 n.00 0.00 0.00 0.00 0.00 0.0 H 000 m.N
.o>< 000 00 0 .m>< 000 00 0 .o>< 000 00 0 .o>< 000 00 0 .o>< 000 00 0 800 000 500 00V.
.0300 .0800 . 0.0.0.0 .0030 .0830 0
.C0E cm .GHE 0N .cfie m0 .C0E OH .GHE m umaaooaoz Hmuoa
GOHumuucoosoo
m u o > 0 > u a m N N
Cm
.oooN .00.m u :0 us moeflu 0cm mcowumuucmosoo
Now ucmpowmwv zufis toumnaosfl mmmwmmmdeHm .pm> mmwm0>wumo .m.mo Hm>0>uam ucooumm .00 magma

Table 15.

Percent survival of g. cerevisiae var. ellipsoideus
incubated with various SO
pH levels, 20°C.

89

 

 

2

concentrations at different

 

 

pH

3.19
3.50
3.80
4.11

4.80

Concentration

SO2

Z Survivors

 

Total

MOlecular

Expt I

Expt II

Expt

III

 

x103M

PPm

 

x104M

PPm

1 2

Average

 

 

2.5

5.0

10.0

20.0

100

 

160
320
640
1,280

6,400

 

 

6.4

6.4

6.4

6.4

6.4

60.29

65.68

62.25

69.60

69.11

 

 

75.92
63.23
66.66
67.15

50.00

 

62.25 66.17
59.31 62.74
63.23 62.74
60.78 57.84

66.17 62.25

 

 

66.17
64.70
64.70
66.17

72.05

 

71.07

67.15

60.78

68.62

63.72

 

67.15

63.72

63.23

65.19

63.72

 

9O

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Average

—.-’--——-

66.17
57.63 '

30.04

Table 16. Percent survival of §-_£9£EX1§1§E.X££-.Slliflfigiégfli
incubated with different 802 concentration at pH=3.19
for 5 min.

SO
2 .
Z Surv1vors

Concentration
Total Molecular Expt I Expt II Expt III

3 4

x10 ppm x10 ppm 1 2 l 2 1

2.5 160 l 6.4 62.06 66.00 72.41 64.21 65.02 69.45
5.0 320 2 12.8 62.65 52.70 64.03 62.06 50.73 52.70

10.0 640 4 25.6 38.91 35.96 25.12 25 61 26.60 27.09

20 1240 8 51.2 0 0 0 0 0

100 6400 40 256 O 0 -&~JZ_M_..m-dLfi.j)

 

 

91

Distribution of 35

 

 

 

 

 

Table 17. S in various fractions of the
yeast §, cerevisiae var. ellipsoideus.
Total activity
(cpm/1.2 3 cells du) 35
Fraction Z S
Exp I Exp II Aver.
Control 14,928 15,516 15,222 100.00
1. Cold trichloroacetic acid
supernatant
a. Inorganic sulfate and sulfite 3 9 6 0.04.
b. Nucleotide S. 2 2 2 0.01
c. Glutathione -4 10 3 0.02
d. Other small molecules 14,861 15,427 15,143 99.48
2. Lipid S. -7 49 21 0.14
3. Alkal-extractable S. 21 29 25 0.16
4. 6N-HC1 hydrolyzate 52 -8 22 0.15

 

 

 

 

 

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