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 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
(1) 5-chloro-2-methyl-4-isothiazolin-3-one (MCI, CMIT, RH-651) and 2-methyl-4-isothiazolin-3-one (MI, MIT, RH-573)

Figure A. Rapid Inhibition of Growth
0.5 ProClin Active Ingredient Level 0ppm 0.4 1ppm 2.5ppm 0.3

Absorbance at 600nm

0.2

0.1

0.0 -0.5 0.0 0.5 1.0 1.5

Hours After Biocide Addition

Figure B. Sites of Inhibition
Glucose Pyruvate 2H CO2 Acetyl-CoA Oxaloacetate Malate Fumarate KREBS CYCLE Citrate [cis-Aconitate] Isocitrate CO2

α-Ketogluatrate Succinate CO2 Succinyl-CoA 2H 2H 2H 2H

NAD Electron Transport Chain Sites of ProClin biocide inhibition

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

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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): 1. 2. Covalent modification via direct electrophilic attack Generation of a secondary electrophile by disulfide exchange and tautomerization to a thioacyl chloride An intracellular generation of free 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) TRIS % Loss of Active ProClin Preservative After 4 weeks at 25 °C 6.3 20.3 8.3 12.0 8.1 19.4 % Loss of Active ProClin Preservative After 4 weeks at 40 °C 4.8 36.8 9.8 45.0 8.2 39.1

3.

Figure C. Cidal Activity of ProClin Preservative (Cell Death)
102

101

5ppm MCI*

pH 7.0 8.0 7.0 8.0 7.0 8.0

% Survivors

100

TRIS HEPES
25ppm MCI

10

-1

HEPES TES

10

-2

TES
100ppm MCI

10

-3

0

15

30

45

60

75

90

Minutes After Biocide Addition

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

Figure D. Cells Killed vs. Protein Thiols Lost
O N HS PROTEIN CI S O CH3 NCH3 S PROTEIN

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.

CI

MCI Disruption of Metabolism

S

RSH RS RS S PROTEIN O

Radical Cascade
O CI SH

NCH3

Irreparable Damage

CI

S

NCH3 OH

Death

PROTEIN

SH NH2

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.

ProClin Preservatives
®

Mechanisms and Stability

Conclusions
1. ProClin preservatives exhibit a unique mechanism of action that results in both inhibition and cell death. ProClin preservatives are immediately bacteriostatic by quickly penetrating cell membranes and inhibiting specific enzymes. ProClin preservatives demonstrate a high degree of specificity by inhibiting only specific key enzymes in the cell. ProClin preservative molecules remain available for more critical target enzymes because they are not consumed by reacting with nonessential enzymes. ProClin preservatives target multiple specific enzymes and reduce the microbe’s ability to mutate and achieve resistance. ProClin preservative treatment results in an accumulation of cell damage beyond the cell’s capacity for repair, and death results.

Ordering Information
Description ProClin 150 Preservative 50 mL bottle 400 mL bottle 3.6 L bottle 15 L pail 110 kg drum (91.7 L) ProClin 200 Preservative 50 mL bottle 400 mL bottle 3.6 L bottle 15 L pail ProClin 300 Preservative 1 x 5 mL vial 50 mL bottle 400 mL bottle 2.0 L bottle 3.6 L bottle 18 L pail 110 kg drum (107.8 L) ProClin 950 Preservative 5 mL ampule 50 mL bottle 400 mL bottle 3.6 L bottle 17 L pail Cat. No. 49376-U 49377-U 49378-U 49379-U 49380-U

2.

3.

4.

48171-U 500380 500399 500402

5.

48934-U 48912-U 48914-U 48915-U 48917-U 48918-U 48919-U

6.

46885-U 46878-U 46879-U 46883-U 46884-U

ProClin Evaluation Kits ProClin Reference Standard 1 mL ampule (1.5% A.I. solution) ProClin 150 — 3 x 5 mL (3 lot numbers) ProClin 150 — 3 x 400 mL (3 lot numbers) ProClin 300 — 3 x 5 mL (3 lot numbers) 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

Cat. No. 33360-U 48121 49381-U 48911-U

48119-U

SAFC Supply Solutions 3050 Spruce St., Saint Louis, MO 63103 800-244-1173 (US Toll Free) +1-314-534-4900 (International) Global Email: safcglobal@sial.com

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. © 2009 SAFC All rights reserved. KVV 04773-508595 0059

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