ProClin® Preservatives Mechanisms and Stability

for Diagnostics Reagents

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ProClin® Preservatives Mechanisms and Stability

for Diagnostic Reagents

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 Preservatives ProClin preservatives are immediately bacteriostatic upon contact with a microbe (Figure A). This is the result of the ability of the active ingredients1 to quickly penetrate cell membranes and inhibit specific enzymes in the cell. Some of these target enzymes are within the central metabolic cycle of the cell, the Krebs cycle.

ProClin preservatives attack the Krebs cycle at four sites: the enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, and NADH dehydrogenase (Figure B). With the Krebs cycle debilitated, cells rapidly lose the ability to produce energy and subsequently die.

Because all bacteria and fungi possess at least part of the Krebs cycle, ProClin preservatives are broad spectrum in their activity. The ability of ProClin preservatives to act on specific enzymes is reflected in the low levels required to control growth. ProClin preservatives also target multiple specific enzymes, reducing the microbes ability to mutate one target site to achieve any level of resistance.

The rapid disruption of cellular metabolism as a result of specific enzyme inhibition severely impairs the ability of the cell to repair damage inflicted upon its components. The accumulation of damage beyond the capacity of the cell for repair results in cell death. Low concentrations of ProClin preservative, which are immediately bacteriostatic, require several hours to kill the cell, while higher concentrations exhibit rapid microbicidal effects (Figure C). This is a reflection of the rate-driven nature of the damage process; i.e. higher concentrations of ProClin

preservative both inflict damage at a greater rate and overwhelm the cell’s repair functions faster than lower concentrations of ProClin preservative.

1ppm

Hours After Biocide Addition

Abs

orba

nce

at 6

00nm

0.2

-0.5 0.0 0.5 1.0 1.5

ProClin ActiveIngredient Level

0.0

0.5

0.4

0.3

0.1

0ppm

2.5ppm

Figure A. Rapid Inhibition of Growth

Glucose

Pyruvate

2H CO2

Acetyl-CoA

Oxaloacetate Citrate[cis-Aconitate]

Isocitrate

α-KetogluatrateSuccinyl-CoA

Succinate

Fumarate

Malate

NAD

Electron Transport Chain

KREBSCYCLE

CO2

CO2

2H 2H2H2H

Sites of ProClin biocide inhibition

Figure B. Sites of Inhibition

(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 possess multiple pathways that result in the lethal loss of protein thiols (Figure D):

Covalent modification via direct 1. electrophilic attack

Generation of a secondary 2. electrophile by disulfide exchange and tautomerization to a thioacyl chloride

An intracellular generation of free 3. radicals as a result of the severe metabolic disruption, which severely stresses the cell’s natural radical defense mechanism

Stability of ProClin Preservatives in Amino Buffers ProClin preservatives exhibit a wide range of chemical compatibility and pH tolerance, making them an ideal choice for preserving a variety of systems. One class of chemicals that is aggressive to the isothiazolone molecules is secondary amines. With their strong nucleophilic activity, secondary amines break the isothiazolones’ sulfur-nitrogen bond, resulting in ring-opening and loss of biocidal activity. Much of this interaction is pH-dependent; at alkaline pH the equilibrium favors amines that are highly aggressive nucleophiles. At neutral pH and below, the amines are protonated and have reduced nucleophilic activity.

Table 1. The Effect of pH on Stability

Buffer(50mM) pH

% Loss of Active ProClin

Preservative After 4 weeks at 25 °C

% Loss of Active ProClin

Preservative After 4 weeks at 40 °C

TRIS 7.0 6.3 4.8

TRIS 8.0 20.3 36.8

HEPES 7.0 8.3 9.8

HEPES 8.0 12.0 45.0

TES 7.0 8.1 8.2

TES 8.0 19.4 39.1

Amino-based buffers are common components of diagnostic reagents. The stability of ProClin preservatives in various amino buffers, and the effect of pH on this stability, have been studied. The results indicate that biocide stability is strongly correlated with buffer pH (Table 1). At 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 also indicated that amine-mediated degradation occurred at a much higher rate when the storage temperature was increased from 25°C to 40°C.

In order to maximize the duration of antimicrobial protection, amino-based buffers should be adjusted to pH 7.0 or lower, whenever possible, prior to adding ProClin 150, 200 or 300 preservatives. If the pH of the reagent system is critical to the diagnostic assay, ProClin 950 should be considered, as well as an alternative buffer system. Buffers based on sterically hindered tertiary amines may be more compatible with ProClin preservatives than those based on primary or secondary amines.

S

RSH

O

CIS

CH3

HSCI

O

NCH3

S

RS S

N

Disruption ofMetabolism

Radical Cascade

Irreparable Damage

Death

CI

O

NCH3

SH

NCH3

O

CI S

SHOH

NH2

RS

MCI

PROTEIN

PROTEIN

PROTEIN

PROTEIN

Figure D. Cells Killed vs. Protein Thiols Lost

Figure C. Cidal Activity of ProClin Preservative (Cell Death)

10 -2

10 -3

10 -1

100

101

102

% S

urvi

vors

06510 45 095703

100ppm MCI

25ppm MCI

5ppm MCI*

Minutes After Biocide Addition

* MCI = methylchloroisothiazolone (primary active ingredient in ProClin preservatives)

KVV04773-5085950059

ProClin® Preservatives Mechanisms and Stability

ConclusionsProClin preservatives exhibit a unique 1. mechanism of action that results in both inhibition and cell death.

ProClin preservatives are immediately 2. bacteriostatic by quickly penetrating cell membranes and inhibiting specific enzymes.

ProClin preservatives demonstrate a 3. high degree of specificity by inhibiting only specific key enzymes in the cell.

ProClin preservative molecules 4. remain available for more critical target enzymes because they are not consumed by reacting with nonessential enzymes.

ProClin preservatives target multiple 5. specific enzymes and reduce the microbe’s ability to mutate and achieve resistance.

ProClin preservative treatment results 6. in an accumulation of cell damage beyond the cell’s capacity for repair, and death results.

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800-244-1173 (US Toll Free) +1-314-534-4900 (International)

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SAFC®, SAFC Supply Solutions® and Sigma-Aldrich® are registered trademarks of Sigma-Aldrich Biotechnology L.P. and Sigma-Aldrich Co.

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

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ProClin Evaluation Kits Cat. No.

ProClin Reference Standard 1 mL ampule (1.5% A.I. solution)

33360-U

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

ProClin 150 — 3 x 400 mL (3 lot numbers)

49381-U

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 aid to select the appropriate ProClin product for a specific application

48119-U

Ordering Information

Description Cat. No.

ProClin 150 Preservative 50 mL bottle400 mL bottle3.6 L bottle15 L pail110 kg drum (91.7 L)

ProClin 200 Preservative 50 mL bottle400 mL bottle3.6 L bottle15 L pail

ProClin 300 Preservative 1 x 5 mL vial50 mL bottle400 mL bottle2.0 L bottle3.6 L bottle18 L pail110 kg drum (107.8 L)

ProClin 950 Preservative 5 mL ampule50 mL bottle400 mL bottle3.6 L bottle17 L pail

49376-U49377-U49378-U49379-U49380-U

48171-U 500380 500399500402

48934-U48912-U 48914-U 48915-U48917-U48918-U48919-U

46885-U46878-U46879-U46883-U46884-U