ProClin Preservatives

Mechanisms and Stability

for Diagnostic Reagents

ProClin Preservatives
®

Mechanisms and Stability

for Diagnostics Reagents

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ProClin Preservatives ®

Mechanisms and Stability

The unique biocidal mechanisms of ProClin preservatives provide a broad

spectrum of activity, strong lethality at low concentration and preclude microbial
resistance by mutation. In addition, the biocidal isothiazolone components
in these preservatives offer a wide range of chemical compatibility and pH
tolerance. Combined, these characteristics make ProClin preservatives an ideal
choice for a variety of diagnostic reagent systems.

The Mechanism of Action of ProClin

Figure A.
Preservatives
Rapid Inhibition of Growth
ProClin preservatives are immediately
bacteriostatic upon contact with a microbe 0.5
ProClin Active
Ingredient Level
(Figure A). This is the result of the ability of
0ppm
the active ingredients1 to quickly penetrate 0.4 1ppm
cell membranes and inhibit specific 2.5ppm

Absorbance at 600nm
enzymes in the cell. Some of these target
0.3
enzymes are within the central metabolic
cycle of the cell, the Krebs cycle.
0.2

ProClin preservatives attack the Krebs

cycle at four sites: the enzymes pyruvate 0.1

dehydrogenase, α-ketoglutarate
dehydrogenase, succinate dehydrogenase, 0.0
and NADH dehydrogenase (Figure B). With -0.5 0.0 0.5 1.0 1.5
the Krebs cycle debilitated, cells rapidly Hours After Biocide Addition

lose the ability to produce energy and

subsequently die.

Because all bacteria and fungi possess Figure B.

at least part of the Krebs cycle, ProClin Sites of Inhibition
preservatives are broad spectrum in their Glucose
activity. The ability of ProClin preservatives Pyruvate
to act on specific enzymes is reflected in
2H CO2
the low levels required to control growth. Acetyl-CoA
ProClin preservatives also target multiple
specific enzymes, reducing the microbes Oxaloacetate Citrate
ability to mutate one target site to achieve Malate KREBS
[cis-Aconitate]

any level of resistance. CYCLE Isocitrate

Fumarate CO2
α-Ketogluatrate
The rapid disruption of cellular metabolism Succinate
Succinyl-CoA CO2
as a result of specific enzyme inhibition
severely impairs the ability of the cell 2H
2H 2H
2H
to repair damage inflicted upon its
components. The accumulation of damage
NAD
beyond the capacity of the cell for repair
results in cell death. Low concentrations Electron Transport Chain
of ProClin preservative, which are Sites of ProClin biocide inhibition
immediately bacteriostatic, require
several hours to kill the cell, while higher
concentrations exhibit rapid microbicidal preservative both inflict damage at a
effects (Figure C). This is a reflection of the greater rate and overwhelm the cell’s repair
rate-driven nature of the damage process; functions faster than lower concentrations
i.e. higher concentrations of ProClin of ProClin preservative.

(1) 5-chloro-2-methyl-4-isothiazolin-3-one (MCI, CMIT, RH-651)

and 2-methyl-4-isothiazolin-3-one (MI, MIT, RH-573)

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for Diagnostic Reagents

Data indicates that ProClin preservatives Stability of ProClin Preservatives in

possess multiple pathways that result in Amino Buffers
the lethal loss of protein thiols (Figure D): ProClin preservatives exhibit a wide
1. Covalent modification via direct range of chemical compatibility and pH
electrophilic attack tolerance, making them an ideal choice
for preserving a variety of systems. One
2. Generation of a secondary class of chemicals that is aggressive to
electrophile by disulfide exchange and the isothiazolone molecules is secondary
tautomerization to a thioacyl chloride amines. With their strong nucleophilic
3. An intracellular generation of free activity, secondary amines break the
radicals as a result of the severe isothiazolones’ sulfur-nitrogen bond,
metabolic disruption, which severely resulting in ring-opening and loss of
stresses the cell’s natural radical biocidal activity. Much of this interaction
defense mechanism is pH-dependent; at alkaline pH the
equilibrium favors amines that are highly
aggressive nucleophiles. At neutral pH and
Figure C. below, the amines are protonated and have
Cidal Activity of ProClin Preservative reduced nucleophilic activity.
(Cell Death)

