1. A method of releasing a reversibly-linked water-soluble polymer from a protein modified by the water-soluble polymer comprising the step of incubating the protein under one or more conditions effective to release the water-soluble polymer.
2. A method of increasing activity of a protein modified with a reversibly-linked water-soluble polymer comprising the step of incubating the protein under one or more conditions effective to release the water-soluble polymer.
3. The method of claim 1 or 2, wherein the condition effective to release the water-soluble polymer comprises increasing pH of a buffer comprising the protein from about pH 6 to about pH 10.
4. The method of claim 3, wherein the condition comprises increasing pH of the buffer from about 6.1 to about 9.8.
5. The method of claim 3, wherein the condition comprises increasing pH of the buffer from about 7.3 to about 9.8.
6. The method of claim 3, wherein the condition comprises increasing pH of the buffer from about 6.5 to about 8.1.
7. The method of claim 1 or 2, wherein the condition effective to release the water-soluble polymer comprises increasing free amine concentration of a buffer comprising the protein.
8. The method of claim 7, wherein the increased free amine concentration of the buffer is the result of an increase in the concentration of lysine.
9. The method of claim 7, wherein the increased free amine concentration of the buffer is the result of an increase in the concentration of histidine.
10. The method of claim 7, wherein the increased free amine concentration of the buffer is the result of an increase in a combination of amines.
11. The method of claim 10, wherein the combination of amines is lysine and histidine.
12. The method of claim 1 or 2, wherein the condition effective to release the water-soluble polymer comprises increasing the temperature of the buffer from about room temperature to about 37\xb0 C.
13. The method of claim 1 or 2, wherein the condition effective to release the water-soluble polymer comprises increasing the temperature of the buffer from about 4\xb0 C. to about 37\xb0 C.
14. The method of claim 1 or 2, wherein the condition effective to release the water-soluble polymer comprises extending a time period for incubating the protein from about 5 minutes to about 168 hours.
15. The method of claim 14, where the time period ranges from about 5 minutes to about 48 hours.
16. A method of assaying quality control of proteins modified with a releasable water-soluble polymer comprising the steps of incubating the proteins from 0 to about 6 hours at 37\xb0 C. in a buffer of about pH 7.3 comprising about 100 mM histidine and about 100 mM lysine, and analyzing protein activity within the first 6 hours.
17. A method of monitoring an increase or regain of activity of a protein modified by a reversibly-linked water-soluble polymer, said method comprising measuring protein activity before and after removing the reversibly-linked water-soluble polymer from the protein.
18. A method of measuring kinetics of water-soluble polymer release from a protein modified by a reversibly-linked water-soluble polymer, said method comprising simultaneously measuring over a period of time in a reaction mixture an amount of free water-soluble polymer and an amount of water-soluble polymer conjugated to a protein, wherein kinetics are determined from a change in the amount of free water-soluble polymer and a change in the amount of water-soluble polymer conjugated to the protein.
19. The method of claim 18, wherein the measuring is based on a fluorescence emission spectra, wherein the simultaneous measuring is carried out using fluorescence at an emission peak of about 350-355 nm for water-soluble polymer-9H-(f)luoren-9-yl(m)eth(o)xy(c)arbonyl conjugates and at an emission peak of about 460-560 nm for water-soluble polymer-dibenzofulvene.
20. The method of claim 18, wherein the measuring is based on high-performance liquid chromatography for free water-soluble polymer-9H-(f)luoren-9-yl(m)eth(o)xy(c)arbonyl and water-soluble polymer-dibenzofulvene.
21. The method of claim 18, wherein the measuring is immunochemically based on an enzyme-linked immunosorbent assay for a water-soluble polymer conjugated protein and for the free protein.
22. The method of any one of claims 18-21 wherein the protein is factor VIII (FVIII).
23. The method of any one of claims 18-21 wherein the protein is von Willebrand Factor (VWF).
24. The method of claim 1, 2, 16, 17, or 18 wherein the water-soluble polymer is reversibly linked to the protein with 9H-(f)luoren-9-yl(m)eth(o)xy(c)arbonyl, dibenzofulvene, or a derivative thereof.
25. The method of claim 24 wherein the water-soluble polymer is linked with 9H-(f)luoren-9-yl(m)eth(o)xy(c)arbonyl, or a derivative thereof.
