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Under extreme conditions, the free amine group can be activated by diazotization and then coupled to protein tyrosine residues in a mild subsequent immobilization step. 4.2.1.4. Acidic Supports. Three methods will be considered for enzyme immobilizations onto carboxylic acid-containing supports. All result in the formation of amide bonds to the primary amines of lysine residues on the protein, but the selectivity for lysine varies with the method used. In the acyl azide method,8 the carboxylic acid group is sequentially converted to its methyl ester, acyl hydrazide and acyl azide (Eq. 4.10): 4:10 The acyl azide is subject to nucleophilic attack, and is most reactive toward primary amino groups (lysine), but will also react with tyrosine hydroxyl, cysteine sulfhydryl, and serine hydroxyl groups (Eq. 4.11): 4:11 Woodward s reagent K9 is a water-soluble dehydrating reagent that activates carboxylic acid groups toward nucleophilic attack. It may be used under mild, aqueous conditions, and sequential activation and coupling steps prevent enzyme crosslinking (Eqs. 4.12 and 4.13):

How we measure availability The failure modes, or typical things that can and do go wrong Situations in which measurements may not be valid

4:12

, to reach a decision about whether or not subsequently to appeal to the market, several conditions need to be brought together: search for a business sponsor interested in the approach (possibly even the reader); identify at least one business or technical domain that seems naturally to call for an application integration solution;.

Throughout this book we use resiliency in terms of overall system availability. We see resiliency as a general term similar to high availability, but without all the baggage that HA carries along with it. High availability once referred to a fairly specific range of system configurations, often involving two computers that monitor and protect each other. During the last few years, however, it has

2

4:13

lost much of its original meaning; vendors and users have co-opted the term to mean whatever they want it to mean. To us, resiliency and high availability mean that all of a system s failure modes are known and well defined, including networks and applications. They mean that the recovery times for all known failures have an upper bound; we know how long a particular failure will have the system down. Although there may be certain failures that we cannot cope with very well, we know what they are and how to recover from them, and we have backup plans to use if our recoveries don t work. A resilient system is one that can take a hit to a critical component and recover and come back for more in a known, bounded, and generally acceptable period of time.

When you discuss availability requirements with a user or project leader, he will invariably tell you that 100 percent availability is required: Our project is so important that we can t have any downtime at all. But the tune usually changes when the project leader finds out how much 100 percent availability would cost. Then the discussion becomes a matter of money, and more of a negotiation process. As you can see in Table 2.1, for many applications, 99 percent uptime is adequate. If the systems average an hour and a half of downtime per week, that may be satisfactory. Of course, a lot of that depends on when the hour and a half occurs. If it falls between 3:00 A.M. and 4:30 A.M. on Sunday, that is going to be a lot more tolerable on many systems than if it occurs between 10:00 A.M. and 11:30 A.M. on Thursday, or every weekday at 2:00 P.M. for 15 or 20 minutes.

If cross-linking is not expected to occur, then Woodward s reagent K may be included in an enzyme solution, and a single-step immobilization reaction may be performed.

Table 2.1 Measuring Availability PERCENTAGE UPTIME 98% 99% 99.8% 99.9% 99.99% 99.999% 99.9999% ( six 9s ) PERCENTAGE DOWNTIME 2% 1% 0.2% 0.1% 0.01% 0.001% 0.0001% DOWNTIME PER YEAR 7.3 days 3.65 days 17 hours, 30 minutes 8 hours, 45 minutes 52.5 minutes 5.25 minutes 31.5 seconds DOWNTIME PER WEEK 3 hours, 22 minutes 1 hour, 41 minutes 20 minutes, 10 seconds 10 minutes, 5 seconds 1 minute 6 seconds 0.6 seconds