102
Table 1. The Effect of pH on Stability
% Loss of % Loss of
Buffer Active ProClin Active ProClin
101 (50mM) pH Preservative After Preservative After
5ppm MCI* 4 weeks at 25 °C 4 weeks at 40 °C
TRIS 7.0 6.3 4.8
100
% Survivors

TRIS 8.0 20.3 36.8

HEPES 7.0 8.3 9.8
-1 25ppm MCI
10
HEPES 8.0 12.0 45.0
TES 7.0 8.1 8.2
-2
10 TES 8.0 19.4 39.1

100ppm MCI Amino-based buffers are common components

10 -3 of diagnostic reagents. The stability of ProClin
preservatives in various amino buffers, and the
0 15 30 45 60 75 90
effect of pH on this stability, have been studied.
Minutes After Biocide Addition
The results indicate that biocide stability is
* MCI = methylchloroisothiazolone (primary active ingredient in
strongly correlated with buffer pH (Table 1). At
ProClin preservatives) pH 7.0, there is a small loss of active biocide
after 4 weeks storage (25°C) in all the buffers
tested. Degradation accelerated significantly
when the pH of the buffers was 8.0. The results
Figure D. also indicated that amine-mediated degradation
Cells Killed vs. Protein Thiols Lost occurred at a much higher rate when the storage
temperature was increased from 25°C to 40°C.
O O
In order to maximize the duration of
HS PROTEIN
CI
N
CH3 CI
NCH3
antimicrobial protection, amino-based
S
MCI
S S PROTEIN
buffers should be adjusted to pH 7.0 or
RSH
lower, whenever possible, prior to adding
Disruption of
Metabolism RS RS S PROTEIN ProClin 150, 200 or 300 preservatives.
O If the pH of the reagent system is critical
Radical Cascade NCH3 to the diagnostic assay, ProClin 950 should
O CI
SH be considered, as well as an alternative
Irreparable Damage CI
S
NCH3
buffer system. Buffers based on sterically
OH hindered tertiary amines may be more
Death
PROTEIN SH
NH2
compatible with ProClin preservatives
than those based on primary or
secondary amines.
 ProClin Preservatives
®

Mechanisms and Stability

Conclusions Ordering Information

1. ProClin preservatives exhibit a unique Description Cat. No.
mechanism of action that results in
ProClin 150 Preservative
both inhibition and cell death. 50 mL bottle 49376-U
400 mL bottle 49377-U
2. ProClin preservatives are immediately
3.6 L bottle 49378-U
bacteriostatic by quickly penetrating
15 L pail 49379-U
cell membranes and inhibiting 110 kg drum (91.7 L) 49380-U
specific enzymes.
ProClin 200 Preservative
3. ProClin preservatives demonstrate a 50 mL bottle 48171-U
high degree of specificity by inhibiting 400 mL bottle 500380
only specific key enzymes in the cell. 3.6 L bottle 500399
15 L pail 500402
4. ProClin preservative molecules
remain available for more critical ProClin 300 Preservative
target enzymes because they are 1 x 5 mL vial 48934-U
not consumed by reacting with 50 mL bottle 48912-U
400 mL bottle 48914-U
nonessential enzymes.
2.0 L bottle 48915-U
5. ProClin preservatives target multiple 3.6 L bottle 48917-U
specific enzymes and reduce the 18 L pail 48918-U
110 kg drum (107.8 L) 48919-U
microbe’s ability to mutate and
achieve resistance. ProClin 950 Preservative
5 mL ampule 46885-U
6. ProClin preservative treatment results
50 mL bottle 46878-U
in an accumulation of cell damage 400 mL bottle 46879-U
beyond the cell’s capacity for repair, 3.6 L bottle 46883-U
and death results. 17 L pail 46884-U

ProClin Evaluation Kits Cat. No.

ProClin Reference Standard

33360-U
1 mL ampule (1.5% A.I. solution)

ProClin 150 — 3 x 5 mL (3 lot numbers) 48121

ProClin 150 — 3 x 400 mL

49381-U
(3 lot numbers)

ProClin 300 — 3 x 5 mL (3 lot numbers) 48911-U

ProClin Variety Kit — 5 mL each of

ProClin 150, 200, 300 and 950 as an
48119-U
aid to select the appropriate ProClin
product for a specific application

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ProClin® is a registered trademark of Rohm and Haas Company.
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