26. The method of any one of claims 1-25 wherein the water-soluble polymer is polyethylene glycol.
27. The method of any one of claims 1-25 wherein the water-soluble polymer is polysialic acid.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A method for eliminating the time delay between transmissions in a time sharing wireless network, comprising:
receiving, from one of a plurality of remote units, a data transmission on a wireless channel;
predicting the end of said data transmission based on the contents of said data transmission; and
sending, to said plurality of remote units at a predetermined time prior to the end of said data transmission, a status message indicating that said wireless channel is idle,
wherein a determination of the predetermined time include consideration of hardware and software latencies in transmitting, receiving, and decoding the status message.
2. The method of claim 1 wherein said message contains a status flag indicating the state of said wireless channel.
3. The method of claim 2 wherein said message is transmitted on a wireless channel that is different from said wireless channel on which said data transmission is being received.
4. The method of claim 3 wherein said data transmission contains a value indicating the total length of the data transmission.
5. A method for controlling traffic in a wireless transmission system wherein information is passed from a remote unit to a base unit along a first wireless channel and information is passed from the base unit to a remote unit along a second wireless channel, comprising:
monitoring said first wireless channel for a transmission;
if a transmission is received:
attempting to decode the transmission,
if said attempt to decode is successful:
predicting the completion time of said transmission,
sending along said second wireless channel, at a predetermined time prior to the end of said transmission, a status message indicating that said first wireless channel is idle,
wherein said predetermined time is based on consideration of hardware and software latencies in transmitting, receiving, and decoding the status message.
6. The method of claim 5 wherein said predicting step is conducted based on information contained in said transmission.
7. The method of claim 6 wherein said transmission contains a value indicating the length of the transmission.
8. The method of claim 6 wherein said predicting step is conducted based on the type of information contained in said transmission.
9. A method for communicating the activity state of a wireless channel and decode status of transmitted messages for use on a time sharing wireless network comprising:
receiving, at a base station, a data transmission from at least one remote unit of a plurality of remote units;
attempting to decode said data transmission;
if said data transmission is successfully decoded, setting a binary status flag to a first value;
if said data transmission is unsuccessfully decoded, setting a binary status flag to a second value;
transmitting a superslot comprised of a plurality of timeslots and including a single status message at the beginning of the superslot, wherein the single status message is said binary status flag, to said plurality of remote units;
wherein if said binary status flag is set to said first value said at least one of said plurality of remote units will continue to transmit said data transmission and the remaining remote units of said plurality of remote units will not attempt to transmit; and
wherein if said binary flag is set to said second value said at least one of said plurality of remote units will cease to transmit said data transmission.
10. The method of claim 9 wherein if said binary status flag is set to said second value said remaining remote units of said plurality of remote units will not refrain from transmitting.
11. A method for controlling traffic in a wireless transmission system wherein information is passed from a remote unit to a base unit along a first wireless channel and information is passed from the base unit to a remote unit along a second wireless channel, comprising:
sending a binary status flag set to a first value in a first timeslot along said second wireless channel;
monitoring said first wireless channel for a plurality of timeslots;
if during said plurality of timeslots a transmission from a remote unit is received:
attempting to decode the transmission from said remote unit,
if said attempt to decode is successful:
transmitting a binary status flag set to a second value along said second wireless channel,
predicting the completion time of said transmission,
sending, at a predetermined time prior to the end of said data transmission, a binary status flag set to said first value along said second wireless channel,
wherein said base unit does not transmit any status messages during said plurality of timeslots and said predetermined time is based on consideration of hardware and software latencies in transmitting, receiving, and decoding the status flag.
12. The method of claim 11 wherein said predicting step is conducted based on information contained in said transmission.
13. The method of claim 12 wherein said transmission contains a value indicating the length of the transmission.
14. The method of claim 12 wherein said predicting step is conducted based on the type of information contained in said transmission.
15. The method of claim 12 wherein said plurality of timeslots cover a time interval that corresponds to a round trip between the remote unit and the base unit.
16. The method of claim 15 wherein said round trip time accounts for signal propagation delay over the first and second wireless channels.
17. The method of claim 15 wherein said round trip time accounts for signal processing at the base unit.
18. The method of claim 17 wherein said round trip time accounts for signal propagation delay over the first and second wireless channels.
19. The method of claim 18 wherein said round trip time accounts for signal processing at the remote unit.